Contents - College of Medical and Dental Sciences - University of ...

The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK 

Workshop programme at a glance 

Scientific programme committee 

Organisers 

Contents 

Confidentiality of results presented at this meeting 

Meeting venue and campus accommodation 

Meeting information including venues of meals 

Travel directions and maps 

Instructions for oral and poster presentations 

Details of excursion to Stratford Upon Avon 

Meeting agenda, including details of coach transport 

Detailed scientific meeting agenda 

Oral presentation abstracts 

Session 1 abstracts 1-7: Latency 

Session 2 abstracts 8-13: Immunology I 

Session 3 abstracts 14-21: Pathogenesis 

Session 4 abstracts 22-28: Virus-Cell Interactions I 

Session 5 abstracts 29-34: Gene Expression I 

Session 6 abstracts 35-41: Lytic Replication 

Session 7 abstracts 42-47: Immunology II 

Session 8 abstracts 48-55: Clinical & Epidemiology 

Session 10 abstracts 56-63: Virus-Cell Interactions II 

Session 11 abstracts 64-67: Gene Expression II 

Poster presentation abstracts P1-P18 

Delegate contact details 

Author index 

Page 

2 

3 

3 

4 

5 

6 

7 

10 

10 

11 

12 

19 

20 

28 

35 

44 

52 

59 

67 

74 

83 

92 

97 

116 

128 

1


Workshop programme at a glance 1 

Tuesday 

Time 

July 22 nd 

Wednesday 

July 23 rd 

Thursday 

July 24 th 

Friday 

July 25th 

7:00-8:00 Breakfast 

Session 1 

Session 4 

Session 6 

8:45-10:30 

Latency 

Virus-Cell 

Interactions I 

Lytic Replication 

(abstracts 1-7) (abstracts 22-28) (abstracts 35-41) 

10:30-11:00 Tea/Coffee 

Session 2 

Session 5 

Session 7 

11:00-12:30 

Immunology I Gene Expression I Immunology II 

Saturday 

July 26 th 

Session 10 

Virus-Cell 

Interactions II 

(abstracts 56-63) 

Session 11 

Gene Expression II 

(abstracts 8-13) (abstracts 29-34) (abstracts 42-47) (abstracts 64-67) 

12:30-1:30 Lunch Workshop ends 

Session 3 

Session 8 

1:30-3:30 

Pathogenesis 

Clinical & 

Excursion to Epidemiology 

(abstracts 14-21) Stratford Upon (abstracts 48-55) 

3:30-4:00 

Poster session 

Avon 

Tea/Coffee 

5:30-8:30 

3:30-6:00 

Sponsored by 

Session 9 

4:00-5:30 Registration (abstracts P1-P18) Geneflow 

Round Table 

& welcome 

Sponsored by 

reception buffet 

Agilent 

Barber Institute Technologies 

Evening 

8:30 

8:00 

7:30 

Indian meal 

BBQ 

Conference 

Shimla Pinks Shackleton House 

dinner 

Sponsored by Edgbaston Cricket 

Miltenyi Biotec 

Ground 

Sponsored by BD 

Biosciences 

1 

Oral sessions 1-11 to be held in the Leonard Deacon lecture theatre, Wolfson Centre, The Medical School, University of Birmingham 

Poster session to be held on the Ground Floor of the Wolfson Centre 

2

Scientific Programme Committee 

David Blackbourn, PhD 

University of Birmingham 

Juergen Haas, MD, PhD 

University of Edinburgh 

Paul Kellam, PhD 

University College London 

Frank Niepel, MD 

University of Erlangen 

Thomas Schulz, MD, PhD 

Hannover Medical School 

Scott Wong, PhD 

Oregon Health &Science University/Vaccine 

& Gene Therapy Institute 

Organisers 

David Blackbourn 

Co-chair, Organising Committee 

Cancer Research UK Institute for Cancer 

Studies 


Vincent Drive 

Edgbaston 

Birmingham 

B15 2TT 

United Kingdom 

Tel. +44 (0) 121 415 8804 

d.j.blackbourn@bham.ac.uk 

Conference Coordinators 

& Organising Committee 

Professional Development Centre 


Birmingham 

B15 2TT 



Ethel Cesarman, MD, PhD 

Weill Cornell Medical College, 

Andrew Hislop, PhD 


John Nicholas, PhD 

Johns Hopkins University 

Rolf Renne, PhD 

University of Florida 

Adrian Whitehouse, PhD 

University of Leeds 

Paul Kellam 

Co-chair, Organising Committee 

Centre for Virology (Bloomsbury) 


46 Cleveland Street 

London 

W1T 4JF 


Tel. +44 (0) 20 7679 9559 

p.kellam@ucl.ac.uk 

3


Confidentiality of results presented at this meeting 

To continue in the sprit of the highly successful Workshops in the past, we hope that this 11 th 

International Workshop on KSHV and Related Agents will provide ample opportunity for informal 

discussions and exchange of ideas, data and reagents. To promote such interactions, and to 

encourage participants to present unpublished results, please treat all scientific presentations and 

abstracts in this book as confidential unpublished research. Abstracts may not be cited. If you want to 

cite results, please do so as a “personal communication”, only with agreement from the respective 

authors. Results presented at this meeting may not be used as the basis of research without the 

explicit permission of the corresponding author. The KSHV field has already benefited from many 

collaborative ventures and a relatively free flow of reagents. Let’s encourage presentation of recent 

data and establish the KSHV workshops as an exciting series of meetings that are worth attending 

and cannot be replaced by scientific journals. 

4


Welcome to the 11 th International Workshop on KSHV and Related Agents at 

the University of Birmingham, Medical School, Birmingham, England, 22-26 

July 2008. 

Meeting Venue 


Medical School 

Vincent Drive 

Edgbaston 

Birmingham 

B15 2TT 

Email – med-pdcbookings@bham.ac.uk 

Tel – +44 (0)121 414 8608 

Scientific sessions will be held in the Wolfson Centre of the Medical School: 

• The oral sessions will be held in the Leonard Deacon lecture theatre, Wolfson Centre 

• The poster session will be held on the Ground Floor of the Wolfson Centre 

Campus Accommodation 

Shackleton 

The Vale 

Edgbaston Park Road 

Birmingham 

B15 3SZ 

Check in from 2pm on 22 nd July 

Internet access available in all rooms, please ask at Shackleton reception 

24 hour reception 

Breakfast will be served from 7am; coaches will then transfer delegates to the conference please be at 

reception for 8am. 

5

Name Badges 


Please wear your name badge at all times during the conference. 

Meals 

The following meals are included in your registration: 

Welcome Reception 

Tuesday, 5.30pm – 8.30pm – Barber Institute, University of Birmingham, Edgbaston, Birmingham, B15 

2TS, Tel 0121 414 7333. 

Lunch 

Wednesday 

Thursday 

Friday 

Evening Meals 

Wednesday, 8pm – 11pm – Shimla Pinks, 214 Broad St, Birmingham, B15 1AY, 

Tel 0121 633 0366 

Thursday, 8pm – BBQ at Rio’s Bar, The Vale, Shackleton, Edgbaston Park Road, Edgbaston, 

Birmingham, B15 3SZ. Generously sponsored by Miltenyi Biotec. 

Friday, 7pm -11pm – Conference dinner at Edgbaston Cricket Ground, County Ground 

Edgbaston Rd, Birmingham, B5 7QU, Tel 0870 062 1902. Generously sponsored by BD Biosciences. 

Transport 

For those staying at Shackleton coaches have been arranged to transport delegates between 

accommodation and restaurant locations. Please wait for the coach at the times stated on the 

agenda. 

Please note coaches have been arranged for those delegates staying at Shackleton only. 

Delegates staying elsewhere must arrange own transport. 

Taxis - TOA – 0121 427 8888 

6

Travel Directions and Maps 


7

By motorway 


Approaching from the north west or south east along the M6: 

• Leave at Junction 6 (signposted Birmingham Central) to join the A38(M) 

• At the end of the motorway, keep to the right, go over a flyover, then through some underpasses 

to join the A38 Bristol Road 

• The University is on your right, two and a half miles from the city centre 

Approaching from the M42 north: 

• Leave at Junction 8 to join the M6 northbound and follow the instructions above 

Approaching from the south west: 

• Leave the M5 at Junction 4 signposted Birmingham (SW) to join the A38 

• The University is approximately eight miles from the motorway 

Approaching from the M40: 

• It is easier to turn south on the M42 and leave at Junction 1, heading north on the A38 Bristol 

Road 

• The University is approximately eight miles from the motorway 

8

By rail 


Most cross-country services to Birmingham arrive at New Street Station. Up to six trains an hour 

depart for the University on the cross-city line (ten minutes to University station, final destination 

Longbridge or Redditch). The centre of the main campus is a five-minute walk from University Station. 

By coach 

There are frequent express coach services to Birmingham from London, Heathrow and Gatwick 

Airports, and many UK cities. The long-distance coach station is in Digbeth in the city centre. 

By bus 

Numbers 61, 62 and 63 travel to the University’s Edgbaston and Selly Oak campuses, while the 21 

and 44 serve the Medical School and Queen Elizabeth Hospital. The services all run frequently from 

the city centre. There is a travel information office outside New Street Station, where you can obtain 

bus timetables and departure point information. Maps can be found throughout the city centre 

indicating bus stop locations. 

By taxi 

There are taxi ranks at New Street Station and throughout the city centre. The journey to the 

University takes about ten minutes. 

By air 

Birmingham International Airport has direct flights from locations in the UK, as well as from the 

USA, Canada, Europe and the Middle East. 

The journey by taxi from the airport to the University takes approximately half an hour. Alternatively, 

Air-Rail Link provides a free, fast connection between the airport terminals and Birmingham 

International railway station. Air-Rail Link operates every two minutes (journey time 90 seconds). 

Birmingham International railway station has frequent services to New Street Station in the city centre 

(journey time around 15 minutes). 

If you are arriving at London, there is a frequent train service from London Euston railway station to 

New Street Station (journey time around 1 hour 30 minutes). 

• From Heathrow Airport. Take the Heathrow Express train to Paddington Station and then the 

Underground or a taxi to Euston Station. Alternatively, an Airbus runs from Heathrow Airport direct 

to Euston Station 

• From Gatwick Airport. Take the Airport Express train to Victoria Station and then the Underground 

or a taxi to Euston Station 

9

Oral Presentations 


Oral presentations are strictly limited to 12 minutes, allowing an additional 1 – 2 minutes for questions. 

Both PC and Macintosh computers are available for oral presentations in the Leonard Deacon lecture 

theatre, where the presentations will be made. 

Powerpoint presentations should be brought on USB stick and loaded by the times stated below. 

Identify the presentation with the abstract number, given previously, and with the last name of 

the presenting author. 

To facilitate smooth running of the scientific sessions, the use of personal laptops for presentations is 

actively discouraged. 

Timetable for uploading presentations to the podium computers in the Leonard Deacon lecture 

theatre: 

Session Abstract numbers Upload by 

1 1-7 8:45 am, Wed July 23 rd 

2 8-13 11:00 am, Wed July 23 rd 

3 14-21 1:30 pm, Wed July 23 rd 

4 22-28 8:45 am, Thurs July 24 th 

5 29-34 11:00 am, Thurs July 24 th 

6 35-41 8:45 am, Fri July 25 th 

7 42-47 11:00 am, Fri July 25 th 

8 48-55 1:30 pm, Fri July 25 th 

10 56-63 8:45 am, Sat July 26 th 

11 64-67 11:00 am, Sat July 26 th 

Poster Viewing and Poster Session 

Generously sponsored by Agilent Technologies 

The poster board dimensions are 1m x 1m. Please identify in the poster title the abstract number, 

given previously. 

Posters can be placed on Wednesday 23 July from 8:30am. The poster boards are located on the 

Ground Floor in the Wolfson Centre. Posters should be removed by 5:00 pm Friday July 25 th . 

Poster and Drinks Reception, Wolfson Centre, Wednesday 23 July, 3:30 pm – 6:00 pm 

Excursion to Stratford Upon Avon 

Generously sponsored by Geneflow 

An excursion to Stratford Upon Avon has been arranged for Thursday 24 July, the coaches will depart 

from the Medical School following the morning session, at 12.30 sharp. A packed lunch will be 

provided, collect this on your way out. Please ensure you and your companions have your passes 

ready to board the coach. 

Upon arrival at Stratford you will be met by guides, the town walk will take in the three Shakespeare 

town houses, Shakespeare's Birthplace, Nahs's House and the site of the New Place and Hall's Croft, 

the Royal Shakespeare Theatres, Shakespeare's Grammar School and Holy Trinity Church, where 

William Shakespeare is buried. It is a very lively, entertaining and informative two hours. 

After the tour you will have the opportunity to explore the town for yourself, enjoying the views and an 

excellent variety of shops, from small individual retailers to major outlets, ideal for window shopping or 

browsing. Alternatively, perhaps you would prefer to relax and enjoy the views by the River Avon. 

Please ensure you are back at the collection point by 4.45pm, when the coaches will return to 

Shackleton. 

10

Agenda 


Date Time Event 

Tuesday 22nd 5.30 - 8.30pm 

Welcome Reception, Barber Institute, University 

Campus 

5.30 – 8.30pm Shuttle coach between Shackleton & Barber Institute 

Wednesday 23rd 8.00am Coach pick up at Shackleton 

8.45 – 10.30am Session 1 – Latency (abstracts 1-7) 

10.30 – 11.00am Break 

11.00am - 12.30pm Session 2 – Immunology I (abstracts 8-13) 

12.30 - 1.30pm Lunch 

1.30 - 3.30pm Session 3 – Pathogenesis (abstracts 14-21) 

3.30 – 6.00pm Posters & Drinks Reception 

6.00pm Coach pick up at Medical School return to Shackleton 

8.00pm Coach pick up at Shackleton 

8.30 – 11.30pm Evening Meal - Shimla Pinks Restaurant 

11.30pm Coach pick up Shimla Pinks, Broad Street 

Thursday 24th 8.00am Coach pick up at Shackleton 

8.45 – 10.30am 

Session 4 – Virus-Cell Interactions 


10.30 - 11am Break 

11.00am - 12.30pm 

Session 5 – Gene Expression 


12.30 - 6pm Excursion - Stratford Upon Avon 

4.45pm Coach pick up Stratford Upon Avon 

8.00pm Evening Meal – BBQ, Shackleton 

Friday 25th 8.00am Coach pick up at Shackleton 

8.45 – 10.30am 

Session 6 – Lytic Replication 


10.30 – 11.00am Break 

11.00 - 12.30pm Session 7 – Immunology II (abstracts 42-47) 

12.30 - 1.30pm Lunch 

1.30 - 3.30pm Session 8 – Clinical & Epidemiology (abstracts 48-55) 

3.30 - 4.30pm Session 9 – Round Table 

5.30pm Coach pick up at Medical School return to Shackleton 

7.00pm Coach pick up at Shackleton 

7.30 – 11.00pm Evening Meal - Edgbaston Cricket Ground 

11.00pm Coach pick up Cricket Ground return to Shackleton 

Saturday 26th 7.30 - 8.30am Coach pick up at Shackleton 

8.45 – 10.30am Session 10 – Virus-Cell Interactions II (abstracts 56-63) 

10.30 - 11am Break 

11.00am - 12.30pm 

Session 11 – Gene Expression II 


12.30pm Conference Close 

11


Detailed Scientific Meeting 

Agenda 

12

Wednesday, July 23 rd 


Session 1: Latency 

8:45 am – 10:30 am 

Chair: Rolf Renne 

Abstract # Title Authors 

1 DELETION OF LANA FROM RHESUS 

RHADINOVIRUS (RRV) GENERATES A 

HIGHLY LYTIC RECOMBINANT VIRUS 

2 DIVERGENCE OF THE NUCLEAR 

LOCALIZATION SIGNAL WITHIN THE 

ORF73 LATENCY-ASSOCIATED NUCLEAR 

ANTIGENS (LANA) OF KSHV AND THE 

MACAQUE RV1 AND RV2 

3 

RHADINOVIRUSES 

NUCLEOPHOSMIN IS A NOVEL 

REGULATOR OF KSHV REPLICATION VIA 

FUNCTIONAL INTERACTIONS WITH VIRAL 

CYCLIN AND LANA 

4 ROLE OF BET PROTEINS IN THE 

FUNCTION OF KSHV AND MHV68 LANA 

5 INTERACTION OF KSHV LANA-1 WITH 

USP7: IMPLICATIONS FOR P53 FUNCTION 

6 KSHV ENCODED LANA INTERACTS WITH 

THE NUCLEAR MITOTIC APPARATUS 

PROTEIN TO REGULATE GENOME 

7 

MAINTENANCE AND SEGREGATION 

MULTIPLE DOMAINS CONTRIBUTE TO THE 

ASSOCIATION OF LANA WITH HOST 

CHROMATIN COMPONENTS 

Session 2: Immunology I 

11:00 am – 12:30 pm 

Chair: Andrew Hislop 


8 INDUCTION OF CYTOKINES BY HHV-8 IN 

MYELOID DENDRITIC CELLS AND B CELLS 

9 KSHV K5 COUNTERACTS TETHERIN, A 

NOVEL COMPONENT OF INNATE 

ANTIVIRAL IMMUNITY, TO FACILITATE 

VIRUS RELEASE 

10 MOLECULAR MECHANISM OF 

11 

BST2/TETHERIN DOWNREGULATION BY 

KSHV-K5 

UPREGULATION OF THE TLR3 PATHWAY 

BY KSHV DURING PRIMARY INFECTION 

OF MONOCYTES 

12 KSHV EVADES INNATE IMMUNITY BY 

SUPPRESSION OF TLR4 EXPRESSION 

13 THE KSHV LYTIC PROTEIN VOX2 AND ITS 

CELLULAR COUNTERPART, CD200, 

INHIBIT EPITOPE-SPECIFIC T CELL 

RESPONSES 

Kwun Wah Wen, Chelsey Hilscher, Dirk P. 

Dittmer and Blossom Damania 

Kellie L. Burnside and Timothy M. Rose 

Grzegorz Sarek, Annika Järviluoma, Henna 

Syrjäkari, Sari Tojkander, Salla Vartia, 

Marikki Laiho, and Päivi M. Ojala 

Magdalena Weidner-Glunde, Matthias 

Ottinger, Daniel Pliquet, Ronald Frank and 

Thomas F. Schulz 

W. Albrecht, E. Gellermann, A. Viejo- 

Borbolla, T. F. Schulz 

Huaxin Si, Subhash C. Verma, Michael 

Lampson, Qiliang Cai, Erle S. Robertson 

Ryo Nasu, Satoko Matsumura, Naoko Tanese 

and Angus Wilson 

Emilee Knowlton, Giovanna Rappocciolo, 

Paolo Piazza, Heather Hensler, Frank J. 

Jenkins, Mariel Jais, Charles R. Rinaldo 

Edward Tsao, Sam Wilson, Claire Pardieu, 

Ben Webb, Imogen Lai, Stuart Neil, Greg 

Towers, and Paul Kellam 

Mandana Mansouri, Janet Douglas, Kasinath 

Viswanathan, Jean Gustin, Ashlee Moses and 

Klaus Früh 

John West, Sean Gregory and Blossom 

Damania 

Dimitris Lagos, Richard James Vart, Fiona 

Gratrix, Samantha Jane Westrop, Ping-Pui 

Wong, Nesrina Imami, Mark Bower, Frances 

Gotch and Chris Boshoff 

Rachel Colman, Karen Misstear, Heather 

Long, Omar Quereshi, David Sansom, 

Andrew Hislop, David J. Blackbourn 

13

Wednesday, July 23 rd 


Session 3: Pathogenesis 

1:30 am – 3:30 pm 

Chair: David Blackbourn 


14 GENERATION OF RHESUS RHADINOVIRUS 

LACKING EXPRESSION OF VGPCR AND 

VCD200 FOR THE ASSESSMENT OF THEIR 

ROLES IN DISEASE DEVELOPMENT IN A 

RHESUS MACAQUE MODEL OF KSHV 

15 

INFECTION 

GENERATION OF VFLIP TRANSGENIC 

MICE: A MODEL TO STUDY KSHV- 

ASSOCIATED LYMPHOMAGENESIS 

16 DEVELOPMENT OF ANIMAL MODELS OF 

KSHV INFECTION AND AIDS-ASSOCIATED 

DISEASE BASED ON MACAQUE 

RHADINOVIRUSES 

17 β ARRESTINS REGULATE KSHV GPCR 

ACTIVITY AND ARE MODULATED BY 

CANNABINOIDS 

18 ACTIVATION OF NF-⎢B BY KSHV K15 

19 

INVOLVES RECRUITMENT OF NIK (NF-ΚB 

INDUCING KINASE) 

KAPOSI'S SARCOMA ASSOCIATED HERPES 

VIRUS (KSHV/HHV-8) INDUCES 

ANGIOGENIN DURING INFECTION OF 

HUMAN DERMAL MICROVASCULAR 

ENDOTHELIAL (HMVEC-D) CELLS THAT IS 

CRITICAL FOR ANTI-APOPTOSIS, CELL 

20 

PROLIFERATION AND ANGIOGENESIS 

ROLE OF K15 IN KAPOSI’S SARCOMA 

HERPESVIRUS INDUCED ENDOTHELIAL 

CELL MIGRATION 

21 DOWNREGULATION OF GALECTIN-3 IN 

KSHV INFECTED DMVEC CELLS AND 

KAPOSI’S SARCOMA: IMPLICATIONS FOR 

TUMORIGENESIS 

Ryan D. Estep, Elisa Cardenas and Scott W. 

Wong 

Gianna Ballon, Amy Chadburn, Yi-Fang Liu, 

Yoshiteru Sasaki, Klaus Rajewsky, Ethel 

Cesarman 

A. Gregory Bruce, Jonathan T. Ryan, 

Courtney Gravett and Timothy M. Rose 

Xuefeng Zhang, Yehoshua Maor, Jerome E. 

Groopman 

Anika Hävemeier, Macel Pietrek and Thomas 

F. Schulz 

Sathish Sadagopan, Neelam-Sharma Walia, 

Mohanan Valiya Veettil, Virginie Bottero, Rita 

Levine and Bala Chandran 

Bernd Hillenbrand, Antje Bürger, Irina 

Fischer, Khaled Alkharsah and Thomas F. 

Schulz 

Donald J. Alcendor Wen Qui Zhu Prashant 

Desai and Gary S. Hayward 

14

Thursday, July 24 th 


Session 4: Virus-Cell Interactions I 

8:45 am – 10:30 am 

Chair: Scott Wong 


22 ANIMAL MODELS OF KAPOSI'S SARCOMA 

REVEAL A ROLE OF KSHV VGPCR AND 

RAC1 IN ANGIOGENESIS AND GENETIC 

INSTABILITY 

23 INSIGHTS INTO KSHV ACTIVATION OF 

THE IKK SIGNALOSOME: CRYSTAL 

24 

STRUCTURE OF A VFLIP-IKKγ COMPLEX 

THE VIRAL INHIBITOR OF APOPTOSIS 

VFLIP/K13 PROTECTS ENDOTHELIAL 

CELLS AGAINST SUPEROXIDE - INDUCED 

CELL DEATH 

25 ROLE OF DEFECTIVE OCT-2 AND OCA-B 

EXPRESSION IN IMMUNOGLOBULIN 

PRODUCTION AND KSHV LYTIC 

REACTIVATION IN PRIMARY EFFUSION 

LYMPHOMA 

26 KAPOSI’S SARCOMA ASSOCIATED 

HERPESVIRUS (KSHV) INDUCES 

TUMORIGENIC CELLULAR MIRNAS IN 

LATENTLY INFECTED ENDOTHELIAL CELLS 

27 KSHV AND CELLULAR MIRNA 

EXPRESSION DURING LATENCY AND 

LYTIC REACTIVATION IN PEL CELLS 


HERPESVIRUS (KSHV/HHV-8) UTILIZES 

MACROPINOCYTIC PATHWAY TO ENTER 

HUMAN DERMAL MICROVASCULAR 

ENDOTHELIAL (HMVEC-D) AND HUMAN 

UMBILICAL VEIN ENDOTHELIAL (HUVEC) 

CELLS 

Session 5: Gene Expression I 

11:00 am – 12:30 pm 

Chair: Adrian Whitehouse 


29 SCREENING FOR XINJIANG KS 

ASSOCIATED GENES AND THE STUDY ON 

THEIR MECHANISMS 

30 HOST CELL AND VIRAL MICRORNA 

(MIRNA) EXPRESSION IN B CELL 

31 

LYMPHOMAS 

CELLULAR AND VIRAL GENE EXPRESSION 

PROFILING 

MONOCYTES 

IN KSHV-INFECTED 

32 PROFILING OF CELLULAR AND VIRAL 

33 

MICRORNAS IN KAPOSI SARCOMA AND 

VIRAL-ASSOCIATED LYMPHOMA 

INEFFICIENT CODON USAGE IN V-FLIP 

MRNA LEADS TO TRANSCRIPT 

34 

INSTABILITY 

ROLE OF MURINE GAMMAHERPESVIRUS- 

68 ORF37 IN MEDIATING INHIBITION OF 

HOST GENE EXPRESSION 

Lucas Cavallin, Qi Ma, Rui Zhang, E. 

Margarita Duran, Pascal J. Goldschmidt- 

Clermont and Enrique A. Mesri 

Claire Bagnéris, Alexander V Ageichik, Nora 

Cronin, Bonnie Wallace, Mary Collins, Chris 

Boshoff, Gabriel Waksman and Tracey Barrett 

Michael Stürzl, Gaby Sander, Nathalie Gonin- 

Laurent, Kristina Weinländer, Elisabeth 

Naschberger, Ramona Jochmann, Khaled R. 

Alkharsah, Thomas F. Schulz, Margot Thome, 

Frank Neipel and Mathias Thurau 

Daniel DiBartolo, Elizabeth Hyjek, Shannon 

Keller, Ilaria Guasparri, Ren Sun, Amy 

Chadburn, Daniel M Knowles, and Ethel 

Cesarman 

Rebecca L. Skalsky Wendell Miley, Rachel 

Bagni, Soo-Jin Han Jianhong Hu, Chang Hee 

Kim, Rolf Renne and Denise Whitby 

Soo-Jin Han Jianhong Hu, Karlie Plaisance, 

Wendell Miley, Rachel Bagni, Chang Hee Kim, 

Denise Whitby and Rolf Renne 

Hari Raghu, Neelam Sharma Walia, Mohanan 

Valiya Veettil, Sathish Sadagopan and Bala 

Chandran 

Lei YANG, Yan ZENG, Dong-Mei LI,Ling-ling 

XIAN,Xiao –Fei ZHOU,Xiao-Hua TAN,Jin 

HUANG,Feng LI,Jian-Xin XIE,Hui 

ZHANG,Sheng Wang,Xian-Dao LUO 

Eve M Coulter, Dan Frampton, Ed Tsao, Paul 

Kellam 

Sean Gregory, Ling Wang, John West, 

Chelsey Hilscher, Dirk P. Dittmer and 

Blossom Damania 

Andrea J. O’Hara, Bruce J. Dezube, William 

Harrington Jr., Blossom Damania, and Dirk P. 

Dittmer 

Priya Bellare, Andrew T Dufresne and Don 

Ganem 

L. Roaden, V. Sheridan, B. Lane, R. Sun, B. 

Dutia and B. Ebrahimi 

15

Friday July 25 th 


Session 6: Lytic Replication 

8:45 am – 10:30 am 

Chair: Thomas Schulz 


35 FUNCTIONAL CHARACTERIZATION OF Yan Wang, Charles Hollow and Yan Yuan 

KAPOSI'S SARCOMA–ASSOCIATED 

HERPESVIRUS 

MUTAGENESIS 

ORF K8 BY BAC-BASED 

36 APPLICATION OF ACTIVE KINOME 

COLLECTION FOR IDENTIFICATION OF A 

NOVEL KINASE FAMILY INVOLVED IN 

37 

REACTIVATION OF KSHV 

NOVEL FUNCTIONS OF K-RTA AND K-BZIP 

AS SUMO-TARGETING LIGASES AND 

38 

EPIGENETIC REGULATORS 

X-BOX BINDING PROTEIN 1 DOES NOT 

INDUCE EBV LYTIC REPLICATION IN 

39 

PRIMARY EFFUSION LYMPHOMA 

RECRUITMENT OF THE COMPLETE HTREX 

COMPLEX IS REQUIRED FOR KSHV 

INTRONLESS MRNA NUCLEAR EXPORT 

40 

AND VIRUS REPLICATION 

DOMAIN STRUCTURE AND FUNCTION OF 

KSHV ORF57 PROTEIN IN PROTEIN- 

PROTEIN 

INTERACTIONS 

AND PROTEIN-RNA 

41 KSHV ORF57 ENHANCES TRANSLATION 

OF VIRAL INTRONLESS MRNAS 

Fang Cheng, Markku Varjosalo, Anne 

Lehtonen, Magdalena Weidner-Glunde, Päivi 

J. Koskinen, Thomas Schulz, Jussi Taipale, 

and Päivi M. Ojala 

Hsing-Jien Kung, Pei-Ching Chang, Latricia 

Fitzgerald, Tom Ellison, Paul Luciw and Yoshi 

Izumiya 

Imogen Yi-Chun Lai and Paul Kellam 

James R. Boyne and Adrian Whitehouse 

Vladimir Majerciak and Zhi-Ming Zheng 

James R. Boyne, Adam Taylor and Adrian 

Whitehouse 

Session 7: Immunology II 

11:00 am – 12:30 pm 

Chair: Paul Kellam 


42 MODULATION BY KSHV OF LIGANDS Alexis Madrid and Don Ganem 

THAT MEDIATE NK CELL RECOGNITION 

43 SCREENING FOR NATURALLY OCCURRING 

ANTI-KCP (KSHV COMPLEMENT CONTROL 

PROTEIN; ORF4) ANTIBODIES IN KSHV- 

INFECTED PATIENTS AND GENERATION 

OF SPECIFIC MONOCLONAL ANTIBODIES 

FOR PATHOGENESIS STUDIES 

44 A KSHV VIRAL HOMOLOGUE OF CELLULAR 

CD200 (VOX2) INDIRECTLY SUPPRESSES 

GRANULOCYTE OXIDATIVE ACTIVITY IN 

HUMAN BLOOD 

45 CD8+ T CELL RECOGNITION OF THE KSHV 

LATENT PROTEIN LANA1 

46 CYTOKINE RESPONSES BY CD8 T 

LYMPHOCYTES TO KSHV/HHV8 LYTIC 

47 

AND LATENCY PROTEINS 

INFECTION OF LYMPHOID CELLS BY KSHV 

IN CULTURED PRIMARY HUMAN 

TONSILLAR LYMPHOID CELLS EX VIVO 

Anna M. Blom, Marcin Okroj, Linda Mark, 

Zoltan Korodi, Rosamaria Tedeschi, Joakim 

Dillner and O. Brad Spiller 

Karen Misstear, Rachel Colman, Hema 

Chahal, Janet Lord, David Blackbourn 

Shereen Sabbah & Andrew D Hislop 

Lauren Lepone, Giovanna Rappocciolo, 

Emilee Knowlton, Paolo Piazza, Mariel Jais, 

Frank J. Jenkins and Charles R. Rinaldo 

Jinjong Myoung and Don Ganem 

16

Friday July 25 th 


Session 8: Clinical & Epidemiology 

1:30 pm – 3:30 pm 

Chair: Ethel Cesarman 


48 PASSIVE TRANSFER OF HHV-8 

ANTIBODIES FROM BLOOD DONORS TO 

TRANSFUSION RECIPIENTS AND 

POSSIBLE 

INFECTION 

PROTECTION FROM HHV-8 

49 HEIGHTENED REDOX STATUS OF 

50 

PRIMARY EFFUSION LYMPHOMA CELLS 

CAN BE EXPLOITED THERAPEUTICALLY BY 

DECREASING RESISTANCE TO OXIDATIVE 

STRESS 

RAPAMYCIN IS EFFECTIVE AGAINST PTEN 

POSITIVE 

MALIGNANCIES 

AIDS-DEFINING 

51 GEOGRAPHIC VARIATION OF THE 

PREVALENCE OF KAPOSI’S SARCOMA- 

ASSOCIATED HERPESVIRUS AND RISK 

FACTORS FOR TRANSMISSION IN WOMEN 

FROM 8 COUNTRIES IN FOUR 

52 

CONTINENTS 

SERUM EPIDEMIOLOGICAL STUDIES ON 

KAPOSI'S SARCOMA IN XINJIANG 

53 KAPOSI’S SARCOMA-ASSOCIATED 

HERPESVIRUS SEROCONVERSION AND 

SEROREVERSION IN A MATERNAL- 

INFANT COHORT IN ZAMBIA 

54 DISTRIBUTION OF KSHV MICRORNA 

POLYMORPHISMS IN AIDS-KS, 

CLASSICAL KS, AND MULTICENTRIC 

CASTLEMAN’S DISEASE 

55 EARLY CHILDHOOD INFECTION BY 

KAPOSI’S SARCOMA-ASSOCIATED 

HERPESVIRUS AND EPSTEIN-BARR VIRUS 

IN ZAMBIA 

4:00 pm – 5:30 pm 

Session 9: Round Table 

Topic and chairs to be confirmed 

Ashley L. Fowlkes, Cedric Brown, Minal M. 

Amin, John Roback, Robert Downing, Esau 

Nzaro, Jonathan Mermin, Wolfgang Hladik, 

Sheila C. Dollard 

Darya Bubman, Utthara Nayar, Balasz 

Csernus, Ilaria Guasparri, Ethel Cesarman 

Debasmita Roy, Sang-Hoon Sin, Ling Wang, 

Blossom A. Damania, Dirk P. Dittmer 

Silvia de Sanjose, Georgina Mbisa, Sussana 

Perez, Sukhon Sukvirach, Nguyen Trong 

Hieu, Hai-Rim Shin, Pham Thi Hoang Anh, 

Jaiye O Thomas, Eduardo Lazcano, Elena 

Matos, Rolando Herrero, Nubia Muñoz, Silvia 

Franceschi, Denise Whitby 

Lei YANG, Xiao-Hua TAN, Jiang-Mei QING, 

Feng LI, Shu-Xia GUO, Jian-Xin XIE, Jin 

HUANG, Dong-Mei LI, Yan ZENG 

Veenu Minhas, Kay L. Crabtree, Janet 

Wojcicki, Tendai J. M’soka, Chipepo Kankasa, 

Charles D. Mitchell and Charles Wood 

Vickie Marshall, Eliza Martró, Elizabeth 

Brown, Dian Wang, Alex Ray, Maria 

Nazzarena Labo, The Classical Kaposi’s 

Sarcoma Working Group, Robert Yarchoan, 

Jordi Casabona, Rolfe Renne and Denise 

Whitby 

Veenu Minhas, Brad P Brayfield, Tendai J. 

M’soka, Chipepo Kankasa, Charles D. Mitchell 

and Charles Wood 

17

Saturday July 26 th 


Session 10: Virus-Cell Interactions II 

8:45 am – 10:45 am 

Chair: John Nicholas 


56 INTRACELLULAR LOCALIZATION MAP OF 

HHV-8 PROTEINS 

57 KSHV INTERFERON REGULATORY FACTOR 

4, A NOVEL CBF1 INTERACTION PARTNER 

58 CHARACTERISATION OF ENDOPLASMIC 

RETICULUM (ER) STRESS INDUCERS THAT 

LEAD TO KSHV REACTIVATION FROM 

59 

LATENCY; IDENTIFYING PHYSIOLOGICAL 

TRIGGERS? 

INTEGRIN αVβ3 BINDS TO THE RGD MOTIF 

OF THE GLYCOPROTEIN B OF KAPOSI’S 

SARCOMA-ASSOCIATED HERPESVIRUS 

(KSHV/HHV8) AND FUNCTIONS AS AN 

RGD-DEPENDENT ENTRY RECEPTOR 


HERPESVIRUS (KSHV/HHV-8) FORMS A 

MULTI-MOLECULAR COMPLEX OF 

INTEGRINS (αVβ5, αVβ3 AND α3β1), CD98 

AND XCT DURING INFECTION OF HUMAN 

DERMAL MICROVASCULAR ENDOTHELIAL 

(HMVEC-D) CELLS 

61 MURINE GAMMAHERPESVIRUS 68 

ESTABLISHES A PERSISTENT INFECTION 

IN ENDOTHELIAL CELLS 

62 TARGETING EXTRACELLULAR HSP90 

REDUCES KSHV GENE EXPRESSION 

DURING DE NOVO INFECTION BY 

INHIBITING 

ACTIVATION 

MAPK PATHWAY 

63 FAK AND SHP2 ARE REQUIRED FOR KSHV 

VGPCR SIGNALING 

Gaby Sander, Andreas Konrad, Mathias 

Thurau, Effi Wies, Rene Leubert, Elisabeth 

Kremmer, Holger Dinkel, Thomas Schulz, 

Frank Neipel and Michael Stürzl 

Katharina Heinzelmann, Barbara Scholz, 

Elisabeth Kremmer, Jürgen Haas, Even 

Fossum, Bettina Kempkes 

Lucy Dalton-Griffin, Ed Tsao and Paul Kellam 

H. Jacques Garrigues, Yelena E. 

Rubinchikova, C. Michael DiPersio and 

Timothy M. Rose 

Mohanan Valiya Veettil, Fu-Zhang Wang, 

Sathish Sadagopan, Neelam Sharma-Walia, 

Hari Raghu, Laszlo Varga and Bala Chandran 

A.L. Suárez and L.F. van Dyk 

Zhiqiang Qin, Jennifer Isaacs and Chris 

Parsons 

Thomas Bakken, Chris Boshoff and Mark 

Cannon 

Session 11: Gene Expression II 

11:15 am – 12:15 pm 

Chair: Dirk Dittmer 


64 DEVELOPMENT OF A COMPREHENSIVE Linda Persson, Scott Millman and Angus 

TRANSCRIPTIONAL NETWORK FOR KSHV Wilson 

65 KSHV ORF57 AND RNA EXPORT FACTORS: 

ROLES OF UAP56, URH49, RBM15, AND 

OTT3 IN ORF57 EXPRESSION AND 

66 

FUNCTION 

DYNAMICS OF K-RTA RECRUITMENT ON 

THE KSHV GENOME REVEAL NOVEL 

67 

REGULATION BY NF-KB 

TYPE I INTERFERONS SUPPRESS MURINE 

GAMMAHERPESVIRUS-68 LYTIC 

INFECTION AT THE VIRUS 

TRANSCRIPTOME LEVEL VIA DIRECT 

MODULATION 

ACTIVITY 

OF RTA PROMOTER 

Merlyn Deng, Vladimir Majerciak, Barbara K. 

Felber, and Zhi-Ming Zheng 

Thomas J. Ellison, Chie Izumiya, Paul A. 

Luciw, Hsing-Jien Kung, Yoshihiro Izumiya 

L. Roaden, B. Manso, B. Lane, E. Arico and 

B. Ebrahimi 

18


Abstracts 

19


Session 1 abstracts 1-7: 

Latency 

20


Latency Abstract 1 

DELETION OF LANA FROM RHESUS RHADINOVIRUS (RRV) GENERATES A 

HIGHLY LYTIC RECOMBINANT VIRUS 

Kwun Wah Wen, Chelsey Hilscher, Dirk P. Dittmer and Blossom Damania 

Department of Microbiology and Immunology and Lineberger Cancer Center, University of 

North Carolina at Chapel Hill, North Carolina 27599. 

Abstract 

Rhesus monkey rhadinovirus (RRV) is a gamma herpesvirus that is closely related to the 

human Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8). RRV serves as an in 

vitro and an in vivo model for KSHV. RRV can be grown to high titers on rhesus 

fibroblasts and the availability of the RRV lytic system facilitates analysis of viral 

replication and the contribution of individual open-reading frames to viral fitness. We 

had previously reported that RRV LANA (R-LANA) can suppress lytic viral replication and 

that R-LANA inhibits Rta/Orf50 transactivation of lytic promoters resulting in lower viral 

titers (Dewire and Damania, J. Virology, 2005). Here we present data on the construction 

of a RRVΔLANA/GFP recombinant virus by homologous recombination. Integrity of the 

recombinant virus was confirmed by Southern blot analysis, restriction digest and PCR. 

We compared replication kinetics of RRVΔLANA/GFP, RRV-GFP, wild-type (WT) RRV, and 

a revertant virus (RRVREV) using one-step growth curves. Viral replication was quantitated 

using traditional plaque assays as well as real-time PCR based genome quantification 

assay. We found that the RRVΔLANA/GFP recombinant virus exhibits highly lytic 

replicative properties compared to RRV-GFP, WT RRV, and the revertant virus. We also 

employed a quantitative real time PCR-based RRV viral array to transcriptionally profile 

lytic gene expression during de novo infection using RRVΔLANA/GFP and RRV-GFP 

recombinant viruses. The RRVΔLANA/GFP virus displayed increased lytic gene expression 

at all time points post-infection compared to RRV-GFP. 

Presenting author E-mail: kenwen@med.unc.edu 

21



DIVERGENCE OF THE NUCLEAR LOCALIZATION SIGNAL WITHIN THE ORF73 

LATENCY-ASSOCIATED NUCLEAR ANTIGENS (LANA) OF KSHV AND THE 

MACAQUE RV1 AND RV2 RHADINOVIRUSES 

Kellie L. Burnside 1 and Timothy M. Rose 1,2 

1 Seattle Children’s Hospital Research Institute and 2 University of Washington, Seattle WA 

Abstract 

KSHV LANA is targeted to the nucleus of infected cells by a discrete lysine/arginine-rich 

nuclear localization signal (NLS; Region I), which allows LANA to enter the nucleus and 

mediate viral episome persistence, interact with cellular proteins and play a role in 

latency and tumorigenesis. Conserved homologs of LANA that also exhibit nuclear 

localization have been identified in KSHV-like macaque herpesviruses belonging to the 

RV1 and RV2 lineages of Old World primate rhadinoviruses. To characterize the signals 

mediating nuclear localization, the N-terminal domains of the LANA homologs of the 

macaque RV1 (RFHVMn) and RV2 (RRV and MneRV2) rhadinoviruses were structurally 

and functionally compared to the KSHV NLS. A distinct lysine/arginine-rich NLS motif 

was identified in RFHVMn LANA (Region II) that was adjacent but evolutionarily unrelated 

to the KSHV NLS. An extended glycine/arginine-rich NLS, spanning 46 residues across 

Regions I and II, was identified within the RRV and MneRV2 LANAs. Although 

evolutionarily distinct, the NLS motifs of the human and macaque RV1 rhadinoviruses, 

KSHV and RFHVMn, both conform to the consensus motif for classical NLSs which interact 

with importin alpha during nuclear localization. In contrast, the NLSs of the more 

distantly-related macaque RV2 rhadinoviruses conform to the structure of a number of 

glycine/arginine-rich NLS motifs which bind directly to importin B during nuclear 

transport. Like other proteins containing glycine/arginine-rich NLSs, the RV2 LANAs are 

targeted to the nucleolus. Our findings suggest that the LANA homologs of the RV1 and 

RV2 rhadinoviruses may be differentially transported and function in different subnuclear 

compartments. 

Presenting author Email: timothy.rose@seattlechildrens.org 

22



NUCLEOPHOSMIN IS A NOVEL REGULATOR OF KSHV REPLICATION VIA 

FUNCTIONAL INTERACTIONS WITH VIRAL CYCLIN AND LANA 

Grzegorz Sarek 1 , Annika Järviluoma 1 , Henna Syrjäkari 2 , Sari Tojkander 2 , Salla Vartia 1 , 

Marikki Laiho 2,3 , and Päivi M. Ojala 1 

1 Genome-Scale Biology Program, Biomedicum Helsinki & Institute of Biomedicine, and 

the Foundation for the Finnish Cancer Institute, 2 Molecular Cancer Biology Program, 

Biomedicum Helsinki & Haartman Institute; University of Helsinki, P.O. Box 63, 00014- 

Univ. of Helsinki, Finland; 3 Department of Radiation Oncology and Molecular Radiation 

Sciences, The Johns Hopkins University School of Medicine, Baltimore, MD 21231 

Abstract 

During Kaposi's sarcoma herpesvirus (KSHV) latency, viral transcription is restricted to a 

subset of latent genes. The latency-associated nuclear antigen (LANA) interacts with 

interphase chromatin together with a variety of cellular factors and it actively represses 

transcription of the KSHV lytic genes. Viral cyclin (v-cyclin), another latent KSHV gene, 

transcribed from the same promoter as LANA, is structurally similar to cellular D-type 

cyclins and forms an active kinase complex with cellular CDK6. Nucleophosmin (NPM) is a 

multifunctional nuclear phosphoprotein implicated in chromatin organization and 

transcription control. We have previously demonstrated that exogenous expression of vcyclin 

causes NPM redistribution from the nucleolus to the nucleoplasm in human 

osteosarcoma cells, suggesting that v-cyclin and NPM may have a functional relationship. 

Here, we present evidence that NPM interacts with LANA and is a novel substrate for vcyclin-CDK6 

kinase complex in primary effusion lymphoma (PEL) cells. Intriguingly, we 

establish the first functional link between latent proteins LANA and v-cyclin by 

demonstrating that phosphorylation of NPM by v-cyclin-CDK6 facilitates interaction 

between NPM and LANA. Silencing of NPM expression in PEL cells induces an increase in 

the acetylation of LANA, decreases its association with chromatin, and leads to 

spontaneous viral lytic reactivation. In addition, we show that NPM depletion in PEL cells 

abolishes interaction between HDAC1 and core histones suggesting that NPM is involved 

in the recruitment of HDAC1 during KSHV latent infection. This work establishes NPM as a 

novel regulator of KSHV replication, which maintains the transcriptional silencing of KSHV 

lytic genes. 

Presenting author Email: grzegorz.sarek@helsinki.fi 

23



ROLE OF BET PROTEINS IN THE FUNCTION OF KSHV AND MHV68 LANA 

Magdalena Weidner-Glunde, Matthias Ottinger, Daniel Pliquet, Ronald Frank* and 

Thomas F. Schulz 

Institute of Virology, Hannover Medical School, Germany 

*HZI Braunschweig, Germany 

Abstract 

KSHV LANA-1 is a multifunctional protein known to be involved in KSHV genome 

maintenance, replication and transcriptional regulation. Its homologue in murine 

herpesvirus 68 has been shown to contribute to establishing latency. Two members of 

the BET protein family, Brd2/RING3 and BRD4/HUNK, which bind to chromatin via 

acetylated histones, were found to interact with KSHV LANA-1and MHV68 orf73 protein. 

In view of the participation of Brd4 in the Mediator transcriptional co-activator complex 

and its implication in HPV-E2-mediated transcriptional regulation, we tested whether 

these two proteins are involved in LANA-1 dependent transcriptional activation and KSHV 

episome replication. 

Using a peptide array covering the MHV68 orf73 we identified aa226-231 as residues 

interacting with recombinant Brd2/RING3. MHV68 orf73 mutants with alanine 

substitutions in this region failed to activate a range of cellular promoters (cyclin E, D1, 

D2). They also showed an altered interaction with cellular chromatin. 

For KSHV LANA-1 we found that siRNAs specific for Brd2 or Brd4 reduced the KSHV 

LANA-1 dependent replication of a plasmid containing the latent origin. In addition, a 

Brd2 fragment comprising the ET domain inhibited episomal replication when 

overexpressed transiently. 

Our data suggest that (I) members of the BRD/BET protein family are involved in 

transcriptional regulation mediated by KSHV LANA-1 and its MHV68 homologue and that 

(II) Brd2/RING3 plays a role in LANA-1-mediated episomal replication. These 

observations could serve as a basis for the development of a peptide inhibitor of the 

interaction between LANA-1 and Brd2 and possibly also of the KSHV latent replication. 

Presenting author Email: weidnerg@allmail.mh-hannover.de 

24



INTERACTION OF KSHV LANA-1 WITH USP7: IMPLICATIONS FOR P53 

FUNCTION 

W. Albrecht (1), E. Gellermann (1), A. Viejo-Borbolla (2), T. F. Schulz (1) 

(1) Institute of Virology, Hanover Medical School, Hanover, Germany 

(2) Dept. of Cell and Molecular Biology, Centro Nacional de Biotecnología, Madrid, Spain 

Abstract 

LANA-1 is constitutively expressed in all latently KSHV-infected cells and displays several 

functions in the persistence and replication of the viral episome. 

We found the ubiquitin-specific protease 7 (USP7) to be a new interaction partner of 

LANA-1. USP7 was originally identified in association with the HSV-1 protein ICP0, an E3 

ligase, and is thought to prevent the proteasomal degradation of autoubiquitinated ICP0. 

The EBV EBNA-1 protein, which is functionally related to LANA-1, also recruits USP7 and 

is thought to absorb USP7 from p53, one of its physiological targets, thereby leading to 

the destabilization of p53. 

USP7 binds to LANA-1 via a sequence motif resembling the USP7-binding site in EBNA-1. 

However, LANA-1 mutants lacking this binding site were not compromised in their ability 

to antagonize p53-mediated transcriptional activation. LANA-1 interacts directly with p53 

and we mapped the binding site to three amino acids in the C-terminus of LANA-1. 

Mutation of the p53-binding site eliminates the ability of LANA-1 to antagonize p53dependent 

transcriptional activation. 

Furthermore, LANA-1 mutants lacking the USP7-binding site showed evidence of 

increased ubiquitination. 

These findings indicate that the model of p53 stabilization by ‚squelching‘ of USP7, as 

developed for EBNA-1, does not apply to LANA-1 and that direct binding of p53 to 

LANA-1 is required for its ability to antagonize p53 function. In contrast, binding of USP7 

by LANA-1 may modulate ubiquitination of LANA-1. 

We currently investigate the functional importance of LANA-1 ubiquitination and the 

properties of LANA-1 mutants deficient in USP7 binding. 

Presenting author Email: albrecht.wiebke@mh-hannover.de 

25 

Comment [W1]: ? 

Comment [W2]: ?



KSHV ENCODED LANA INTERACTS WITH THE NUCLEAR MITOTIC APPARATUS 

PROTEIN TO REGULATE GENOME MAINTENANCE AND SEGREGATION 

Huaxin Si 1 , Subhash C. Verma 1 , Michael Lampson 2 , Qiliang Cai 1 , Erle S. Robertson 1 

1Department 

of Microbiology and the Tumor Virology Program of the Abramson 

Comprehensive Cancer Center, University of Pennsylvania, School of Medicine, 

Philadelphia, Pennsylvania, 19104 

2 

Department of Biology, University of Pennsylvania, School of Medicine, Philadelphia, 

Pennsylvania, 19104 

Abstract 

Kaposi’s sarcoma associated herpesvirus (KSHV) genomes are tethered to the host 

chromosomes and partitioned faithfully into daughter cells with the host chromosomes. 

The latency associated nuclear antigen (LANA) is important for segregation of the newly 

synthesized viral genomes to the daughter nuclei. Here we report that the Nuclear Mitotic 

Apparatus protein (NuMA) and LANA can associate in KSHV infected cells. In 

synchronized cells, NuMA and LANA are colocalized in interphase cells and separate 

during mitosis at the beginning of prophase, reassociating again at the end telophase and 

cytokinesis. Silencing of NuMA expression by siRNA and expression of LGN and a 

dominant negative of dynactin (P150-CC1) which disrupts the association of NuMA with 

microtubule resulted in the loss of KSHV terminal repeats plasmids containing the major 

latent origin. Thus, NuMA is required for persistence of the KSHV episomes to daughter 

cells. This interaction between NuMA and LANA is critical for segregation and 

maintenance of the KSHV episomes through a temporally controlled mechanism of 

binding and release during specific phases of mitosis. 

Presenting author Email: vermas@mail.med.upenn.edu 

26



MULTIPLE DOMAINS CONTRIBUTE TO THE ASSOCIATION OF LANA WITH HOST 

CHROMATIN COMPONENTS 

Ryo Nasu, Satoko Matsumura, Naoko Tanese and Angus Wilson 

Department of Microbiology & NYU Cancer Institute, New York University School of 

Medicine, New York, NY 10016, USA 

Abstract 

The latency-associated nuclear antigen (LANA, LANA1, LNA1) encoded by ORF73 is 

expressed in all KSHV infected cells and performs a variety of functions associated with 

maintenance of the viral genome and proliferation of the infected host cell. As its name 

indicates, LANA accumulates in the nucleus where it is associated with host chromosomes 

throughout the cell cycle. Direct interactions with a number of chromatin components 

have been described but the functional significance of these interactions is not fully 

understood. We have focused on the association of LANA with methyl-CpG binding 

protein 2 (MeCP2) and core histones. Evidence will be presented showing that both the 

N- and C-terminal domains of LANA contribute to interaction with MeCP2 and this can 

direct LANA to regions of pericentric heterochromatin. Localization is dependent on the 

methyl-CpG binding (MBD) and adjacent trans-repression (TRD) domains of MeCP2. The 

chromatin binding motif (CBM) at the extreme N-terminus of LANA (residues 5-11) 

influences subcellular localization but is not strictly required for MeCP2 binding. Instead, 

the CBM recognizes the four histones that form the nucleosome core. In addition to 

interactions with histones H2A/H2B previously described by the Kaye and Luger 

laboratories, we will present evidence for additional interactions with histones H3 and H4. 

Sedimentation studies employing recombinant proteins show that the LANA CBM is 

capable of assembling isolated nucleosomes into compacted structures that may serve to 

regulate host and viral gene expression. 

Presenting author Email: wilsoa02@med.nyu.edu 

27



Immunology I 

28


Immunology I Abstract 8 

INDUCTION OF CYTOKINES BY HHV-8 IN MYELOID DENDRITIC CELLS AND B 

CELLS 

Emilee Knowlton, Giovanna Rappocciolo, Paolo Piazza, Heather Hensler, Frank J. Jenkins, 

Mariel Jais, Charles R. Rinaldo 

Dept of Infectious Diseases and Microbiology, Graduate School of Public Health, 

University of Pittsburgh, PA 

Abstract 

Objectives 

Myeloid dendritic cells (DC) and activated B lymphocytes are professional antigen 

presenting cells susceptible to HHV-8 infection following DC-SIGN receptor binding. 

However, the virus undergoes productive infection in B cells but not in DC. We examined 

whether these different pathways taken by the virus were related to differential cytokine 

production in DC and B cells. 

Methods 

Monocyte derived DC and activated B cells were infected with purified HHV-8 and 

supernatants were examined for cytokines at 0, 6, 12, 18, 24, and 48 hours postinfection 

by the Cytometric Bead Array (BD). 

Results 

Peak levels of TNF-α, IL-6, IL-8, IL-10, MIP-1α and MIP-1β of 30 (IL-10) to 2.6 x 10 4 

(MIP-1β) pg/ml were detected in B cell cultures by 24-48h of infection, which were 4 to 

22 fold higher than in uninfected B cell cultures. In contrast, DC produced peak amounts 

of IP-10, RANTES, MIP-1α and MIP-1β of 20 to 3.5 x 10 4 pg/ml by 24-48h, which were 

1.8-18 fold higher than in uninfected DC. Moreover, infected DC did not produce higher 

levels of IL-8, IL-12p70 or TNF compared to uninfected DC. 

Conclusions 

Elevated levels of several inflammatory cytokines and MIP chemokines were detected in 

HHV-8 infected B cells. In contrast, HHV-8 infected DC produced MIP chemokines, 

RANTES and IP-10, but not IL-12 p70. Distinct cytokine production by HHV-8 infected B 

cells and DC could play a role in viral replication and functional abilities in these antigen 

presenting cells. 

Presenting author Email: ERK21@pitt.edu 

29



KSHV K5 COUNTERACTS TETHERIN, A NOVEL COMPONENT OF INNATE 

ANTIVIRAL IMMUNITY, TO FACILITATE VIRUS RELEASE 

Edward Tsao 1 , Sam Wilson 1 , Claire Pardieu 1 , Ben Webb, Imogen Lai, Stuart Neil 2 , Greg 

Towers 1 , and Paul Kellam 1 

1. MRC Centre for Medical Molecular Virology, Department of Infection, UCL, 46 

Cleveland Street, London W1T 4JF; 2. Peter Gorer Department of Immunobiology, 2nd 

Floor, Borough Wing, Guy's, King's and St Thomas' Hospitals, King's College London, 

Great Maze Pond, London SE1 9RT 

Abstract 

Tetherin (also called CD317, BST2, or HM1.24) has been identified as an interferoninducible 

human factor that inhibits the release of retroviruses, and its antiviral effect can 

be antagonised by HIV-1 vpu. It has been postulated that tetherin, a membraneassociated 

protein, is present both on plasma membranes and viral envelopes (acquired 

from the host membrane through viral budding). The interaction between virion- and 

cell-associated tetherin molecules not only prevents virus release, but may also lead to 

endocytotic internalisation of the virions and their subsequent degradation. This model 

suggests that tetherin would be effective against all enveloped viruses. A recent 

proteomic study revealed that the levels of tetherin were strongly reduced in HeLa cells 

expressing KSHV K5. Here we show that 1) tetherin is expressed in PELs, that 2) KSHV 

K5 is required for the efficient release of virions from cells, and 3) in the absence of K5, 

the reduction of tetherin expression using shRNA rescues virus release. This is the first 

study to show that tetherin is active against enveloped DNA viruses, supporting the 

notion that it is an indiscriminate antiviral factor acting against enveloped viruses. 

Presenting author Email: e.tsao@ucl.ac.uk 

30



MOLECULAR MECHANISM OF BST2/TETHERIN DOWNREGULATION BY KSHV-K5 

Mandana Mansouri, Janet Douglas, Kasinath Viswanathan, Jean Gustin, Ashlee Moses and 

Klaus Früh 

Vaccine and Gene Therapy Institute, Oregon Health and Science University, 505 

NW185th Ave, Beaverton, OR, 97001, USA 

Abstract 

K5 (MIR2) targets cellular immunostimulatory proteins for degradation by ubiquitinating 

their cytoplasmic tails. Using quantitative membrane proteomics we previously showed 

that bone marrow stromal antigen 2 (BST2) is downregulated by K5 (Bartee, 2006, PLoS 

Pathogens 2:e107). In similar experiments, we also observed that BST2 is 

downregulated by the HIV-1 immunomodulator Vpu. Recent reports showed that BST2 is 

an interferon-induced transmembrane glycoprotein that prevents egress of mature HIV-1 

virions by tethering them to the cell membrane, an antiviral activity that is overcome by 

Vpu (Neil, 2008, Nature 451). We determined the molecular mechanisms through which 

KSHV-K5 downregulates BST2/Tetherin in comparison to Vpu. In dermal microvascular 

endothelial cells, IFN-induced expression of BST2 is inhibited by K5. Upon infection with 

KSHV, BST2 expression is further increased in the presence of K5-specific siRNA 

suggesting that K5 downregulates virus-induced BST2 during de novo infection. We 

further show that Vpu downregulates BST2 from the cell surface by sequestering it in the 

Golgi whereas K5 targets BST2 for degradation since steady state levels of total BST2 or 

cell surface BST2 are dramatically reduced in K5-expressing cells. Taken together with 

other data to be presented, we conclude that BST2 is a bona-fide target of K5. The role 

of BST2/Tetherin in modulating KSHV-infection is unknown. We will present data to 

address whether, in the absence of K5, BST2 interferes with egress of KSHV virions. Our 

data suggest that BST2 is part of the innate antiviral response to viral infection and that 

viruses of different genera have developed distinct countermeasures against this protein. 

Presenting author Email: dougljan@ohsu.edu 

31



UPREGULATION OF THE TLR3 PATHWAY BY KSHV DURING PRIMARY INFECTION 

OF MONOCYTES 

John West, Sean Gregory and Blossom Damania 


North Carolina at Chapel Hill, North Carolina 27599 

Abstract 

Kaposi’s sarcoma-associated herpesvirus (KSHV) is associated with several different 

human malignancies including Kaposi’s sarcoma, primary effusion lymphoma, and 

multicentric Castleman’s disease. KSHV establishes lifelong latency in the host and 

modulates the host immune response. Innate immunity is critical for controlling de novo 

viral infection. Toll like receptors (TLRs) are key components of the innate immune 

system and they serve as pathogen recognition receptors that stimulate the host antiviral 

response. In particular, TLR3 has been implicated in RNA virus recognition. 

Currently there is no information on how KSHV infection modulates any TLR pathway. We 

report the first evidence showing that KSHV upregulates TLR3 expression in human 

monocytes sixteen hours post-infection. This is also the first demonstration of a human 

DNA tumor virus upregulating TLR3, a TLR that thus far has been associated with the 

recognition of RNA viruses. We found that KSHV upregulates the TLR3 pathway and 

induces TLR3-specific cytokines and chemokines, including IFN-β1 and CXCL10 (IP-10). 

SiRNAs directed against TLR3 greatly reduced the ability of KSHV to upregulate IFN-β1 

and CXCL10 upon infection. TLR3 upregulation was monitored at different time-points 

post-infection. We found that TLR3 expression levels were highest 16 hours postinfection 

and slowly declined thereafter returning to baseline by 120 hours post-infection 

as the virus established latency. 

Presenting author Email: john_west@med.unc.edu 

32



KSHV EVADES INNATE IMMUNITY BY SUPPRESSION OF TLR4 EXPRESSION 

Dimitris Lagos 1 , Richard James Vart 1 , Fiona Gratrix 1 , Samantha Jane Westrop 2 , Ping-Pui 

Wong 1 , Nesrina Imami 2 , Mark Bower 3 , Frances Gotch 2 and Chris Boshoff 1 

1 

Cancer Research UK Viral Oncology Group, UCL Cancer Institute, Paul O’Gorman 

Building, Huntley Street, University College London, WC1E 6BT, London, U.K. 

2 

Department of Immunology, Chelsea and Westminster Hospital, Imperial College 

London, London, U.K. 

3 

Imperial College School of Medicine, Chelsea and Westminster Hospital, Imperial College 

London, London, UK. 

Abstract 

Toll-like receptors (TLRs) play a critical role in innate immune responses against invading 

pathogens. However, the role and regulation of TLRs during KSHV infection remain poorly 

understood. We demonstrate that TLR4 mediates innate response against KSHV and that 

KSHV targets TLR4 expression as a mechanism of immune evasion. In vitro, mouse 

macrophages or human lymphatic endothelial cells lacking TLR4 are more susceptible to 

KSHV infection, whereas activation of TLR4-mediated signalling with bacterial 

lipopolysaccharide protects cells from KSHV infection. In vivo, HIV-1-infected individuals 

who carry a non-functional TLR4 allele appear more likely to have multicentric 

Castleman’s disease, a lymphoproliferation associated with enhanced KSHV replication. 

Furthermore, we show that KSHV has evolved mechanisms to evade the TLR4-mediated 

response. The KSHV-encoded vIRF1 and vGPCR co-operate to suppress TLR4 expression 

in human lymphatic endothelial cells. Notably, ERK activation by vGPCR or by viral 

attachment to the cell membrane results in rapid TLR4 down-regulation. Our findings 

reveal the first example of viral immune evasion through suppression of TLR4 expression 

and suggest the potential use of TLR4 agonists for treatment of KSHV-related neoplasms. 

Presenting author Email: d.lagos@ucl.ac.uk 

33



THE KSHV LYTIC PROTEIN VOX2 AND ITS CELLULAR COUNTERPART, CD200, 

INHIBIT EPITOPE-SPECIFIC T CELL RESPONSES 

Rachel Colman, Karen Misstear, Heather Long, Omar Quereshi^, David Sansom^, 

Andrew Hislop, David J. Blackbourn 

CRUK Institute for Cancer Studies and ^Division of Immunity and Infection, University of 

Birmingham, Birmingham B15 2TT, UK 

Abstract 

Background & Objectives: The KSHV lytic protein vOX2 shares 36% identity with 

human cellular CD200. An engineered soluble derivative of vOX2 inhibited neutrophil 

activity in vitro and had anti-inflammatory activity in vivo, suggesting the protein inhibits 

innate immune responses. In the present study we determined whether adaptive 

immune responses were modulated by native, membrane-bound vOX2 and CD200. 

Methods: Antigen presenting cells (APC) were engineered to express either vOX2 or 

CD200 on their surface by retroviral transduction and membrane expression was 

confirmed by flow cytometry and IFA. APC loaded with epitope peptides were incubated 

with appropriate epitope-specific CD4+ or CD8+ T cell clones and T cell responses were 

measured by IFN-γ release. Ex-vivo restimulation of epitope-specific CD8+ T cells was 

performed by coculturing peripheral blood mononuclear cells (PBMC) with autologous APC 

engineered to express either vOX2 or CD200 on their surface. T cell function was 

measured by IFN-γ release. 

Results: Expression of vOX2 or CD200 on the surface of APCs inhibited IFN-γ secretion 

by co-cultured CD4+ and CD8+ T cell clones up to 50% when compared to vector only 

transduced APCs. Restimulation of PBMC was similarly inhibited. Neither HLA class I, nor 

CD80 and CD86 costimulatory molecule expression levels were downregulated on APCs 

by vOX2 or CD200 expression. 

Conclusion: These data demonstrate that both KSHV vOX2 and cellular CD200 inhibit 

antigen-specific T-lymphocyte activity. They suggest vOX2 suppresses adaptive immune 

responses against KSHV lytically infected cells, facilitating virus replication and 

dissemination in vivo. 

Presenting author Email: r.colman@bham.ac.uk 

34



Pathogenesis 

35


Pathogenesis Abstract 14 

GENERATION OF RHESUS RHADINOVIRUS LACKING EXPRESSION OF VGPCR 

AND VCD200 FOR THE ASSESSMENT OF THEIR ROLES IN DISEASE 

DEVELOPMENT IN A RHESUS MACAQUE MODEL OF KSHV INFECTION 

Ryan D. Estep 1 

, Elisa Cardenas 3 , and Scott W. Wong 1,2,3 

Vaccine and Gene Therapy Institute1 , Oregon Health & Science University West Campus, 

Division of Pathobiology and Immunology2 , Oregon National Primate Research Center, 

Beaverton, Oregon 97006; 

Department of Molecular Microbiology and Immunology3 , Oregon Health & Science 

University, Portland, Oregon 97201. 

Abstract 

RRV strain 17577, the rhesus macaque homologue of KSHV, was recently cloned as an 

infectious Bacterial Artificial Chromosome (BAC). The RRV BAC system allows for the 

rapid genetic manipulation of the RRV genome in bacteria, and the subsequent 

production of modified forms of RRV. Currently, we are using the RRV BAC system to 

modify genes that have been proposed to be important in the development of disease 

associated with KSHV infection in humans. KSHV and RRV both encode a viral G proteincoupled 

receptor (vGPCR) and viral CD200 homologue (vCD200), proteins which possess 

oncogenic and immunomodulatory functions, respectively. Although extensively analyzed 

in vitro, the roles that the KSHV and RRV vGPCR and vCD200 proteins play during a de 

novo infection in vivo are currently unknown. Therefore, we have used the RRV BAC 

system to disrupt expression of the vGPCR and vCD200 proteins in RRV, through the 

insertion of specific stop mutations in ORF74 and R15. Thus far, we have successfully 

generated viruses that no longer express these proteins during infection, and preliminary 

analyses indicate that viruses devoid of vGPCR or vCD200 expression possess similar 

growth properties to wild-type RRV in vitro. Other ongoing studies with these viruses 

include examination of the effects of vGPCR and/or vCD200 expression on virus-induced 

signaling and immunoregulatory properties in vitro, and importantly, how lack of these 

proteins may affect the development of viral-induced disease in vivo. Further plans 

include the generation of chimeric versions of RRV expressing KSHV vGPCR and/or 

vCD200. 

Presenting author Email: estepr@ohsu.edu 

36



GENERATION OF VFLIP TRANSGENIC MICE: A MODEL TO STUDY KSHV- 

ASSOCIATED LYMPHOMAGENESIS 

Gianna Ballon 1 , Amy Chadburn 1 , Yi-Fang Liu 1 , Yoshiteru Sasaki 2 , Klaus Rajewsky 2 , Ethel 

Cesarman 1 

1 

Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New 

York, NY 10021, USA 

2 

CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA 02115, USA 

Abstract 

Primary effusion lymphoma (PEL) is a distinct subtype of aggressive non-Hodgkin’s 

lymphoma (NHL), specifically associated with infection by Kaposi's sarcoma-associated 

herpesvirus (KSHV). Several in vitro observations suggest that vFLIP, a protein 

expressed during latency, is an important viral oncogene. It is essential for the survival 

of KSHV-infected PEL cells, mainly by constitutively activating the NF-kB pathway. In 

order to assess the role of vFLIP in the pathogenesis of PEL, we developed transgenic 

mouse models expressing vFLIP in B-cells. The experimental approach used has been a 

conditional recombinant activation of vFLIP, by using the ROSA26 knock-in system. A 

specifically restricted expression of the transgene in CD19+ B-cells has been achieved by 

crossing the ROSA26.vFLIP knock-in mice with other mice expressing cre recombinase 

under the control of the CD19 promoter. These mice have also been crossed with the 

LANA transgenic mice to assess a potential synergistic effect between these two KSHV 

latent proteins in the lymphomagenic process of PEL. vFLIP expression in the CD19+ Bcells 

results in splenomegaly, with an increase in both T and B-cells, and with a relative 

increase of the T versus B-cell ratio. Although primary follicles were enlarged, the 

expression of vFLIP in the CD19+ B-cells results in lack of germinal center formation in 

the spleen, lymph nodes and intestine, and in partially impaired class-switching 

recombination. These results indicate that, by constitutively activating the NF-kB 

pathway in pre-germinal center B-cells expressing CD19, the normal B-cell differentiation 

is impaired, and provide clues about possible aberrant differentiation in PEL cells. 

Presenting author Email: gib2004@med.cornell.edu 

37



DEVELOPMENT OF ANIMAL MODELS OF KSHV INFECTION AND AIDS- 

ASSOCIATED DISEASE BASED ON MACAQUE RHADINOVIRUSES 

A. Gregory Bruce 1 , Jonathan T. Ryan 1 , Courtney Gravett 1,2 , and Timothy M. Rose 1,2 , 

1 Seattle Children’s Hospital Research Institute and 2 University of Washington, Seattle WA 

Abstract 

Two related viral lineages within the Rhadinovirus genus of gammaherpesviruses have 

been identified in macaques and other Old World non-human primates. In macaques, 

the prototypes of these lineages were first identified in the rhesus macaque (Macaca 

mulatta); retroperitoneal fibromatosis-associated herpesvirus (RFHVMm) and rhesus 

rhadinovirus (RRV) members of the Rhadinovirus-1 (RV1) and RV2 lineages, respectively. 

Each macaque species is host to a distinct pair of RV1 and RV2 rhadinoviruses, 

distinguishable by molecular sequence. Both RV1 and RV2 rhadinoviruses have been 

implicated in diseases in macaques that closely resemble cancers and proliferative 

diseases associated with KSHV in humans, including Kaposi sarcoma (KS), lymphoma 

and multicentric Castleman’s disease. Thus, the characterization of the macaque 

rhadinoviruses, including the development of specific reagents and assays that enable 

the evaluation of their biology and life cycles, is an important aspect in the development 

of KSHV-related macaque models of disease. We have developed protocols for 

experimental infection of macaques with both RV1 and RV2 macaque rhadinoviruses, and 

are developing reagents and analytical approaches to follow the resulting infections and 

host responses to infection in vivo. We will present the current status of the first small 

pilot study examining the experimental infection of naïve macaques with infectious 

rhadinoviruses from saliva of infected macaques and subsequent chemical and viral 

treatments to activate virus by inducing immunosuppression. These studies will provide 

the groundwork for establishing relevant animal models of human disease associated 

with KSHV. 

Presenting author Email: greg.bruce@seattlechildrens.org 

38



β ARRESTINS REGULATE KSHV GPCR ACTIVITY AND ARE MODULATED BY 

CANNABINOIDS 

Xuefeng Zhang, Yehoshua Maor, Jerome E. Groopman 

Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Harvard 

Medical School, Boston, MA 02115 USA 

Abstract 

Kaposi’s sarcoma (KS) is a frequent neoplasm among immuno-compromised patients 

such as those with AIDS and after organ transplantation. Among certain populations, 

recreational use of cannabinoids is common and medicinal use of cannabinoids is 

increasing as well. Cannabinoids bind to CB1 and CB2 GPCRs on the endothelial cell 

surface. In our previous study, we found that low doses of Δ 9 -tetrahydrocannabinol (Δ 9 - 

THC), the major psychoactive ingredient of marijuana, induced KSHV GPCR expression 

and endothelial transformation. Using immunoprecipitation and confocal microscopy, we 

then found that KSHV GPCR was constitutively associated with β arrestins. β arrestins are 

key molecules contributing to desensitization of GPCRs. We now report that knockdown 

of β arrestins using RNAi technology increased KSHV GPCR expression. This resulted in 

phosphorylation of focal adhesion kinase (FAK) and inhibition of p53 expression. Such 

changes in FAK and p53 may contribute to endothelial transformation by KSHV GPCR. We 

also found that knockdown of β arrestins significantly induced NFκB (p65) expression in 

KSHV-infected HMVEC. The NFκB pathway has been observed to participate in KSHV 

GPCR-induced transformation. Our results indicate that β arrestins may exert inhibitory 

effects on KSHV GPCR activity, and when KSHV-infected cells are exposed to a strong 

GPCR stimulus like cannabinoids, cytosolic arrestins are preferentially recruited to 

activated GPCRs like CB1 and CB2. This would significantly decrease association of KSHV 

GPCR with β arrestins, increasing activity of KSHV GPCR and enhancing KS 

transformation. 

Presenting author Email: xzhang3@bidmc.harvard.edu 

39



Activation of NF-κB by KSHV K15 involves recruitment of NIK (NF-κB inducing 

kinase) 

Anika Hävemeier, Macel Pietrek and Thomas F. Schulz 

Institute of Virology, Medical School Hannover, Hannover, Germany 

Abstract 

ORF K15 is the positional homologue of EBV LMP2A. Like LMP2A, the K15 protein 

contains 12 predicted transmembrane and a cytoplasmic domain involved in signaling. 

The cytoplasmic domain interacts with several cellular proteins, including TRAF and Src 

kinases, and activates NF-kB as well as the MAPKs c-Jun-N-terminal kinase (JNK) and 

extracellular signal-regulated kinase (Erk). Mutation of tyrosine 481 in the cytoplasmic 

tail of K15 (K15 Y 481 F) abolishes the ability of K15 to induce these pathways. 

Activation of NF-kB by several stimuli occurs via two distinct pathways: the classical 

(canonical) or the alternative (noncanonical) pathway. We found that K15 induces the 

alternative NF-kB pathway via NIK, leading to the processing of p100 and the 

translocation of p52 into the nucleus. Transfection of a dominant negative NIK and NIK 

siRNA decreases the K15 mediated induction of NF-kB, shown by western blot analysis or 

luciferase reporter experiments. Using alanin scanning mutants the binding site for NIK 

localised to six aminoacids near the last transmembrane domain of K15. NIK-binding 

deficient K15 mutants show a decreased NF-kB activation, but appear to retain the ability 

to activate other K15-induced promoters. 

Direct recruitment of NIK represents a novel way for a viral protein to activate NF-kB. In 

activating NF-kB via NIK, KSHV K15 shows functional similarities to the Lymphotoxin 

beta-receptor, rather than the B-cell receptor, whose function is thought to be mimicked 

by EBV LMP2A. 

Presenting author Email: haevemeier.anika@mh-hannover.de 

40



KAPOSI'S SARCOMA ASSOCIATED HERPES VIRUS (KSHV/HHV-8) INDUCES 

ANGIOGENIN DURING INFECTION OF HUMAN DERMAL MICROVASCULAR 

ENDOTHELIAL (HMVEC-D) CELLS THAT IS CRITICAL FOR ANTI-APOPTOSIS, 

CELL PROLIFERATION AND ANGIOGENESIS 

Sathish Sadagopan, Neelam-Sharma Walia, Mohanan Valiya Veettil, Virginie Bottero, Rita 

Levine and Bala Chandran 

H.M. Bligh cancer research laboratories, Department of Microbiology and Immunology, 

Chicago Medical School, Rosalind Franklin University of Medicine and Science, Chicago, IL 

Abstract 

De novo KSHV infection of HMVEC-d cells results in increased expression and secretion of 

angiogenin, a multifunctional angiogenic protein. KS tissue sections were positive for 

angiogenin highlighting the importance of angiogenin in KS pathogenesis. Viral gene 

expression was critical for the sustained angiogenin secretion during primary infection. 

KSHV ORF 73 (latent) and ORF 74 (lytic) genes induced an increased secretion of 

angiogenein. TIVE cells latently infected with KSHV secreted high levels of angiogenin. 

Angiogenin bound to surface actin, internalized in a microtubule independent manner, 

translocated into the nucleus when the cells were semi-confluent and became nucleolar 

in sub-confluent cells. In the nucleolus, angiogenin bound to the upstream sequence of 

45SrRNA promoter and increased 45SrRNA transcription. Angiogenin increased the antiapoptosis 

and cell proliferation of KSHV infected endothelial cells, and these activities 

were blocked by neomycin which blocked the nuclear translocation of angiogenin. 

Upregulation of angiogenin lead to an increased activation of urokinase plasminogen 

activator and generation of active plasmin from inactive plasminogen. Plasmin generation 

aided the migration of endothelial cells towards chemoattractant including angiogenin, 

and chemotaxis was prevented by the inhibition of angiogenin nuclear translocation. 

Tube formation of endothelial cells was significantly inhibited by treating infected cell 

supernatants with anti-angiogenin antibodies. Inhibition of nuclear translocation of 

angiogenin also blocked VEGF-C expression. Collectively, these results suggest that KSHV 

induced angiogenin plays multiple roles during infection such as increase in 45SrRNA 

synthesis, proliferation, cell migration and angiogenesis, and blocking angiogenin could 

have a therapeutic value in treating KSHV infection and KS. 

Presenting author Email: bala.chandran@rosalindfranklin.edu 

41



ROLE OF K15 IN KAPOSI’S SARCOMA HERPESVIRUS INDUCED ENDOTHELIAL 

CELL MIGRATION 

Bernd Hillenbrand, Antje Bürger, Irina Fischer, Khaled Alkharsah and Thomas F. Schulz 

Institute of Virology, Hanover Medical School, Hanover, 30625, Germany 

Abstract 

Kaposi’s sarcoma herpesvirus (KSHV) is the cause of Kaposi’s sarcoma, a highly 

vascularized tumour. Others have previously proposed a role for IL-8 in endothelial cell 

migration induced by KSHV-infected cells (Lane et al., J.Virol. 2002; 76:11570-11583). 

We previously showed that the K15 protein of KSHV induces increased IL-8 secretion in 

epithelial cells. In this study we addressed the role of K15 for virus induced endothelial 

cell migration in the context of the whole viral genome. We constructed a K15 deficient 

recombinant virus genome (KSHVΔK15) by using a bacterial artificial chromosome (BAC) 

system. We generated stable HEK293 cells carrying the BAC36 (wt) or BAC ΔK15. 

Following the induction of the lytic replication cycle reduced IL-8 mRNA levels were found 

by gene expression microarray in BAC ΔK15 cells in comparison to BAC36 cells. This 

reduction was confirmed by IL-8 specific ELISA of BAC36 and BAC ΔK15 conditioned 

media. In endothelial cell migration assays the migration of primary human vascular 

endothelial cells (HUVEC) in response to conditioned culture supernatants from KSHV 

ΔK15-infected cells was significantly reduced in comparison to supernatants from 

KSHVwt-infected cells. In contrast, supernatants from cells infected with a recombinant 

KSHV lacking the viral FLIP (BAC ΔFLIP), like K15 an inducer of NFκB, induced the same 

degree of endothelial cell migration as KSHVwt–infected cells. These results suggest an 

involvement of K15 in KSHV induced endothelial cell migration and possibly angiogenesis. 

Presenting author Email: hillenbrand.bernd@mh-hannover.de 

42



DOWNREGULATION OF GALECTIN-3 IN KSHV INFECTED DMVEC CELLS AND 

KAPOSI’S SARCOMA: IMPLICATIONS FOR TUMORIGENESIS 

Donald J. Alcendor 1 Wen Qui Zhu 1 Prashant Desai 2 and Gary S. Hayward 2 

1 Meharry Medical College, School of Medicine, Center for AIDS Health Disparities 

Research and Department of Microbial Pathogenesis and Immune Response, 1005 Dr. 

D.B. Todd Jr. Blvd., Hubbard Hospital, Rm. 5025, Nashville, Tennessee, 37208-3599, 

USA, 2 Department of Viral Oncology and Pharmacology, Molecular Virology Laboratories, 

Sidney-Kimmel Cancer Research Center, Johns Hopkins University School of Medicine, 

Baltimore, Maryland 21231-1000 USA 

Abstract 

The Galectins are a family of proteins that share an affinity for b-galactoside containing 

glycoconjugates. Galectin-3 has been implicated in tumor progression and metastasis. 

Down regulation of Galectin-3 is associated with malignancy in prostate, ovarian and 

breast cancer. Kaposi’s sarcoma (KS) is characterized as an angioproliferative tumor of 

vascular endothelial cells and produces rare B cell lymphoproliferative diseases in the 

form of primary effusion lymphomas (PEL) and some forms of Multicentric Castleman’s 

Disease (MDC). We have observed suppression of galectin-3 expression in KSHV infected 

dermal microvascular endothelial cells (DMVEC) and KS tumor tissue. Here we 

demonstrate that galectin-3 protein expression is downregulated 20-fold in KSHV 

infected DMVEC cells by immunoblot analysis. In infected DMVEC, we show absence of 

galectin-3 staining by dual labeled immunofluorescence in latency associated nuclear 

antigen (LANA) positive spindle cells. There is also transcriptional suppression of 

Galectin-3 message in KSHV infected DMVEC cells as observed by RT-PCR. Furthermore 

we find reduced levels of galectin-3 expression in LANA positive spindle cell regions in 

archival KS tissue by dual labeled immunohistochemistry. Transfection studies reveal 

KSHV vFLIP is likely the viral gene that targets galectin-3 downregulation in HeLa cells. 

Finally, in uninduced PEL cells lines galectin-3 expression levels vary with levels of KSHV 

replication. The search for novel host cell factors that may contribute to the overall 

pathogenesis of KS is essential for early detection of KS and development of innovative 

therapies for treatment. 

Presenting author Email: dalcendor@mmc.edu 

43



Virus-Cell Interactions I 

44


Virus-Cell Interactions I Abstract 22 

ANIMAL MODELS OF KAPOSI'S SARCOMA REVEAL A ROLE OF KSHV VGPCR AND 

RAC1 IN ANGIOGENESIS AND GENETIC INSTABILITY 

Lucas Cavallin 1,2 , Qi Ma 1 , Rui Zhang 1,2 , E. Margarita Duran 1,2 , Pascal J. Goldschmidt- 

Clermont 1 and Enrique A. Mesri 1,2 

1 University of Miami Miller School of Medicine and 2 Viral Oncology Program, Sylvester 

Comprehensive Cancer Center. Miami, FL 33136 

Abstract 

Kaposi’s sarcoma (KS) is a viral malignancy characterized by angiogenesis and 

proliferation of KSHV-infected spindle cells. Spindle cells explanted from tumors lose 

KSHV and are not tumorigenic. However, tumorigenic KS cells could be isolated from 

advanced tumors where is more frequent to find cellular oncogenic alterations. We 

described a new animal model of KS (Mutlu et al. 2007. Cancer Cell 11:245-258): 

KSHVBac36-tranfected mouse endothelial lineage cells (mECK36) induce KSHV-infected 

KS-like tumors in nude mice, while mECK36 that lose the KSHV episome lose their 

tumorigenicity. shRNA silencing showed that vGPCR, a KSHV angiogenic gene 

upregulated in mECK36 tumors, is essential for angiogenesis and tumorigenicity. We 

found that mECK36 explanted from tumors that lose the KSHV episome are tumorigenic, 

indicating that, as KSHV tumorigenesis progresses in vivo, the malignant phenotype 

becomes irreversible and KSHV-independent. Explanted mECK36 displayed 3 times more 

foci of DNA repair than cells never grown in tumors indicating that DNA damage is 

increased in vivo. We now find that expression of constitutively activated Rac1, a 

mediator of vGPCR pathogenesis over-expressed in KS lesions, is sufficient to induce KSlike 

tumors in transgenic mice by a mechanism involving PTEN inactivation and R.O.S.mediated 

oxidative DNA damage. These results points to a key role for Rac1 induced 

R.O.S. production in KS pathogenesis. They indicate that in addition to angiogenesis, 

vGPCR activation of Rac1 can increase mutation rate to foster a multi-step carcinogenesis 

process driven by oxidative genetic damage. 

Presenting author Email: emesri@med.miami.edu 

45



INSIGHTS INTO KSHV ACTIVATION OF THE IKK SIGNALOSOME: CRYSTAL 

STRUCTURE OF A VFLIP-IKKγ COMPLEX. 

Claire Bagnéris, Alexander V Ageichik, Nora Cronin, Bonnie Wallace, Mary Collins, Chris 

Boshoff, Gabriel Waksman and Tracey Barrett 1 

1 

Institute of Structural and Molecular Biology, School of Crystallography, Birkbeck 

College, Malet Street, London WC1E 7HX, UK. 

Abstract 

During the latent phase of Kaposi Sarcoma Herpes virus (KSHV) infection, a limited 

number of genes are expressed that are pivotal to viral propagation and survival. 

Amongst them is ks-vFLIP whose over-expression has been directly linked to Kaposi 

Sarcoma (KS) and other lymphoproliferative malignancies. ks-vFLIP shares considerable 

homology with the human cellular FLIPs that have a key anti-apoptotic role, but by 

contrast operates via a mechanism that involves subversion of the canonical NF-κB 

transcriptional pathway (implicated in diverse cellular processes that include immune 

activation and cellular growth). This is achieved through a direct interaction with IKKγ, 

the regulatory component of the IKK complex or signalosome, and results in the normally 

tightly regulated pathway being rendered constitutively active. As a consequence, the 

expression of genes associated with anti-apoptosis, inflammation and oncogenesis are 

aberrantly up-regulated. In order to establish the molecular basis underpinning ks-vFLIP 

constitutive activation of the canonical NF-κB pathway, we have determined the crystal 

structure of ks-vFLIP bound to an IKKγ fragment incorporating the ks-vFLIP recognition 

motif. This first structure of a ks-vFLIP-IKKγ complex reveals an extensive ks-vFLIP-IKKγ 

interface that has been probed using site directed mutagenesis to confirm the roles of 

key amino acids in mediating the ks-vFLIP-IKKγ interaction. In addition, these have been 

shown to be essential for ks-vFLIP induced activation of the canonical NF-κB pathway. 

Our structure thus provides a framework for the design of novel therapeutics aimed at 

the treatment of KS and related pathologies. 

Presenting author Email: t.barrett@mail.cryst.bbk.ac.uk 

46



THE VIRAL INHIBITOR OF APOPTOSIS VFLIP/K13 PROTECTS ENDOTHELIAL 

CELLS AGAINST SUPEROXIDE - INDUCED CELL DEATH 

Michael Stürzl(1), Gaby Sander(1), Nathalie Gonin-Laurent(1), Kristina Weinländer(1), 

Elisabeth Naschberger(1), Ramona Jochmann(1), Khaled R. Alkharsah(2), Thomas F. 

Schulz(2), Margot Thome(3), Frank Neipel(4) and Mathias Thurau(1) 

(1) Department of Surgery, Division of Molecular and Experimental Surgery, University of 

Erlangen-Nuremberg, Erlangen, Germany. (2) Medical School Hannover, Department of 

Virology, Hannover, Germany. (3) Department of Biochemistry, University of Lausanne, 

Epalinges, Switzerland. (4) Institute for Clinical and Molecular Virology, University of 

Erlangen-Nuremberg, Erlangen, Germany 

Abstract 

Human herpesvirus-8 (HHV-8) is the etiological agent of Kaposi’s sarcoma (KS). The 

gene K13 of HHV-8 encodes for the anti-apoptotic viral FLICE-inhibitory protein 

vFLIP/K13. It is expressed in the latent phase of the viral life cycle and its expression in 

KS lesions is inversely related to the rate of apoptosis. The mechanisms by which 

vFLIP/K13 inhibits cell death have to be elucidated. By using a comparative proteome 

analysis we identified manganese superoxide dismutase (MnSOD), a mitochondrial 

antioxidant and an important anti-apoptotic enzyme, as the most strongly up-regulated 

protein by vFLIP/K13 in endothelial cells. MnSOD expression was also up-regulated in 

endothelial cells upon infection with HHV-8. The induction of MnSOD expression by 

vFLIP/K13 was dependent on NF-kappaB activation, occurred in a cell intrinsic manner, 

and correlated with decreased intracellular superoxide accumulation and increased 

resistance of endothelial cells against superoxide-induced death. These findings show a 

novel function of vFLIP/K13 in cell death inhibition and in regulation of mitochondrial 

redox-balance via the upregulation of MnSOD expression. 

Presenting author Email: michael.stuerzl@uk-erlangen.de 

47



ROLE OF DEFECTIVE OCT-2 AND OCA-B EXPRESSION IN IMMUNOGLOBULIN 

PRODUCTION AND KSHV LYTIC REACTIVATION IN PRIMARY EFFUSION 

LYMPHOMA 

Daniel DiBartolo 1 , Elizabeth Hyjek 1 , Shannon Keller 1 , Ilaria Guasparri 1 , Ren Sun 2 , Amy 

Chadburn 1 , Daniel M Knowles 1 , and Ethel Cesarman 1 

Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New 

York, NY, 1 Molecular Biology IDP, University of California at Los Angeles, Los Angeles, 

CA 2 

Abstract 

PEL cells do not express surface or cytoplasmic immunoglobulin (Ig) despite having a 

genotype and gene expression signature of highly differentiated B cells. We show the 

lack of Oct-2 and OCA-B transcription factors to be responsible, at least in part, for this 

defect in Ig production. ORF50/Rta, the major regulator of KSHV lytic reactivation, 

contains an octamer motif within its promoter. Thus, we also examined the impact that 

the lack of Oct-2 and OCA-B have on lytic reactivation mediated by ORF50. Previous 

studies had shown that binding of Oct-1 to the ORF50 promoter significantly enhances 

ORF50 transactivation. However, we found that Oct-2, on the other hand, inhibits ORF50 

expression and consequently lytic reactivation. Chromatin immunoprecipitation assays 

showed that endogenous Oct-1 associates with ORF50 promoter in uninduced cells and 

transfection of ORF50 further enhances its binding. Using PEL cells with inducible Oct-2 

expression we found that induction of Oct-2 results in Oct-2 association with the ORF50 

promoter and decreased Oct-1 binding suggesting that Oct-2 competes with Oct-1 for the 

octamer site in the ORF50 promoter, explaining mechanistically the observed inhibitory 

effect on lytic reactivation. The consistent absence of Oct-2 in PEL leads us to speculate 

that this deficiency is selected for in order to maintain the ability to efficiently transition 

from latency to lytic reactivation. 

Presenting author Email: ecesarm@med.cornell.edu 

48



KAPOSI’S SARCOMA ASSOCIATED HERPESVIRUS (KSHV) INDUCES 

TUMORIGENIC CELLULAR MIRNAS IN LATENTLY INFECTED ENDOTHELIAL CELLS 

1Rebecca L. Skalsky 2 Wendell Miley, 2 Rachel Bagni, 1 Soo-Jin Han 1 Jianhong Hu, Chang 

Hee Kim 3 , 1 Rolf Renne and 2* Denise Whitby 

1. Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, 

Florida. 2. Viral Oncology Section, AIDS and Cancer Virus Program, SAIC-Frederick, NCI- 

Frederick, Frederick MD. 3. Laboratory of Molecular Technology, Advanced Technology 

Program, SAIC-Frederick, NCI-Frederick, Frederick, MD 

Abstract 

MicroRNAs are small non-coding RNA molecules which post-transcriptionally regulate 

gene expression by binding to 3’UTRs of target genes thereby contributing to important 

biological processes. Recently, a subset of the more than 450 human miRNAs has been 

shown to be aberrantly expressed in many human tumors. Kaposi’s sarcoma associated 

herpesvirus (KSHV) encodes 12 miRNAs, some of which target host cellular genes 

thereby affecting angiogenesis, apoptosis, and B cell development. So far reports on 

KSHV-encoded miRNAs have focused solely on lymphoma cells and not on endothelial 

cells from which KS tumours arise. 

To address this question, we utilized a custom miRNA array to investigate both viral and 

the host cellular miRNA expression pattern in endothelial cells in the presence and 

absence of latent KSHV infection. 

We detected a limited set of KSHV miRNAs in endothelial cells from two different lineages 

- overall viral miRNA expression was significantly lower in endothelial cells compared to 

PEL cell lines. Additionally, we observed significant changes in host cellular miRNA 

expression in latently infected cells. Importantly, the hsa-miR 17/92 cluster, hsa miR16, 

hsa-miR155 and several let-7 family members were significantly up-regulated. Aberrant, 

expression of miR155 and the miR17/92 cluster has been reported in many lymphoid and 

solid tumours. Moreover, these cellular miRNAs were the first for which oncogenic activity 

was demonstrated in transgenic mouse models. 

Our data suggests that KSHV usurps miRNA pathways not only by expression of viral 

miRNAs but also by inducing significant changes in the host cellular miRNA expression 

pattern. 

Presenting author Email: whitbyd@ncifcrf.gov 

49



KSHV AND CELLULAR MIRNA EXPRESSION DURING LATENCY AND LYTIC 

REACTIVATION IN PEL CELLS 

*1Soo-Jin Han * 1 Jianhong Hu, * 1 Karlie Plaisance * 2 Wendell Miley 2 Rachel Bagni, Chang 

Hee Kim 3 , 2 Denise Whitby and 2 Rolf Renne 

1. Department of Molecular Genetics and Microbiology, University of Florida, Gainesville, 

Florida 32610, 2.Viral Oncology Section, AIDS and Cancer Virus Program, SAIC- 

Frederick, NCI-Frederick, Frederick MD 21702 3. Laboratory of Molecular Technology, 

Advanced Technology Program, SAIC-Frederick, NCI-Frederick, Frederick, MD 21702 

(*equal contribution). 

Abstract 


gene expression by binding to 3’UTRs of target genes thereby contributing to important 

biological processes. Kaposi’s sarcoma associated herpesvirus (KSHV) encodes 12 

miRNAs within the KSHV latency associated region (KLAR), some of which target host 

cellular genes thereby affecting angiogenesis, apoptosis, and B cell development. 

Originally, KSHV miRNAs were cloned from BCBL-1 cells in the presence and absence of 

TPA induction, suggesting that miRNAs may contribute to both phases of infection. 

The KLAR region is expressed from at least three different promoters giving rise to singly 

and multiply spliced transcripts. To further elucidate the complexity of this locus with 

emphasis on transcripts that could serve as pri-miRNAs, we performed a detailed RT-PCR 

and RNase protection analysis and identified several new transcripts that could give rise 

to miRNA expression. We identified novel multiply-spliced transcripts originating from the 

LANAp and LTI promoters upstream of LANA, which contained 3’ exons within the 

Kaposin locus. Furthermore, the observed splicing patterns from these long KLAR 

encompassing transcripts changed during lytic replication, suggesting that both LANAp 

and LTI could drive miRNA expression. 

Next, we utilized a custom miRNA array containing both viral and human miRNAs to 

investigate miRNA output in PEL cells during latency and reactivated by Ad-ORF50. 

Surprisingly, while we identified marked changes in the splicing pattern in this region, 

viral miRNA expression was not significantly altered in PEL cells 48 hours post 

reactivation. Cellular miRNA expression signatures during both conditions will also be 

discussed. 

Presenting author Email: rrenne@ufscc.ufl.edu 

50



KAPOSI’S SARCOMA ASSOCIATED HERPESVIRUS (KSHV/HHV-8) UTILIZES 

MACROPINOCYTIC PATHWAY TO ENTER HUMAN DERMAL MICROVASCULAR 

ENDOTHELIAL (HMVEC-D) AND HUMAN UMBILICAL VEIN ENDOTHELIAL 

(HUVEC) CELLS 

Hari Raghu, Neelam Sharma Walia, Mohanan Valiya Veettil, Sathish Sadagopan and Bala 

Chandran 

H.M. Bligh cancer research laboratories, Department of Microbiology and Immunology, 

H.M. Bligh Cancer Research Laboratories, Chicago Medical School, Rosalind Franklin 

University of Medicine and Science, 3333 Green Bay Road, North Chicago, IL. USA 

Abstract 

KSHV utilizes the clathrin mediated endocytic pathway for its infectious entry into human 

foreskin fibroblast (HFF) cells (J.Virology. 2003. 77: 7978-7990). Here, we characterized 

KSHV entry in primary HMVEC-d and HUVEC cells. Similar to HMVEC-d cells, KSHV 

infection of HUVEC cells also resulted in the initial high level lytic ORF50 and K8 gene 

expression and subsequent decline, while the latent gene expression persisted. In 

contrast to HFF cells, cytochalasin D affecting actin polymerization significantly blocked 

virus entry and gene expression in both endothelial cell types tested. Chlorpromazine, a 

clathrin mediated endocytosis inhibitor which inhibited KSHV entry in HFF cells did not 

have any effect on KSHV binding, internalization and gene expression in HMVEC-d and 

HUVEC cells. No significant inhibition was observed in both the endothelial cell types with 

filipin, a caveolar endocytosis inhibitor. In contrast, internalization and gene expression 

was significantly inhibited in both the endothelial cell types by macropinocytosis 

inhibitors EIPA and Rottlerin. Internalized virus particles enclosed in large vesicles were 

seen by electron microscopy. Confocal microscopy localized the viral capsid with KSHV 

envelope glycoprotein gpK8.1A at 5’ and 10’ post infection. Inhibition of macropinocytosis 

resulted in the distribution of viral capsids at the cell periphery and very little association 

with microtubules. Internalized viral glycoprotein gpK8.1A was associated with dextran, a 

marker for macropinocytosis, and KSHV entry in HMVEC-d cells was dynamin 

independent. Although KSHV was not associated with the early endosome marker EEA-1, 

internalized KSHV was associated with Rab5 and Rab34 GTPases that are known to 

regulate macropinocytosis. Taken together, these findings suggest that for its infectious 

entry in HMVEC-d and HUVEC cells, KSHV utilizes a macropinocytic pathway. 


51



Gene Expression I 

52


Gene Expression I Abstract 29 

SCREENING FOR XINJIANG KS ASSOCIATED GENES AND THE STUDY ON THEIR 

MECHANISMS 

Lei YANG, Yan ZENG, Dong-Mei LI,Ling-ling XIAN,Xiao –Fei ZHOU,Xiao-Hua TAN,Jin 

HUANG,Feng LI,Jian-Xin XIE,Hui ZHANG,Sheng Wang,Xian-Dao LUO 

Key Laboratory of Xinjiang Endemic and Ethnic Disease, Shihezi University,Shihezi, 

XinJiang, 832002, China 

Abstract 

In China, classical KS mainly occurs in Uygur and Kazak ethic group in Xinjiang 

Autonomous Region. The occurrence and development of sarcoma is linked with the 

abnormality of genes. For comprehensive clarifying the pathogenesis of KS in Xinjiang, 

We screened the differentially expressed genes between KS’ tissues and normal skin 

tissues from an classical patient of Uygur by Suppression subtractive hybridization (SSH). 

We cloned and sequenced them and established differential expression cDNA library on 

the base of SSH.Then we enlarged the samples and selected 4 classic patients to do the 

further screening by human genome U133 plus 2.0 microarray (Affymetrix). According to 

the bioinformatics analysis,we got the congenerous differentially expressed genes 

nearly 80. The function of them are focused on signal transduction and correlated with 

apoptosis or tumor occurrence. These studies identified a large number of genes whose 

expression was altered in KS. we have verified some of these differentially expressed 

genes by Real-time PCR and Immunohistochemistry, it’s consistent with the SSH and 

gene chip results.We have carried on some functional study on these genes,such as 14- 

3-3β, HO-1, Neuritin, EGFR, MMP14, LYN, et al. We detected the expression level of KS 

associated genes in BC-3 cells and Human umbilical vein endothelial cells to analyze if 

infection of HHV8 have effect on the expression of these genes .Most of these genes were 

related with the infection of HHV8 and participate in cell proliferation, apoptosis and this 

might show particular relation in the pathogenesis of KS. 

Presenting author Email: zengyan910@yahoo.com.cn 

53



HOST CELL AND VIRAL MICRORNA (MIRNA) EXPRESSION IN B CELL 

LYMPHOMAS 

Eve M Coulter, Dan Frampton, Ed Tsao, Paul Kellam 

MRC Centre for Medical Molecular Virology, Department of Infection, University College 

London, 46 Cleveland Street, London, England W1T 4JF 

Abstract 

Micro RNAs (miRNAs) are a class of approximately 21- nucleotide non-coding small RNAs 

that post-transcriptionally down-regulate the expression of mRNAs bearing 

complementary target sequences. Several hundred miRNAs have been predicted within 

the human genome and their key role as regulators of important biological pathways 

such as development, proliferation, apoptosis and viral infection is becoming clear. 

Recent discovery of virus-encoded miRNAs, especially miRNAs encoded by herpesviruses, 

indicates that viruses also use this fundamental mode of gene regulation. Using miRNA 

microarray technology, (470 human and 64 human viral miRNAs represented), we have 

determined the pattern of expression of human, Epstein Barr virus (EBV) and Kaposi’s 

sarcoma virus (KSHV) encoded miRNAs in B cell tumour cell lines associated with 

different stages of normal B cell development. Here we show that only a small proportion 

of human miRNAs are expressed in different B cell tumours cell lines and that difference 

in viral miRNA expression are evident between different herpesvirus positive tumour 

types. miR-155 and other human miRNAs are differentially expressed across different Bcell 

lymphomas including PEL. This suggests potential functional roles for their target 

genes in PEL biology. These data show that like virus and host cell gene expression, virus 

and host cell miRNA expression is associated with the underlying cell type. 

Presenting author Email: rebmeco@ucl.ac.uk 

54



CELLULAR AND VIRAL GENE EXPRESSION PROFILING IN KSHV-INFECTED 

MONOCYTES 

Sean Gregory, Ling Wang, John West, Chelsey Hilscher, Dirk P. Dittmer and Blossom 

Damania 


North Carolina at Chapel Hill, North Carolina 27599. 

Abstract 

Kaposi's sarcoma (KS)-associated herpesvirus is the etiologic agent of Kaposi's sarcoma, 

primary effusion lymphoma and multicentric Castleman's disease. KSHV can infect a 

number of cell types including B cells, endothelial cells, epithelial cells and monocytes, 

macrophages and dendritic cells. We report the establishment of a monocytic cell line 

that is latently infected with KSHV (KSHV-THP-1). To study the host response to KSHV 

infection, we compared cellular gene expression in KSHV-THP-1 cells to uninfected THP-1 

cells. We found that approximately six percent of cellular genes demonstrated a greater 

than two fold increase or decrease in gene expression in the KSHV-THP-1 cells compared 

to uninfected controls. Differences in gene expression of a select panel of genes were 

also confirmed by quantitative RT-PCR. We found that several genes involved in the host 

immune response were selectively downregulated in the KSHV-infected monocytes. 

These included tumor necrosis factor (TNF), interleukin-1b, CXCL10 and CCL4. 

Interestingly, the activation markers, CD86 and CD83 were also downregulated in KSHVinfected 

monocytes. Additionally we found several factors involved in gene expression 

and signal transduction to be upregulated in the KSHV-THP1 cells compared to the 

uninfected cells. We also profiled eighty-four viral genes in the KSHV-THP-1 cells, and 

found that KSHV predominantly exhibited a latent gene profile. Latency associated genes 

such as LANA, v-Cyclin and v-FLIP were expressed in the KSHV-infected monocytes. 

Presenting author Email: sgregory@med.unc.edu 

55



PROFILING OF CELLULAR AND VIRAL MICRORNAS IN KAPOSI SARCOMA AND 

VIRAL-ASSOCIATED LYMPHOMA 

Andrea J. O’Hara*, Bruce J. Dezube # , William Harrington Jr.^, Blossom Damania*, and 

Dirk P. Dittmer^* 

*Department of Microbiology and Immunology, Lineberger Comprehensive Cancer 

Center, and Curriculum in Genetics and Molecular Biology, University of North Carolina at 

Chapel Hill, Chapel Hill, NC 27599; # Beth Israel Deaconess Medical Center; ^Sylvester 

Comprehensive Cancer Center, University of Miami. 

Abstract 

MicroRNAs are regulated by gene alteration at the DNA level, transcriptional regulation, 

and mature miRNA processing via Dicer. Thus far, few studies have simultaneously 

assessed all three levels of regulation. Using real-time quantitative polymerase chain 

reaction (QPCR)-based arrays, changes in gene copy number, pre-miRNA and mature 

miRNA levels for a large set of primary effusion lymphomas (PELs) and primary Kaposi 

Sarcoma biopsies (KS) has been determined; this includes miRNA gene alterations and 

concordant changes in pre-miRNA and mature miRNA expression levels. The real-time 

QPCR based approach confirmed many of the KSHV viral and cellular miRNAs previously 

cloned from PEL. However, array-based profiling also uncovered many novel PEL–specific 

miRNAs, since cloning-based approaches are not always saturating. The miRNA 

expression pattern for neither viral nor cellular miRNAs has hitherto been determined for 

KS. Furthermore, comprehensive SNP analysis of the viral miRNAs has been profiled to 

further examine the effects of sequence and processing. This defines the miRNA 

signature of PEL, KS, KSHV-infected cancers and experimental models. It shows that the 

transcriptional regulation of pre-miRNA as well as mature miRNA levels contribute nonredundant 

information that can be used in the classification of human tumors. 

Presenting author Email: ohara@unc.edu 

56



INEFFICIENT CODON USAGE IN V-FLIP MRNA LEADS TO TRANSCRIPT 

INSTABILITY 

Priya Bellare, Andrew T Dufresne and Don Ganem 

HHMI and G.W. Hooper Foundation, University of California, San Francisco, California 

94143-0552, USA 

Abstract 

KSHV v-FLIP is a latent gene product that activates the NF-κB pathway, prolonging the 

survival of PEL cells and causing the characteristic spindled morphology of latently 

infected endothelial cells. The expression of v-FLIP is unusual in two regards. (i) The 

gene is expressed from a bi or tricistronic message that also encodes v-cyclin and LANA 

genes; to date, no monocistronic v-FLIP mRNA has been consistently detected in infected 

cells. (ii) The level of v-FLIP protein in infected or transfected cells is extremely low, even 

when a monocistronic mRNA is artificially engineered. Here we show that the low 

abundance of v-FLIP mRNA can be attributed to instability of the v-FLIP message, which 

in turn is a consequence of its sub-optimal codon usage. We transfected 293 cells with a 

vector designed to produce a monocistronic v-FLIP mRNA expressing Flag-tagged v-FLIP. 

As expected, v-FLIP protein levels were nearly undetectable; surprisingly, however, v- 

FLIP mRNA levels were also extremely low. Examination of codon usage in v-FLIP showed 

it to be dominated by poorly used codons throughout the length of the ORF. Expression 

of a re-engineered v-FLIP gene with efficient codon usage not only generated abundant 

v-FLIP protein but also restored mRNA accumulation to levels readily detected by 

Northern blotting. Analysis of chimeras of optimal and sub-optimal v-FLIP coding 

sequences reveals that mRNA stability cannot be mapped to a unique region of the 

mRNA; however, when an unstable v-FLIP construct is placed 3’ to a well expressed ORF, 

the resulting bicistronic mRNA is stable. These results explain many, but perhaps not all, 

of the prior conundrums of v-FLIP expression, and provide a striking example of 

translational regulation of RNA accumulation, the mechanism of which is now under 

study. 

Presenting author Email: Priya.Bellare@ucsf.edu 

57


Gene Expression II Abstract 34 

ROLE OF MURINE GAMMAHERPESVIRUS-68 ORF37 IN MEDIATING INHIBITION 

OF HOST GENE EXPRESSION 

L. Roaden 1 , V. Sheridan 1 , B. Lane 1 , R. Sun 2 , B. Dutia 3 and B. Ebrahimi 1 

1 Division of Medical Microbiology, School of Infection & Host Defence, University of 

Liverpool, Liverpool L69 3GA, UK, 2 Molecular Biology Institute, University of California, 

Los Angeles, California, USA, and 3 Centre for Infectious Diseases, Division of Veterinary 

Biomedical Sciences, University of Edinburgh, UK. 

Abstract 

In lytic infection, Murine gammaherpesvirus-68 (MHV-68) and Kaposi’s sarcoma 

associated herpesvirus (KSHV) and Epstein-Barr virus have been shown to mediate a 

global inhibition of host gene expression. g-herpesviruses, unlike a-herpesviruses, do 

not encode RNases. Recently, however, the KSHV ORF37 (an alkaline DNA exonuclease, 

also known as SOX) was shown to promote this shutdown by enhancing the degradation 

of cellular mRNAs. Similar observations have been made with the EBV homologue of 

SOX. Interestingly, SOX does not appear to possess any apparent RNase activity. The 

murine gammaherpesvirus-68 (MHV-68) encodes an ORF37 highly homologous to KSHV 

ORF37 both at DNA (55% identity) and protein (44% identity) levels. Here we show that 

MHV-68 is capable of orchestrating a global repression of cellular gene expression. Using 

transient transfection assays and engineered reporters with very rapid half-lives, we 

show that ORF37 can independently cause rapid loss of these transcripts when expressed 

in transient transfection assays. Using point mutations, we have been able to dissect key 

amino acids which may relate to the RNase activity of MHV-68 ORF37. Furthermore, we 

have generated a stop mutant of MHV-68 ORF37 to assess the potential shutdown 

properties of this viral protein during lytic infection in vitro and in vivo and its potential 

role in host gene shut-off during latency and reactivation from latency in vivo. 

Presenting author Email: ebrahimi@liv.ac.uk 

58



Lytic Replication 

59


Lytic Replication Abstract 35 

FUNCTIONAL CHARACTERIZATION OF KAPOSI'S SARCOMA–ASSOCIATED 

HERPESVIRUS ORF K8 BY BAC-BASED MUTAGENESIS 

Yan Wang, Charles Hollow and Yan Yuan 

Department of Microbiology, University of Pennsylvania School of Dental Medicine, 

Philadelphia, Pennsylvania 19104 

Abstract 

The open reading frame K8 of KSHV encodes a bZip protein, namely K8 protein or 

replication-associated protein (RAP) or K-bZip. Three functions have been reportedly 

associated with this protein. (i) K8 / RAP was found to bind to the origin of viral lytic DNA 

replication (ori-Lyt) of KSHV and its binding is absolutely required for the DNA 

replication. (ii) K8 / RAP causes cell cycle arrest at G1 phase through induction of C/EBP 

and p21. (iii) K8 / RAP interacts with virally encoded replication and transcription 

activator (RTA) and represses the transcription of viral delayed-early genes by RTA. But 

the mechanisms behind these activities of K8 still remain elusive. Especially most of data 

regarding K8 function were obtained in transient assays but not in the context of the 

virus. To study the role of K8 in viral life cycle, we generated K8-null recombinant viruses 

with bacterial artificial chromosome (BAC) and the recombineering technique. Stable 

293T cells carrying BAC-cloned wild type KSHV (BAC-36) and K8-null viral genomes 

(BAC- K8 and BAC-stopK8) were generated. When monolayers of 293T-BAC36, 293T- 

BAC- K8 and 293T-BAC-stop45 cells were induced with tetradecanoyl-phorbol-13acetate, 

no significant difference was found between them in overall viral gene 

expression. To our surprise, lytic DNA replication and extracellular virion particles were 

detected in the K8-null mutant viruses in the levels comparable to the wild type, 

suggesting that K8 is not absolutely required for viral DNA replication and viral 

propagation. The effects of the K8 mutations on host gene expression were also 

investigated. 

Presenting author Email: yuan2@pobox.upenn.edu 

60



APPLICATION OF ACTIVE KINOME COLLECTION FOR IDENTIFICATION OF A 

NOVEL KINASE FAMILY INVOLVED IN REACTIVATION OF KSHV 

Fang Cheng 1 , Markku Varjosalo 1 , Anne Lehtonen 1 , Magdalena Weidner-Glunde 2 , Päivi J. 

Koskinen 3 , Thomas Schulz 2 , Jussi Taipale 1 , and Päivi M. Ojala 1 . 

1 Genome-Scale Biology Program, Biomedicum Helsinki & Institute of Biomedicine, 

University of Helsinki, Helsinki, Finland, 2 Institute of Virology, Hannover Medical School, 

Hannover, 3 Turku Center for Biotechnology, Turku, Finland 

Abstract 

Infection by KSHV displays two different phases: latent and lytic. During latency, the 

viral genome is episomal, with only few viral genes expressed. Upon induction of the lytic 

cycle, extensive viral DNA replication and viral gene expression is initiated (viral 

reactivation), which leads to production of new viral particles. Host signal-transduction 

pathways are involved in the switch between latency and productive infection. By using a 

gain-of-function human kinome screen, we identified two new kinases, Pim-1 and -3, to 

be involved in KSHV reactivation. Ectopic expression of Pim-1 and Pim-3 Kinases induces 

viral lytic replication and production of progeny viruses. Silencing of Pim-1 and Pim-3 by 

RNA interference demonstrate that Pim-1 and Pim-3 are required for KSHV lytic 

reactivation. By performing experiments in both de novo and naturally infected KSHV cell 

models we were able to identify the molecular mechanism for Pim-1/3 induced viral 

reactivation. The identification of this novel cellular kinase family regulating the 

gammaherpesvirus life cycle will facilitate a deeper understanding of KSHV reactivation 

and could represent a potential novel target for therapeutic intervention. 

Presenting author Email: cheng.fang@helsinki.fi 

61



NOVEL FUNCTIONS OF K-RTA AND K-BZIP AS SUMO-TARGETING LIGASES AND 

EPIGENETIC REGULATORS 

Hsing-Jien Kung, Pei-Ching Chang, Latricia Fitzgerald, Tom Ellison, Paul Luciw* and Yoshi 

Izumiya 

Dept. of Biochemistry and Molecular Medicine, *Dept of Pathology, UC Davis Cancer 

Center, Sacramento CA 95864 

Abstract 

SUMO modification emerges as a major post-translational modification of viral and 

cellular proteins. Sumoylation in concert with histone methylation at H3K9 are hallmarks 

of heterochromatin. Like phospho-tyrosines, sumoylated lysines also serve as signals 

linking components of signal transduction. SUMO interacting motifs (SIM) have been 

identified in a number of cellular regulatory proteins including PML, DAXX and RNF4. 

RNF4, a SUMO-targeting ubiquitin ligase, preferentially facilitates the degradation of 

sumoylated proteins. Here we report the identification of K-Rta and K-bZIP as SUMO 

binding proteins, and have mapped the SIMs in these molecules. We have previously 

shown that K-bZIP behaves like a SUMO-ligase, via its ability to bind Ubc9 E2 SUMO 

conjugation enzyme (which is itself sumoylated). Studies from the labs of Hayward and 

Wood showed that K-Rta behaves like a ubiquitin ligase. We reported that K-Rta 

decreases the overall level of sumoylated proteins, and is counteracted by K-bZIP. We 

now show that K-Rta preferentially degrades sumoylated interacting protein. Taken in the 

present context, these data are consistent with the notion that K-bZIP is a SUMOtargeting 

SUMO ligase, and K-Rta, a SUMO-targeting Ubiquitin ligase. If true, they are 

the first SUMO-targeting viral ligases identified. The ability of K-Rta and K-bZIP to 

modulate the activity and stability of sumoylated proteins raises the possibility of their 

roles in the epigenetic regulation of viral latency. Data will be presented that JMJD2A, a 

H3K9me3 histone demethylase, is sumoylated and its demethylase activity is directly 

blocked by K-bZIP. 

Presenting author Email: yizumiya@ucdavis.edu 

62



X-BOX BINDING PROTEIN 1 DOES NOT INDUCE EBV LYTIC REPLICATION IN 


Imogen Yi-Chun Lai and Paul Kellam 

MRC Centre for Medical Molecular Virology, Department of Infection, University College 

London. 46 Cleveland Street, London W1T 4JF 

Abstract 

The plasma cell differentiation factor XBP-1 has been shown to transactivate the KSHV 

ORF50 promoter in primary effusion lymphoma (PEL) cells, leading to reactivation of 

KSHV from latency. PEL is a plasmablast lymphoma, arrested immediately before 

terminal differentiation into antibody secreting plasma cells. PEL are negative for the 

spliced transcriptional active form of XBP-1, namely XBP-1s. All PEL cells are infected 

with Karposi’s Sarcoma-associated Herpesvirus (KSHV), with about 75% also co-infected 

with Epstein - Barr virus (EBV). However little is known about the reactivation of EBV 

from latency in PEL cells. XBP-1s has been suggested to transactivate the immediate 

early gene BZLF-1 of EBV. Here we investigate the effect of different inducers of EBV 

lytic reactivation in PEL cells. By using Western blot, we show that XBP-1s does not 

induce the lytic reactivation of EBV defined by lack of expression of BZLF-1, despite 

inducing lytic reactivation of KSHV. 

Presenting author Email: imogen.lai@ucl.ac.uk 

63



RECRUITMENT OF THE COMPLETE HTREX COMPLEX IS REQUIRED FOR KSHV 

INTRONLESS MRNA NUCLEAR EXPORT AND VIRUS REPLICATION 

James R. Boyne and Adrian Whitehouse 

Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United 

Kingdom 

Abstract 

The majority of pre-mRNAs in higher eukaryotes contain introns and are exported via 

recruitment of the conserved export machinery, TREX, which occurs in a splicingdependent 

manner. In contrast, most Kaposi’s sarcoma associated herpesvirus (KSHV) 

mRNAs lack introns and are exported by the KSHV ORF57 protein. Herein, we show that 

ORF57 functions to recruit the human TREX (hTREX) complex to intronless viral mRNAs 

and that this recruitment, but not recruitment of the exon-junction complex, is essential 

for nuclear export. The formation of the ORF57-hTREX complex is mediated by a direct 

interaction between ORF57 and the export adapter, Aly. We show that a point mutation 

in ORF57 which disrupts the ORF57-Aly interaction leads to a failure in both the ORF57mediated 

recruitment of hTREX to viral mRNA and their subsequent nuclear export. 

Furthermore, using a KSHV replication system we demonstrate that expression of a 

trans-dominant Aly mutant disrupts hTREX assembly but retains an ORF57-Aly-TAP 

complex on the viral mRNA. Strikingly, in the absence of UAP56 and hTHO-complex we 

observed a dramatic decrease in intronless viral mRNA export and virus replication. 

These data provide the first direct evidence that the entire hTREX complex is essential for 

the export of viral intronless mRNAs and in turn, KSHV replication. 

Presenting author Email: bmbjb@bmb.leeds.ac.uk 

64



DOMAIN STRUCTURE AND FUNCTION OF KSHV ORF57 PROTEIN IN PROTEIN- 

PROTEIN AND PROTEIN-RNA INTERACTIONS 

Vladimir Majerciak and Zhi-Ming Zheng 

HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, 

10 Central Dr. 6N106, Bethesda, MD 20892-1868, USA 

Abstract 

Kaposi’s sarcoma-associated herpesvirus (KSHV) ORF57 is a multifunctional regulator in 

the expression of viral lytic genes. ORF57 enhances the expression of both intronless and 

intron-containing viral genes and is essential for virus replication. However, the 

molecular mechanism of ORF57 action remains unclear. Our analysis of ORF57 amino 

acid (aa) sequence identified two distinctive parts of ORF57 based on secondary structure 

prediction: the N-terminal part has no obvious secondary structure (coil), but bears a 

strong antigenic epitope; the C-terminal part consists of several alpha-helixes and betasheets, 

with all weak antigenic epitopes. In vivo ORF57 was found in a multiprotein 

complex enriched in RNA-binding proteins including RNA helicase A, hnRNP U, nucleolin, 

and Aly/REF in association with RNA. The relaxed N-terminal region, in particular the 

nuclear localization signal 2 (NLS2, aa 121-130) that overlaps the antigenic epitope, was 

mapped to mediate these interactions. The interaction of ORF57 with nucleolin depends 

on RNAs. In lyticaly infected B cells, KSHV ORF57 interacts with nucleolin in nucleoplasm, 

but not colocalizes with nucleolin concentrated in nucleoli. The C-terminal structural 

region of ORF57 contains a domain for ORF57 dimerization. The dimer formation between 

mutant and wild-type ORF57 leads the mt ORF57 normally localized in the cytoplasm to 

translocate in the nucleus in co-transfected cells. Data indicate that KSHV ORF57 

associates with many protein partners through its different domains to diversify its 

functions in regulating expression of viral intronless or intron-containing genes at 

posttranscriptonal level. 

Presenting author Email: majerciv@mail.nih.gov 

65



KSHV ORF57 ENHANCES TRANSLATION OF VIRAL INTRONLESS MRNAS 

James R. Boyne, Adam Taylor and Adrian Whitehouse 

Institute of Molecular and Cellular Biology, University of Leeds, Leeds, LS2 9JT, United 

Kingdom 

Abstract 

The majority of Kaposi’s sarcoma associated herpesvirus (KSHV) mRNAs lack introns and 

are exported by the ORF57 protein. We have shown that ORF57 binds specifically to 

intronless viral mRNAs and functions to recruit the human TREX (hTREX) complex, in 

contrast to the exon-junction complex, allowing efficient nuclear export of the viral 

intronless mRNAs. The failure of ORF57 to recruit EJC components to viral intronless 

mRNAs presents KSHV with an intriguing problem, as the EJC enhances translation of 

cellular spliced mRNAs. The EJC specifically recruits the translation machinery onto the 

mRNA via an interaction with the cellular protein PYM, thereby enhancing mRNA 

translation. Herein, we demonstrate that KSHV ORF57 functions in translation 

enhancement, bypassing the EJC, by recruiting essential cellular translation factors. We 

shown, using an in vitro translation assay, that recombinant KSHV ORF57 can enhance 

translation of an in vitro transcribed KSHV intronless RNA. Moreover, coimmunoprecipitation 

assays demonstrate that KSHV ORF57 interacts with PYM and 48S 

preinitiation components in an RNA-independent manner in both transfected and 

reactivated BCBL cells. Furthermore, GST-pulldown analysis shows that ORF57 only 

associates directly with PYM and no other members of the 48S preinitiation complex. This 

suggests that KSHV ORF57 enhances translation by recruiting the 48S preinitiation 

complex onto a viral intronless mRNA lacking an EJC, via its direct interaction with PYM. 

This data highlights a new role for the ORF57 protein in the KSHV lytic replication cycle. 

Presenting author Email: A.Whitehouse@leeds.ac.uk 

66



Immunology II 

67


Immunology II Abstract 42 

MODULATION BY KSHV OF LIGANDS THAT MEDIATE NK CELL RECOGNITION 

Alexis Madrid and Don Ganem 

HHMI and Dept of Microbiology, UCSF, San Francisco, CA 94143-0552 

Abstract 

Natural Killer (NK) cells express an array of activating and inhibitory receptors on their 

surfaces. The inhibitory receptors generally recognize MHC class I (MHC-I) molecules on 

healthy cells, which are therefore not targeted for destruction. Activating receptors 

recognize a variety of cellular ligands, many of which are upregulated in pathologic 

conditions. The balance of activating and inhibitory signals within the NK cell determines 

whether a target cell will be destroyed and an immune response mounted. KSHV 

encodes proteins that downregulate MHC-I on infected cells, thus potentially exposing 

these cells to NK cell mediated lysis. Therefore we hypothesized that KSHV would also 

have evolved mechanisms to evade killing by NK cells, and one potential way to evade 

killing is through downregulation of NK activating ligands. To test this hypothesis, we 

infected several human cell lines with KSHV, and measured the cell surface levels of the 

various NK activating ligands. The surface display of most NK ligands tested was not 

affected during infection. However, we found that one NK activating ligand, NKp44-L, is 

consistently downregulated during both latent and lytic KSHV infection. We then 

screened a KSHV ORF library in order to search for the viral gene product responsible for 

this activity, and identified this protein as Kaposin B. However, Nkp44-L downregulation 

is not due to Kaposin B’s known function in activating the p38/MK2 signaling pathway, 

and likely reflects an additional activity of the molecule. Studies of the mechanism by 

which Kaposin B downregulates NKp44-L are ongoing and will be reported. 

Presenting author Email: Alexis.madrid@ucsf.edu 

68



SCREENING FOR NATURALLY OCCURRING ANTI-KCP (KSHV COMPLEMENT 

CONTROL PROTEIN; ORF4) ANTIBODIES IN KSHV-INFECTED PATIENTS AND 

GENERATION OF SPECIFIC MONOCLONAL ANTIBODIES FOR PATHOGENESIS 

STUDIES 

Anna M. Blom 1a , Marcin Okroj, 1a Linda Mark, 1a Zoltan Korodi, 1b Rosamaria Tedeschi, 2 

Joakim Dillner 1b and O. Brad Spiller 3 

a Department of Laboratory Medicine, b Department of Medical Microbiology, 1 Lund 

University, University Hospital Malmö, S-205 02, Malmö, Sweden. 2 Department of 

Microbiology, Oncological Center, Aviano 33081, Italy. 3 Department of Child Health, 

Cardiff University, School of Medicine, Cardiff CF14 4XN, United Kingdom. 

Abstract 

ORF4 encodes a complement regulating protein expressed in the late virus replication 

phase, referred to as the KSHV Complement Control Protein (KCP). KCP is expressed on 

the virion surface and can be induced on the surface of KSHV-positive primary effusion 

lymphoma (PEL) B-lymphocytes. KCP mediates protection from complement attack and 

an integral heparin binding motif enables virion KCP to enhance virus infection through 

extracellular matrix binding. Using recombinant soluble KCP and transfected cells hyperexpressing 

various recombinant portions of KCP, serum from different KSHV-infected 

patient groups were investigated for anti-KCP reactivity. Specific anti-KCP IgG, but not 

IgM, was found predominantly in patients that had high titres of antibodies against other 

lytic KSHV proteins and the second (CCP2) of the 4 complement control domains in KCP 

was found to be the dominant epitope recognised. Patient anti-KCP antibodies enhanced 

complement attack on KCP-expressing CHO cells, suggesting neutralisation. Anti-KCP 

antibodies were found in all KSHV-infected lymphoma patients except one examined, but 

only 3/16 KS patients, suggesting differential KCP expression between these two 

diseases. Recombinant soluble and cell-expressed KCP were used to generate and 

characterise monoclonal anti-KCP antibodies against each CCP domain. These 

monoclonal antibodies differentially regulated decay-accelerating factor activity, cofactor 

activity and heparin binding. Primary conjugated anti-KCP monoclonal antibodies 

labelled with FITC, PE, APC or HRP have now been created and await appropriate KSHV 

patient groups for longitudinal flow cytometric and histological assessment through 

disease progression and treatment. 

Presenting author Email: spillerb@Cardiff.ac.uk 

69



A KSHV VIRAL HOMOLOGUE OF CELLULAR CD200 (VOX2) INDIRECTLY 

SUPPRESSES GRANULOCYTE OXIDATIVE ACTIVITY IN HUMAN BLOOD 

Karen Misstear 1 , Rachel Colman 1 , Hema Chahal 2 , Janet Lord 2 , David Blackbourn 1 

CRUK Institute for Cancer Studies 1 and Department of Immunity and Infection 2 , 

University of Birmingham, Edgbaston, Birmingham B15 2TT, UK 

Abstract 

Background: Kaposi’s sarcoma-associated herpesvirus (KSHV) encodes a homologue 

(vOX2) of CD200, a transmembrane protein with known immunoregulatory activities 

exerted via its receptor, CD200R. vOX2 shares approximately 36% identity with CD200, 

suggesting similar roles, though vOX2 functions are incompletely understood. In vivo, an 

engineered, soluble vOX2:Fc reduced inflammation in the murine carrageenan footpad 

model, suggesting it suppresses neutrophil activity. vOX2 and CD200 bind CD200R with 

similar affinity, though a putative integrin binding domain indicates a larger receptor 

repertoire for vOX2 is likely. 

Methods: vOX2, CD200 and an inactive KSHV protein, KCPmut, were expressed with an 

in-frame carboxyl-terminal Fc domain of human IgG1. Human primary neutrophil 

activation was quantified by superoxide and myeloperoxidase release. vOX2:Fc and 

CD200:Fc activity in whole blood was determined by flow cytometric analysis of 

granulocyte oxidative activity. CD200R expression by leukocyte subpopulations was 

quantified by flow cytometry. 

Results and conclusions: vOX2:Fc and CD200:Fc exerted no effect upon isolated 

neutrophils, but suppressed granulocytic oxidative activity in whole blood by 19.13% and 

24.34% respectively. These results suggest that vOX2:Fc acts upon granulocytes 

indirectly, via another leukocyte population(s). Natively expressed vOX2 and CD200 

inhibited the secretion of IL-8, a potent neutrophil chemoattractant, from a monocytic 

cell line. The expression of CD200R on major leukocyte subpopulations, including 

granulocytes, suggests that although vOX2:Fc ligates CD200R, it may not invoke 

signalling by this receptor. 

Presenting author Email: kxm694@bham.ac.uk 

70



CD8+ T CELL RECOGNITION OF THE KSHV LATENT PROTEIN LANA1 

Shereen Sabbah & Andrew D Hislop 

CRUK Institute for Cancer Studies, The University of Birmingham, Edgbaston, 

Birmingham, B15 2TT, UK 

Abstract 

Kaposi’s sarcoma-associated herpesvirus (KSHV) has been implicated in the development 

of the endothelial malignancy, Kaposi’s sarcoma, and the B cell malignancies primary 

effusion lymphoma and multicentric Castleman’s disease. In these malignancies as well 

as KSHV infected cells, the viral genome maintenance protein latency associated nuclear 

antigen 1 (LANA1) is expressed. Potentially then, LANA1 is an attractive immune target. 

Similar to what is seen in the genome maintenance protein of the other human gammaherpesvirus, 

Epstein-Barr virus EBNA1, LANA1 contains extensive amino acid repeat 

sequences. In the case of EBNA1, these repeats reduce the efficiency of presentation of 

EBNA1 derived epitopes to CD8+ T cells. To determine whether the repeats of LANA1 

afford a similar protection, we established a model system to examine the ability of 

CD8+ T cells to recognise target cells expressing either LANA1 or a LANA1 construct 

deleted of the repeat region (LANA1-delta), both of which encode an inserted human 

CD8+ T cell epitope. We find that cells expressing the LANA1-delta protein express 

higher levels of this protein compared to the full length protein, yet CD8+ T cells 

recognise both target cells types efficiently. In this model system then, the LANA1 

repeat sequences give no protection against CD8+ T cell recognition. We are now 

mapping CD8+ T cell epitopes contained in LANA1 and will establish CD8+ T cell clones 

specific for identified epitopes. These T cell clones will be used to examine their ability to 

recognise natively processed and presented LANA1 epitopes on KSHV-infected cells. 

Presenting author Email: sxs463@bham.ac.uk 

71



CYTOKINE RESPONSES BY CD8 T LYMPHOCYTES TO KSHV/HHV8 LYTIC AND 

LATENCY PROTEINS 

Lauren Lepone, Giovanna Rappocciolo, Emilee Knowlton, Paolo Piazza, Mariel Jais, Frank 

J. Jenkins and Charles R. Rinaldo 

Dept of Infectious Diseases and Microbiology, Graduate School of Public Health, 

University of Pittsburgh, Pittsburgh, PA 

Abstract 

Objectives: CD8 T cells producing more than one cytokine or cytotoxicity factor, i.e. 

polyfunctional, have been shown to relate to control of HIV-1 infection. Based on our 

previous findings that dendritic cells (DC) are optimally required as antigen presenting 

cells to reveal MHC class I epitopes of KSHV/HHV-8 glycoprotein B (gB) (Blood 99:3360, 

2002), we determined CD8 T cell cytokine responses to multiple lytic and latency viral 

proteins. 

Methods: T cells were stimulated with DC from HHV-8 seropositive, HLA A*0201 donors 

that had been loaded with overlapping peptides derived from gB and K8.1 lytic proteins, 

and K12 and LANA latency proteins, and screened for IFNγ production by ELISPOT. 

Positive peptides were assessed for IFNγ, IL2, TNFα, MIP1β, and CD107a production by 

flow cytometry. 

Results: ELISPOT and flow cytometry revealed new, potential HLA A*0201 9mer 

epitopes for gB (5), K8.1 (4), K12 (2) and LANA (5). In HHV-8 seropositive healthy 

donors controlling infection, the predominant CD8 T cell response to protein epitopes was 

monofunctional, with a portion of CD8 T cells being polyfunctional. 

Conclusions: This demonstrates that HHV-8 proteins predominantly induce single 

cytokine or cytotoxicity factors in CD8 T cells from healthy, HHV-8 seropositive, HIV-1 

negative adults, with a portion of T cells producing 2-3 of these factors. These 

immunogenic regions of the viral proteins could be important in immunopathogenesis of 

KSHV/HHV-8 infection and progression to Kaposi’s sarcoma. 

Presenting author Email: LML33@pitt.edu 

72



INFECTION OF LYMPHOID CELLS BY KSHV IN CULTURED PRIMARY HUMAN 

TONSILLAR LYMPHOID CELLS EX VIVO 

Jinjong Myoung and Don Ganem 

HHMI and Departments of Microbiology and Medicine, UCSF 

Abstract 

KSHV is known to infect B cells in vivo and is a causative agent of rare human B cell 

lymphomas (PELs). Paradoxically, however, established B cell lines are resistant to 

infection in vitro, which has greatly retarded progress in understanding the biology of 

KSHV in its natural lymphoid compartment. We prepared primary human lymphoid 

aggregate cultures (HLACs) from tonsils, and examined their infectability by KSHV. 

Recombinant virus (rKSHV.219) expressing GFP under the EF1α promoter was prepared 

from induced Vero cells latently infected with this virus. Tonsil cells were infected with 

rKSHV, then subjected to flow cytometric analysis. When tonsil cells were infected with 

an MOI of 0.75, 15% of CD19+ cells became GFP-positive. Surprisingly, in the same 

culture many more T cells (54% of CD3+ cells) were shown to be GFP+; CD8+ T cells 

were consistently more susceptible than CD4+ T cells. This difference in infectivity in T 

cell subsets became more prominent when PHA was used to activate the T cells prior to 

infection. When exposed to chemical inducers of lytic growth, infected HLAC cultures 

displayed enhanced production of infectious KSHV.214, demonstrating that HLACs are 

competent to support the complete lytic cycle of KSHV. No immortalization or blastic 

transformation has yet been observed in infected HLACs. However, GFP+ B cells were 

significantly enriched when assessed at d13 post-infection, while the proportion of GFP+ 

T cells decreased significantly by d13 PI. These data suggest that KSHV infection 

provides survival advantage in B cells, but not in T cells. 

Presenting author Email: jinjong.myoung@ucsf.edu 

73



Clinical & Epidemiology 

74


Clinical & Epidemiology Abstract 48 

PASSIVE TRANSFER OF HHV-8 ANTIBODIES FROM BLOOD DONORS TO 

TRANSFUSION RECIPIENTS AND POSSIBLE PROTECTION FROM HHV-8 

INFECTION 

Ashley L. Fowlkes 1 , Cedric Brown 1 , Minal M. Amin 1 , John Roback 2 , Robert Downing 3 , Esau 

Nzaro 4 , Jonathan Mermin 5 , Wolfgang Hladik 3 , Sheila C. Dollard 1 

1Centers for Disease Control and Prevention (CDC), Atlanta, GA. 2 Emory University 

School of Medicine, Atlanta, GA, 3 Global AIDS Program, CDC, Uganda; 4 Mulago Hospital, 

Kampala, Uganda 5 Coordinating Office for Global Health, CDC Kenya 

Abstract 

Human herpesvirus 8 (HHV-8) has been found to be transmissible by blood transfusion. 

Examination of serially collected serum specimens from patients following blood 

transfusion with HHV-8 seropositive blood revealed that antibodies to HHV-8 appeared in 

several patients following transfusion then declined rapidly over a matter of weeks. The 

source of this antibody was determined to be the transfused blood. We compared the 

frequency of passive antibody transfer by donor serostatus, the frequency of HHV-8 

infection in the absence of passive antibody, and evaluated the risk of infection as a 

function of the number of seropositive transfusions and recipient antibody levels. Of 542 

recipients included, passive antibody was found in 69% of patients transfused with high 

antibody titer blood. Patients who received multiple HHV-8 seropositive transfusions and 

those with highest levels of anti-HHV-8 antibodies were least likely to contract HHV-8 

infection from transfusion. Our study indicates that HHV-8 antibody is passively 

transferred by blood transfusion, waning within 40 days. Furthermore, higher levels of 

HHV-8 antibody in donor blood may have conferred protection from infection in 

transfused patients, or indicated donors less likely to have infectious virus in the blood. 

Presenting author Email: sgd5@cdc.gov 

75



HEIGHTENED REDOX STATUS OF PRIMARY EFFUSION LYMPHOMA CELLS CAN BE 

EXPLOITED THERAPEUTICALLY BY DECREASING RESISTANCE TO OXIDATIVE 

STRESS 

Darya Bubman 1 , Utthara Nayar 2 , Balasz Csernus 3 , Ilaria Guasparri 3 , Ethel Cesarman 3 

1= Program in Pharmacology, Weill Cornell Graduate School of Medical Sciences, 2= 

Program in Immunology and Microbial Pathogenesis, Weill Cornell Graduate School of 

Medical Sciences, 3= Department of Pathology and Laboratory Medicine, Weill Cornell 

Medical College 

Abstract 

Intracellular reactive oxygen species (ROS) levels are maintained in a fine balance; high 

levels can induce an oxidative stress response and cell death, while chronic moderately 

high levels may promote neoplastic growth and proliferation. Specifically, a high level of 

the superoxide (O2 - ) species is associated with cellular transformation. We have shown 

that PEL cells have higher levels of ROS, particularly superoxide, than KSHV-negative 

cells. Furthermore, we showed that vFLIP is responsible for these high levels of 

superoxide, by both overexpression and knockdown approaches. PEL cells also express 

high levels of superoxide dismutases (SODs), specifically MnSOD, which is probably 

necessary for PEL cells to eliminate superoxide and avoid oxidative stress. We therefore 

determined whether oxidative stress could be used as an approach for the treatment of 

PEL. 2-Methoxyestradiol (2ME2), or Panzem (Entremed), is an endogenous metabolite of 

estrogen that has antiangiogenic and antitumor effects, and is currently in early clinical 

studies for a variety of neoplasms. While 2ME2 acts through multiple mechanisms of 

action, these include inhibition of SOD activity and disruption of angiogenesis through the 

inhibition of hypoxia inducible factor-1 alpha (HIF-1α), a protein required for 

angiogenesis and cell survival under hypoxic and oxidative stress. We therefore treated 

cells with 2ME2 in vitro, and demonstrated that PELs, but not normal lymphocytes, were 

extremely sensitive to drug. We also determined whether this drug is effective in vivo, by 

using a mouse PEL xenograft model with in vivo imaging, following oral administration of 

clinically formulated 2ME2. 

Presenting author Email: utn2001@med.cornell.edu 

76



RAPAMYCIN IS EFFECTIVE AGAINST PTEN POSITIVE AIDS-DEFINING 

MALIGNANCIES 

Debasmita Roy, Sang-Hoon Sin, Ling Wang, Blossom A. Damania, Dirk P. Dittmer 

University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA 

Abstract 

Primary Effusion Lymphoma (PEL), a B-cell lymphoma characteristic of AIDS and 

immune-compromised patients, is tightly correlated with Kaposi’s Sarcoma-associated 

Herpes Virus (KSHV) infection. In this study we investigate the effect of inhibition of 

mTor signaling (mammalian target of Rapamycin) in PEL. The anti-tumor potential of 

Rapamycin has been the subject of intense investigations and we have shown previously 

that it is effective against PEL both in culture and our murine xenograft model via downregulation 

of cytokines, IL-6 and IL-10 (Sin et. al. Blood 2007. 109(5)). Here we show 

that the PEL-specific inhibitory effect of Rapamycin is also mediated by reduction of proangiogenic 

Vascular Endothelial Growth Factor (VEGF). We further find that the genetic 

status of the mTOR signaling cascade is intact in PEL cells, without mutations in negative 

regulators such as Phosphatase and Tensin Homolog (PTEN), Tuberous Sclerosis (TSC) 

Factor-1 and TSC-2. Typically, Akt activation results from deletion of the PTEN gene 

resulting in hyperactivation of mTor; instead we observe that Akt is activated and PTEN 

inactivated post-translationally, presumably by viral factors. Specifically, we show the 

presence of wildtype PTEN, but that it is silenced epigenetically, the mechanism of which 

differs between PEL and KSHV-infected endothelial cells. This suggests that in the special 

case of HIV-AIDS defining malignancies, Rapamycin can be effective in PTEN wild type 

tumors, contrary to previous reports of Rapamycin being effective exclusively in PTEN 

deleted tumors. 

Presenting author Email: debasmita_roy@med.unc.edu 

77



GEOGRAPHIC VARIATION OF THE PREVALENCE OF KAPOSI’S SARCOMA- 

ASSOCIATED HERPESVIRUS AND RISK FACTORS FOR TRANSMISSION IN 

WOMEN FROM 8 COUNTRIES IN FOUR CONTINENTS 

Silvia de Sanjose 1 , Georgina Mbisa 2 , Sussana Perez 1 , Sukhon Sukvirach 3 , Nguyen Trong 

Hieu 4 , Hai-Rim Shin 5 , Pham Thi Hoang Anh 4 , Jaiye O Thomas 6 , Eduardo Lazcano 7 , Elena 

Matos 8 , Rolando Herrero 9 , Nubia Muñoz 10 , Silvia Franceschi 5 , Denise Whitby 2 

1. Institut Català d’Oncologia, Barcelona, Spain; 2. NCI-Frederick, Frederick, MD, USA; 3. 

Nacional Institute, Bangkok; 4. Hung Vuong Hospital, Ho Chi Minh City, Vietnam; 5. 

IARC, Lyon, France; 6. University of Ibadan, Ibadan, Nigeria; 7. Instituto Nacional de 

Salud Publica, Cuernavaca, Mexico; 8. Instituto de Oncologia Angel H Roffo, Universidad 

de Buenos Aires, Argentina; 9. Proyecto Epidemiologico Guanacaste, Fundación 

INCIENSA, San José, Costa Rica; 10. Instituto Nacional de Cancerologia, Bogota, 

Colombia 

Abstract 

Transmission routes of Kaposi’s sarcoma-associated herpes virus (KSHV) in the general 

population are poorly understood. Sexual transmission appears to be common in 

homosexual men but heterosexual transmission has not been clearly documented. This 

study aims to estimate the prevalence of KSHV in the female general populations of 

Argentina, Colombia, Costa Rica, Nigeria, Spain, Vietnam, Thailand and Korea to explore 

geographical variation and potential heterosexual transmission. Samples and 

questionnaire data were available from a study organized by the International Agency for 

Research on Cancer (IARC) to estimate the prevalence of distinct sexually transmitted 

infections. The study includes 10963 women from 10 centers with questionnaire 

information available on socio-demographic, reproductive and sexual lifetime 

experiences, smoking habits. HPV DNA detection was previously measured .Antibodies 

against KSHV encoded K8.1 and orf73 were determined. Prevalence of antibodies to any 

of the two antigens k8.1 or orf73 was 13.9% with an important geographical variation 

(range= Nigeria 46%- 3.8% in Spain). Antibodies increased with increasing age 

particularly in high prevalent countries such as Nigeria, Colombia and Costa Rica. KSHV 

was not related to education, age at first sexual intercourse, number of sexual partners, 

number of children, patterns of use of oral contraceptives or presence of cervical HPV 

DNA. A decreased prevalence was observed with increasing number of cigarettes smoked 

per day ( p=0.000). 

The study provides reliable and comparable estimates of KSHV in diverse cultural settings 

across four continents and provides a powerful indication of absence of heterosexual 

transmission of KSHV. 

Presenting author Email: whitbyd@ncifcrf.gov 

78



SERUM EPIDEMIOLOGICAL STUDIES ON KAPOSI'S SARCOMA IN XINJIANG 

Lei YANG, Xiao-Hua TAN, Jiang-Mei QING, Feng LI, Shu-Xia GUO, Jian-Xin XIE, Jin 

HUANG, Dong-Mei LI,Yan ZENG 

Key Laboratory of Xinjiang Endemic and Ethnic Disease, Shihezi University,Shihezi, 

XinJiang, 832002, China 

Abstract 

Kaposi’s Sarcoma (KS) is a multiple malignant idiopathic pigmented sarcoma. KS has 

obvious characteristic of endemicity and ethnology. In China, Xinjiang Uygur Autonomous 

Region is one of the highest incidence regions of KS.A 1:4 matched case-control study 

was conducted to assess the role of environment and life style factor on the pathogenesis 

of KS in Xinjiang. We collected blood samples from17 cases of KS patients and 68 cases 

of non-KS patients as control. Infectious factors, some cytokines’ serum level and 

immune state IL-6, TNF-α, IFN-γ, VEGF, Neopterrin, β2-MG were tested for both group 

using ELISA. We tested the infection rate of Human herpesvirus 8(HHV-8) by nested 

PCR. The strength of association of risk factors was determined using x 2 test. We found 

KS show no relationship to area of residence and environment factors. The level of VEGF, 

TNF-α, IL-6, neopterin and β2-miroglobin were significantly higher among cases than 

control. The positive infection rate of HHV-8 in KS patient (91.66%) was significantly 

higher (p



KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS SEROCONVERSION AND 

SEROREVERSION IN A MATERNAL-INFANT COHORT IN ZAMBIA 

Veenu Minhas, 1 Kay L. Crabtree, 1 Janet Wojcicki, Tendai J. M’soka, 2 Chipepo Kankasa, 2 

Charles D. Mitchell, 3 and Charles Wood 1 

1 Nebraska Center for Virology, School of Biological Sciences, University of Nebraska 

Lincoln, Lincoln, NE USA; 2 Department of Paediatrics and Child Health, University 

Teaching Hospital, Lusaka, Zambia; 3 Department of Pediatrics, University of Miami 

School of Medicine, Miami, FL USA. 

Abstract 

Zambia is currently experiencing an HIV-1 epidemic leading to an increase in the 

incidence of Kaposi’s sarcoma among adults and children. This is a prospective study 

designed to investigate Kaposi’s sarcoma-associated herpesvirus (KSHV) seroincidence 

and associated risk factors for KSHV seroconversion and seroreversion in a mother-infant 

cohort, by following them for 48 months after delivery. Our results show that overall 

KSHV seroincidence in KSHV negative Zambian women was 0.7 per 100 person-months 

which was lower as compared to children followed in the same cohort. We observed that 

up to 40% of Zambian children in this cohort may be infected by 48 months of age, 

indicating that primary infection occurs mostly in early childhood. HIV-1 infection was 

associated with higher risk for seroconversion in women and children. In adult women, 

KSHV seroconversion was not associated with most socio-economic factors, sexual habits 

or medical history. This indicates that sexual transmission during adulthood may not be 

an important route of transmission in this cohort. 

Our laboratory has earlier reported that KSHV antibodies may not persist over time in 

children and that seroreversion leading to partial or complete loss of KSHV antibodies is 

prevalent. We observed seroreversion in up to 55% of women and this was more 

common in single women and in women delivering for the first time. These results 

demonstrate that multiple longitudinal samples per patient may be required to accurately 

determine KSHV infection status. 

Presenting author Email: veenu@bigred.unl.edu 

80



DISTRIBUTION OF KSHV MICRORNA POLYMORPHISMS IN AIDS-KS, CLASSICAL 

KS, AND MULTICENTRIC CASTLEMAN’S DISEASE 

Vickie Marshall, 1 Eliza Martró, 2 Elizabeth Brown, 3 Dian Wang, 1 Alex Ray, 1 Maria 

Nazzarena Labo, 1 The Classical Kaposi’s Sarcoma Working Group, Robert Yarchoan, 4 

Jordi Casabona, 2 Rolfe Renne, 5 and Denise Whitby 1 

1 NCI-Frederick, Frederick, MD, USA; 2 Hospital Universitari Germans Trias i Pujol, 

Badalona, Spain; 3 University of Alabama at Birmingham, Birmingham, AL, USA; 4 Division 

of Clinical Services, NCI, NIH, Bethesda, MD, USA; 5 University of Florida Shands Cancer 

Center, Gainsville, FL, USA 

Abstract 

MicroRNAs are short regulatory RNAs of approximately 19-24 nucleotides in length that 

bind specifically to mRNA in a process that interferes with translation or promotes 

degradation. KSHV encodes twelve microRNAs, all located within the latency associated 

transcript which is highly expressed in KSHV-associated malignancies. 

We amplified, cloned, and sequenced a 2,712 bp non-coding region containing a cluster 

of 10 microRNAs and a 646 bp fragment of the K12/T0.7 gene which contains two 

microRNAs. We successfully characterized KSHV microRNA sequences from lymphocytes 

collected as part of two Kaposi’s sarcoma case-control studies (one AIDS-KS, one 

Classic–KS) and MCD clinic attendees from the NCI. We have completely or partly 

characterized all twelve KSHV microRNAs from a total of 37 Kaposi sarcoma cases, 21 

KSHV positive controls, and 8 MCD patients. 

Phylogenetic analysis of both regions demonstrates extreme conservation of microRNAs 

overall. However, several distinct polymorphisms were discovered in this study, some 

occurring within the mature microRNA transcript. Phylogenetic characterization of the 

KSHV K1 and viral microRNA sequences in this study indicated all were subtypes A/C. 

However, we did detect four microRNA polymorphisms we previously described as 

specific to KSHV viral subtypes A5/B. Some polymorphisms appear to be more common 

in Kaposi’s sarcoma and MCD cases than in KSHV positive controls. 

Our data demonstrate that most KSHV microRNAs are highly conserved suggesting that 

they contribute to the biological activity and possibly viral pathogenesis of KSHV. 

However, some KSHV microRNAs have distinct polymorphisms which may affect 

processing, function, and mRNA target recognition. 

Presenting author Email: marshall@ncifcrf.gov 

81



EARLY CHILDHOOD INFECTION BY KAPOSI’S SARCOMA-ASSOCIATED 

HERPESVIRUS AND EPSTEIN-BARR VIRUS IN ZAMBIA 

Veenu Minhas, 1* Brad P Brayfield, 1 Tendai J. M’soka, 2 Chipepo Kankasa, 2 Charles D. 

Mitchell, 3 and Charles Wood, 1 

1 Nebraska Center for Virology, School of Biological Sciences, University of Nebraska 

Lincoln, Lincoln, NE USA; 2 Department of Paediatrics and Child Health, University 

Teaching Hospital, Lusaka, Zambia; 3 Department of Pediatrics, University of Miami 

School of Medicine, Miami, FL USA. 

Abstract 

Significant differences exist between developed and developing countries in the 

prevalence rate, age of infection and the observed clinical manifestations for Kaposi’s 

sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) infections. Both are 

oncogenic gamma-herpesviruses that have been associated with Kaposi’s sarcoma and 

AIDS-related non-Hodgkin lymphomas, respectively. Therefore, it is of interest to 

understand the natural history of infection of these two gamma-herpesviruses in an 

endemic area, their potential interaction and the role of human immunodeficiency virus 

(HIV) infection and other risk factors that may be associated with early childhood 

infection. This study was conducted on a cohort of 12 month old infants from Lusaka, 

Zambia. A set of pre-tested standardized questionnaires was used to gather data from 

the caregivers and multivariate analysis was performed to assess the association of a 

range of risk factors to seroprevalence. We observed a significantly higher 

seroprevalence for EBV (60.7%) as compared to KSHV (15.4%). HIV infected children 

were at a significantly higher risk of being infected with KSHV. The HIV positive status of 

their mothers was a significant risk factor for increased risk of EBV but not KSHV 

transmission to children. Presence of rashes was significantly associated with higher risk 

of seroconversion for KSHV and EBV. Most other medical and socio-economic factors 

were not associated with increased risk. High viral burdens during early childhood 

especially in HIV infected children may be contributing to higher morbidity, mortality or 

risk of developing childhood cancers in young children. 

Presenting author Email: veenu@bigred.unl.edu 

82



Virus-Cell Interactions II 

83


Virus-Cell Interactions II Abstract 56 

INTRACELLULAR LOCALIZATION MAP OF HHV-8 PROTEINS 

Gaby Sander 1 , Andreas Konrad 1 , Mathias Thurau 1 , Effi Wies 2 , Rene Leubert 3 , Elisabeth 

Kremmer 4 , Holger Dinkel 5 , Thomas Schulz 6 , Frank Neipel 2 and Michael Stürzl 1 

1 Division of Molecular and Experimental Surgery, Department of Surgery, University of 

Erlangen-Nuremberg, Schwabachanlage 10, D-91054 Erlangen, Germany; 2 Institute of 

Clinical and Molecular Virology, University of Erlangen-Nuremberg, Schlossgarten 4, D- 

91054 Erlangen, Germany; 3 Department of Virus-induced Vasculopathy, GSF-National 

Research Center for Environment and Health, Ingolstädter Landstrasse 1, D-85764 

Neuherberg, Germany; 4 GSF-Service Unit Monoclonal Antibodies and Cell Sorting, GSF- 

National Research Center for Environment and Health, Marchioninistr. 25, D-81377 

Munich, Germany; 5 Division of Bioinformatics, Institute of Biochemistry, University of 

Erlangen-Nuremberg, Fahrstrasse 17, D-91054 Erlangen, Germany; 6 Medical School 

Hannover, Department of Virology, Carl-Neubergstrasse 1, D-30625 Hannover, Germany 

Abstract 

Human herpesvirus-8 (HHV-8) is the etiological agent of Kaposi’s sarcoma. We present a 

localization map of 85 HHV-8-encoded proteins in mammalian cells. Viral open reading 

frames were cloned with a Myc-tag in expression plasmids, confirmed by full-length 

sequencing, and expressed in HeLa cells. Protein localizations were analyzed by 

immunofluorescence microscopy. 51% of all proteins were localized in the cytoplasm, 

22% in the nucleus, and 27% were found in both compartments. Surprisingly, we 

detected vFLIP in the nucleus and in the cytoplasm, whereas cellular FLIPs are generally 

localized exclusively in the cytoplasm. This suggested that vFLIP may exert additional or 

alternative functions as compared to cellular FLIPs. In addition, it has been shown 

recently that K10 can bind to at least 15 different HHV-8 proteins. We noticed that K10 

but only five of its 15 putative binding factors were localized in the nucleus when 

expressed in HeLa cells individually. Interestingly, in co-expression experiments K10 colocalized 

with 87% (13 of 15) of its putative binding partners. Co-localization was 

induced by translocation of either K10 alone or of both proteins. These results indicate 

active intracellular translocation processes in virus infected cells. Specifically in this 

framework, the localization map may provide a useful reference to further elucidate the 

function of HHV-8-encoded genes in human diseases. 

Presenting author Email: gaby.sander@uk-erlangen.de 

84



KSHV INTERFERON REGULATORY FACTOR 4, A NOVEL CBF1 INTERACTION 

PARTNER 

Katharina Heinzelmann 1+ , Barbara Scholz 1*+ , Elisabeth Kremmer 2 , Jürgen Haas 3 , Even 

Fossum 3 , Bettina Kempkes 1 

Helmholtz Zentrum München, Institute of Clinical Molecular Biology and Tumor Genetics 1 , 

Institute of Molecular Immunology 2 , Munich, Germany; Max-von-Pettenkofer-Institute 3 , 

Ludwig-Maximilians-University, Munich, Germany; 

+ These authors contributed equally. 

Abstract 

Activation of the Notch transmembrane receptor by ligand binding induces the proteolytic 

cleavage and release of an intracellular Notch fragment which can bind to the CBF1 

protein. CBF1 (also named CSL, Su(H), RBP-Jk) is a sequence specific DNA binding 

protein, which can recruit corepressor complexes to target genes and thereby repress 

transcription. Notch binding results in corepressor release and assembly of a coactivator 

complex. The KSHV proteins RTA and LANA-1 have been recently shown to bind to CBF1. 

CBF1/RTA interactions are critical for the lytic life cycle of the virus, while the 

CBF1/LANA-1 interaction might establish a regulatory loop which controls lytic 

reactivation. 

We have identified a novel CBF1 interacting KSHV protein, the viral interferon regulatory 

factor 4 (vIRF4). The KSHV genome encodes four viral proteins, which share homology to 

cellular interferon regulatory (cIRF) transcrition factors. Although vIRFs in contrast to 

cIRFs lack DNA binding functions, three vIRFs (vIRF1-3) can still modulate the interferon 

response by diverse mechanisms. For vIRF4 such activities have not been reported. We 

show, that vIRF4 is the only CBF1 interacting vIRF. Within vIRF4 we have mapped two 

distinct CBF1 binding regions. CBF1 mutants, which are deficient for Notch binding, no 

longer bind vIRF4 suggesting that Notch and vIRF4 binding sites might overlap. In Gal4fusion 

based reporter gene assays vIRF4 scores as a repressor. Since vIRF4 is induced 

upon lytic cycle reactivation, the CBF1/vIRF4 interaction might be an additional critical 

factor controlling the balance of lytic and latent life cycle of KSHV. 

Presenting author Email: kempkes@helmholtz-muenchen.de 

85



CHARACTERISATION OF ENDOPLASMIC RETICULUM (ER) STRESS INDUCERS 

THAT LEAD TO KSHV REACTIVATION FROM LATENCY; IDENTIFYING 

PHYSIOLOGICAL TRIGGERS? 

Lucy Dalton-Griffin, Ed Tsao and Paul Kellam 

MRC centre for Medical Molecular virology, Department of Infection, 

University College London, 46 Cleveland Street, London, W1T 4JF, UK 

Abstract 

Kaposi’s Sarcoma-Associated Herpesvirus (KSHV) like other herpesviruses has two stages 

to its life cycle; latency and lytic replication. KSHV is known to be required for 

development of Kaposi’s Sarcoma, a tumour of endothelial origin and is associated with 

the B-cell tumours, Primary Effusion Lymphomas (PELs) and the B-cell polyclonal 

expansion Multicentric Castleman’s Disease (MCD). Recently we and others have shown 

that the transcription factor X-box binding protein-1 (XBP-1) is a physiological trigger of 

KSHV lytic reactivation in PEL. XBP-1 is responsible for the terminal differentiation of Bcells 

into plasma cells (PCs) as it is a major regulator of the unfolded protein response 

(UPR) and therefore allows production of large quantities of immunoglobulin in these 

cells. We have previously shown that PEL are blocked in differentiation by the absence of 

active XBP-1; when provided the PEL cells differentiate towards a PC and induce KSHV 

lytic replication. Using an RFP lytic reactivation reporter assay we show that the ER stress 

inducer DTT activates the KSHV lytic cycle in an XBP-1 dependent manner. Recent 

studies have also indicated a role for hypoxia in reactivation via the hypoxia inducible 

factors (HIFs). Hypoxia can also result in XBP-1 activation as it is an ER stress inducer. 

Here we have investigated the roles of HIF-1α, HIF-2α and XBP-1 in activating the KSHV 

lytic cycle. 

Presenting author Email: ucbclda@ucl.ac.uk 

86



INTEGRIN αVβ3 BINDS TO THE RGD MOTIF OF THE GLYCOPROTEIN B OF 

KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (KSHV/HHV8) AND 

FUNCTIONS AS AN RGD-DEPENDENT ENTRY RECEPTOR 

H. Jacques Garrigues 1 , Yelena E. Rubinchikova 1 , C. Michael DiPersio 2 

and Timothy M. Rose 1,3 

1 Seattle Children’s Hospital Research Institute, Seattle WA, 2 Albany Medical College, 

Albany, New York, and 3 University of Washington, Seattle WA 

Abstract 

The virion-associated glycoprotein B (gB) of KSHV is involved in the initial steps of 

binding cells during KSHV infection. Glycoprotein B contains an RGD motif reported to 

bind the integrin α3β1 during virus entry. Although the ligand specificity of α3β1 has been 

controversial, current literature indicates that α3β1 ligand recognition is independent of 

RGD. We compared α3β1 to αVβ3, a known RGD-binding integrin, for binding to envelopeassociated 

gB and a peptide, gB(RGD), containing the RGD motif. Adhesion assays 

demonstrated that β3-CHO cells overexpressing αVβ3 specifically bound gB(RGD), whereas 

α3-CHO cells overexpressing α3β1 did not. Function-blocking antibodies to αVβ3 inhibited 

adhesion of HT1080 fibrosarcoma cells to gB(RGD), while antibodies to α3β1 did not. 

Using affinity-purified integrins and confocal-microscopy, we determined that αVβ3 bound 

to gB(RGD) and KSHV virions demonstrating direct receptor-ligand interactions. Specific 

αVβ3 antagonists, including cyclic and di-cyclic RGD peptides and αVβ3 function-blocking 

antibodies, strongly inhibited KSHV infection. Keratinocytes from α3-null mice lacking 

α3β1 were fully competent for infection by KSHV and reconstitution of α3β1 function by 

transfection with α3 cDNA reduced KSHV infectivity. Additional inhibitory effects of α3β1 

were detected on the expression of αVβ3 and on αVβ3-mediated adhesion of α3-CHO cells 

overexpressing α3β1, consistent with previous reports of transdominant inhibition of 

αVβ3function by α3β1. These observations may explain previous reports of an inhibition of 

KSHV infection by soluble α3β1. Our studies demonstrate that αVβ3 is a cellular receptor 

mediating both cell adhesion and entry of KSHV into target cells through binding the RGD 

motif of the virion-associated gB. 

Presenting author Email: timothy.rose@seattlechildrens.org 

87



KAPOSI’S SARCOMA ASSOCIATED HERPESVIRUS (KSHV/HHV-8) FORMS A 

MULTI-MOLECULAR COMPLEX OF INTEGRINS (αVβ5, αVβ3 AND α3β1), CD98 AND 

XCT DURING INFECTION OF HUMAN DERMAL MICROVASCULAR ENDOTHELIAL 

(HMVEC-D) CELLS 

Mohanan Valiya Veettil, Fu-Zhang Wang, Sathish Sadagopan, Neelam Sharma-Walia, 

Hari Raghu, Laszlo Varga and Bala Chandran 

Presenting author. H.M. Bligh cancer research laboratories, Department of Microbiology 

and Immunology, Chicago Medical School, Rosalind Franklin University of Medicine and 

Science, 3333 Green Bay Road, North Chicago, IL 60064, USA 

Abstract 

Baculovirus expressed purified KSHV-gB mediated the adhesion of HMVEC-d, HFF, CV-1 

and HT-1080 cells. Anti-αV and β1 integrin antibodies inhibited the cell adhesion. 

Variable levels of neutralization of HMVEC-d and HFF cell infection was observed with 

anti- αVβ3 and αVβ5 integrin antibodies, and variable levels of inhibition of virus entry in 

four adherent cell types was observed with soluble integrins. Virus binding and DNA 

internalization studies suggest that αVβ3 and αVβ5 integrins play roles in KSHV entry. 

We observed time dependent temporal KSHV interactions with HMVEC-d cell integrins, 

CD98 and xCT. Integrin αvβ5 interaction with CD98 predominantly occurred by 1’ postinfection 

(PI) and dissociated at 10’ PI, whereas α3β1-CD98 interaction was maximum at 

10’ PI which dissociated at 30’ PI, and αvβ3 -CD98 interaction was maximum at 10’ PI 

and retained at the observed 30’ PI. Confocal microscopy studies demonstrated the 

association of CD98/xCT with α3β1 and KSHV. Pre-incubation of KSHV with soluble 

heparin and α3β1 significantly inhibited this association. KSHV infection activated FAK 

and Src co-localized with CD98 at the plasma membranes of infected cells. Anti-CD98 

and xCT antibodies did not block virus binding and entry; however, viral gene expression 

was significantly inhibited suggesting that CD98-xCT play roles in post entry-stage of 

infection. Together, these studies suggest that KSHV interacts with functionally related 

integrins (αvβ5, α3β1 and αvβ3) and CD98/xCT molecules to form a multi-molecular 

complex during the early stages of endothelial cell infection probably mediating multiple 

roles in entry, signal transduction and viral gene expression. 


88



MURINE GAMMAHERPESVIRUS 68 ESTABLISHES A PERSISTENT INFECTION IN 

ENDOTHELIAL CELLS 

A.L. Suárez and L.F. van Dyk 

University of Colorado Denver School of Medicine, Department of Microbiology, 12800 E 

19 th Ave, RC1 North Bldg P18-9401A, Aurora, CO 80010, USA 

Abstract 

Gammaherpesvirus associated diseases occur predominately in immunocompromised 

individuals. Gammaherpesvirus infection of cells usually results either in productive lytic 

infection, characterized by expression of all viral genes and complete cell lysis, or in 

latent infection, in which few viral genes are expressed and no cell lysis occurs. 

Herpesvirus infections must traverse endothelial cell (EC) barriers, and infection of ECs is 

important to the pathogenesis of both human cytomegalovirus and Kaposi’ sarcoma 

associated virus. The role of ECs in the mouse model system, gammaherpesvirus 68 

(gHV68), has not been previously characterized. Recently, we demonstrated that ECs 

were unique in that a significant proportion of the cells escaped lysis, remained viable 

and proliferated in culture, but continued to produce infectious virus. Infected ECs were 

altered in adherence, morphology and surface marker expression. However, infected ECs 

maintained MHC class I expression, suggesting that EC contribution to pathogenesis is 

likely limited to immunodeficient states. Primary ECs also supported productive infection 

with enhanced survival, and we are now investigating ECs as a source of persistent 

infection in vivo. From a panel of gHV68 mutants, we identified several viral oncogenes 

which promote survival of infected ECs. Intriguingly, the viral genes which promote EC 

survival have previously been characterized as important in latency, but dispensable for 

lytic infection. Our data reveal a cell type specific outcome of gHV68 infection which can 

provide an ongoing source of virus production. gHV68 infection of ECs provides a facile 

screen for viral persistence genes and investigation of EC infection in pathogenesis. 

Presenting author Email: andrea.suarez@uchsc.edu 

89



TARGETING EXTRACELLULAR HSP90 REDUCES KSHV GENE EXPRESSION 

DURING DE NOVO INFECTION BY INHIBITING MAPK PATHWAY ACTIVATION 

Zhiqiang Qin 1 , Jennifer Isaacs 2 , and Chris Parsons 1,3 

Departments of 1 Medicine, 2 Pharmacology and 3 Microbiology/Immunology 

Hollings Cancer Center, Medical University of South Carolina 

Charleston, SC, USA 

Abstract 

Heat shock protein 90 (Hsp90) acts as a molecular chaperone orchestrating the folding of 

intracellular signaling proteins. Although extracellular (EC) domains for Hsp90 have been 

identified, the functional consequences of engaging EC Hsp90 are largely unknown. 

Signal transduction induced during KSHV receptor binding and entry is critical for de novo 

viral gene expression. Using PCR and immunofluorescence assays, we found that 

treatment of HeLa cells with a non-permeable compound targeting EC Hsp90, DNo, 

inhibited de novo expression of latent KSHV transcripts and LANA. Pre-treatment of cells 

with DNo did not reduce intracellular KSHV DNA content, and DNo maintained an 

inhibitory effect when treatment was initiated following viral incubation. In addition, no 

effect was observed following KSHV pre-incubation with two soluble Hsp90 protein 

isoforms or a mAb targeting a different EC Hsp90 domain. These results suggest that 

DNo interferes with post-entry events important for viral gene expression. Immunoblot 

analyses of signal transduction intermediates indicated that mitogen-activated protein 

kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) phosphorylation 

were selectively inhibited in a dose- and time-dependent manner by DNo. Furthermore, 

DNo blocked KSHV-mediated induction of MEK and ERK phosphorylation corresponding to 

a reduction in LANA expression. Finally, constructs conferring constitutive expression of 

P-MEK and P-ERK, but not an ERK dominant negative construct, rescued KSHV gene 

expression in DNo-treated cells. These results suggest that EC Hsp90 plays an important 

role in KSHV-initiated signal transduction events and viral gene expression and that 

strategies targeting EC Hsp90 may have therapeutic benefit for KSHV-related disease. 

Presenting author Email: parsonch@musc.edu 

90



FAK AND SHP2 ARE REQUIRED FOR KSHV VGPCR SIGNALING 

Thomas Bakken*, Chris Boshoff**, Mark Cannon* 

*CIDMTR, Department of Medicine, University of Minnesota, ** C.R.U.K. Viral Oncology 

Group, University College London 

Abstract 

The KSHV vGPCR is a constitutively active homologue of CXCR1 and CXCR2. It shows 

potential both in vitro and in vivo to contribute to the proliferative, angiogenic and 

inflammatory components of KS biology. In transgenic mice, vGPCR causes KS-like 

tumors and potentiates the tumorgenicity of other viral proteins. These effects are likely 

a combination of direct vGPCR signaling and paracrine effects via vGPCR-induced 

elaboration of various growth and angiogenic factors. Inhibition of vGPCR function is a 

promising anti-KSHV strategy. 

We focus on identifying host cell pathways that are disrupted by vGPCR and could be 

targeted to interfere with vGPCR function. The protein phosphatase, Shp2, and the focal 

adhesion-associated kinase (FAK) are signal integration proteins with adaptor and 

enzymatic functions. Up-regulation of both is associated with various malignancies. 

Furthermore, both are the target of newly developed pharmacologic inhibitors (FAK 

inhibition in particular has shown promise in a mouse ovarian cancer model). Using 

shRNA knock-down and pharmacologic inhibition in HEK293, we find that vGPCR 

activation of MEK requires both FAK and Shp2. vGPCR-induced NFκB and AP-1 activation 

are also FAK-dependent, although only the former requires Shp2. These studies are 

being taken into primary endothelial cells in which the role of Shp2 and FAK in vGPCRmediated 

signaling and phenotypic changes will be assessed. 

Interfering with pathways that are upregulated by vGPCR is a promising anti-KSHV 

approach. We have identified two signaling integration proteins that are receiving 

attention in the medicinal chemistry field and that are required for important vGPCRmediated 

events. 

Presenting author Email: canno101@umn.edu 

91



Gene Expression II 

92



DEVELOPMENT OF A COMPREHENSIVE TRANSCRIPTIONAL NETWORK FOR KSHV 

Linda Persson, Scott Millman and Angus Wilson 

Department of Microbiology & NYU Cancer Institute, New York University School of 

Medicine, New York, NY 10016, USA 

Abstract 

Viral transcription factor RTA (product of ORF50) plays an essential and far-reaching role 

in control of KSHV replication and pathogenesis. In latently infected cells, the silent 

ORF50 promoter responds to a variety of reactivation stimuli, leading to rapid 

accumulation of RTA protein and initiation of the lytic program. A principal function of 

RTA is to direct the transcription of more than 80 genes required for the lytic replication 

and virion assembly. Several RTA targets encode positive and negative regulators which 

further mold the basic program into a cascade of sequential viral gene expression. 

Paradoxically, RTA may also contribute to the establishment of latency during de novo 

infection, acting through an inducible promoter (LTi) upstream of the K12-ORF73 latency 

gene cluster. To better understand the intricate network of regulatory interactions that 

control lytic replication and determine how the program can be modified by 

environmental parameters, we embarked on a comprehensive analysis of the RTAdirected 

transcriptome. We began with a functional screen to identify RTA-responsive 

sequences irrespective of their position in the genome. A pilot study covering 37,300-bp 

between K8.1 and ORF66 revealed 8 separate RTA-responsive elements, corresponding 

to a mix of new and known lytic promoters. Currently, this unbiased mapping approach is 

being applied to the entire KSHV genome. Mutational analysis and bioinformatics 

methods are being used to dissect the individual response elements and identify points at 

which cellular signaling pathways intersect with RTA to fine tune the lytic program or 

express only a subset of viral genes. 

Presenting author Email: wilsoa02@med.nyu.edu 

93



KSHV ORF57 AND RNA EXPORT FACTORS: ROLES OF UAP56, URH49, RBM15, 

AND OTT3 IN ORF57 EXPRESSION AND FUNCTION 

Merlyn Deng 1 , Vladimir Majerciak 1 , Barbara K. Felber 2 , and Zhi-Ming Zheng 1 

1HIV 

and AIDS Malignancy Branch, National Cancer Institute, NIH, Bethesda, MD. USA. 

2 

Human Retrovirus Pathogenesis Section, Vaccine Branch, National Cancer Institute, 

Frederick, MD, USA 

Abstract 

KSHV ORF57 promotes the expression of a subset of viral lytic genes at 

posttranscriptional level and is essential for virus production. To investigate how ORF57 

might function to promote RNA export of its targets, we examined the cellular export 

factors UAP56, URH49, RBM15 and OTT3 for their possible roles in ORF57-enhanced 

expression of KSHV ORF59. We found that reducing the expression of each of these 

proteins with gene-specific siRNAs significantly reduced the expression of ORF59 in 

cotransfection experiments. Surprisingly, we also found that all four proteins affected 

ORF57 expression, indicating that the reduced ORF59 expression was likely due to the 

reduction of ORF57. The effect became most severe when two genes were knocked down 

simultaneously. To investigate whether the downregulation of ORF57 expression was due 

to a reduction of ORF57 mRNA export, we fractionated the nuclear RNA from the 

cytoplasmic RNA of cells transfected with an ORF57 expression vector and UAP56 

and/URH49 siRNAs. A substantial reduction of cytoplasmic ORF57 RNA was observed 

along with substantial accumulation of nuclear ORF57 RNA. A greater reduction of 

cytoplasmic ORF57 RNA was noticed in double-knockdown cells. Although overexpression 

of UAP56 had no effect on ORF57 or ORF59, the presence of RBM15 or OTT3 greatly 

increased expression of ORF59, but suppressed ORF57 expression. Collectively, our data 

suggest that ORF57 expression is controlled by the four RNA export factors analyzed. 

The role of ORF57 is likely to substitute cellular RBM15 and OTT3 in promotion of ORF59 

expression by sequestering them from RNA export machinery. 

Presenting author Email: zhengt@exchange.nih.gov 

94



DYNAMICS OF K-RTA RECRUITMENT ON THE KSHV GENOME REVEAL NOVEL 

REGULATION BY NF-KB 

Thomas J. Ellison 1 , Chie Izumiya 1 , Paul A. Luciw , Hsing-Jien Kung 1 , Yoshihiro Izumiya 1 

1 nd 2 

UC Davis Cancer Center, 4645 2 Ave. Sacramento, California 95817, USA. 

Department of Pathology, UC Davis, 1 Sheilds Ave. Davis, Davis, California 95616, USA. 

Abstract 

KSHV is regulated epigenetically and transcriptionally, subject to cellular factors 

including NF-kB. K-Rta and K-bZIP are two key factors that control reactivation and lytic 

replication. In this work, we performed genome-wide chromatin immunoprecipitation 

anaylsis with a viral promoter-chip (ChIP-on-Vchip) containing all 83 putative KSHV 

promoter regions. The recruitment of K-Rta and K-bZIP were examined in BCBL-1, as 

well as association with acetylated histone 3 as a marker for chromatin state. K12 and 

Ori-RNA promoters were major sites for recruitment of K-Rta, and a number of K-bZIP 

binding sites were also identified. To examine the dynamics of recruitment, ten viral 

promoters were selected for a time-course. K-Rta recruitment was most evident at 

intermediate-strength target promoters, whereas the primary sites of K-bZIP binding 

were its repression targets, to which it was co-recruited with K-Rta by 4-12 hours post 

induction. Because NF-kB has been implicated in K-Rta transactivation, it was also 

examined using the viral promoter library. Interestingly, the only two viral promoters 

not responsive to NF-kB mediated inhibition were the K-Rta major binding promoters. 

Overexpression of NF-kB strongly inhibited recruitment of K-Rta to the ORF57 and KbZIP 

promoters but not the K12 promoter during viral reactivation. These results were 

further tested by in vitro DNA binding assay using RBPjk, RelA, NF-kB1, and K-Rta. NFkB 

sequestered RBP-jk from the ORF57 promoter, and was found to form a complex with 

RBP-jk through their Rel homology domains. These studies set the stage for further 

analysis of regulation of KSHV reactivation. 

Presenting author Email: yizumiya@ucdavis.edu 

95



TYPE I INTERFERONS SUPPRESS MURINE GAMMAHERPESVIRUS-68 LYTIC 

INFECTION AT THE VIRUS TRANSCRIPTOME LEVEL VIA DIRECT MODULATION 

OF RTA PROMOTER ACTIVITY 

L. Roaden 1 , B. Manso 1 , B. Lane 1 , E. Arico 2 and B. Ebrahimi 1 

1 Division of Medical Microbiology, School of Infection & Host Defence, University of 

Liverpool, Duncan Building, Daulby Street, Liverpool L69 3GA, UK, and 2 Laboratory of 

Virology, Istituto Superiore di Sanità, Viale Regina Elena n.299 0161 Rome, Italy 

Abstract 

Type I interferons (IFN) are potent suppressors of herpesvirus lytic infections, however, 

their mode of action during early stages post-infection and reactivation from latency is 

not entirely clear. This is partly because IFN modulate a number of cellular pathways 

which interfere with viral infections. In this study, we used the murine 

gammaherpesvirus (MHV-68) as a model to understand how type I IFNs control 

productive phase of MHV-68 at different stages immediately post infection. IFN 

treatment prevented the formation of infectious virus particles. Although IFN treatment 

was capable of reducing virus entry, the virus was still capable to enter cells and uncoat 

and gain entry into the nucleus as evidenced by virus genomic DNA copy numbers and 

electron microscopy. IFN-mediated suppression was also evident at the level of viral 

protein expression; viral lytic antigens were absent in IFN-treated cells. Using a virusspecific 

microarray platform, IFN-mediated suppression was most profound at the level of 

virus transcriptome including viral tRNAs and miRNAs. Bioinformatics analysis 

highlighted a number of key IFN-related transcription factor binding sites within the 

ORF50 promoter. Using transient transfection assays, we show that direct suppression of 

ORF50 promoter by IFN is a major regulatory mechanism in suppression of MHV-68 lytic 

infection. Since the ORF50 protein product, Rta, is a key lytic cycle switch in MHV-68 

and KSHV, direct manipulation of its promoter would explain virus transcriptome shutdown 

by IFN. The implications of these findings in relation to other gammaherpesviruses 

are discussed. 

Presenting author Email: ebrahimi@liv.ac.uk 

96


Poster session abstracts 

P1-P18 

97


Poster Session Abstract P1 

THE LOCALIZATION OF LANA TO THE NUCLEAR MATRIX FRACTION IS LINKED 

TO THE KSHV GENOME REPLICATION 

Eriko Ohsaki, Tohru Suzuki, Keiji Ueda 

Department of Infectious Diseases, University of Hamamatsu School of Medicine, 1-20-1 

Handayama, Hamamatsu, Shizuoka 431-3192, Japan. 

Abstract 

Kaposi’s sarcoma-associated herpesvirus (KSHV) has mechanisms of the replication that 

synchronizes a cell cycle and of the genome segregation during the cell division. Latent 

replication of the KSHV genome requires the latency-associated nuclear antigen (LANA) 

and terminal repeat (TR) as a replication origin (Ori-P), and it is thought that prereplication 

complexes (pre-RCs) are recruited to the TR region in a LANA-dependent 

manner. 

In this report, cell fractionation experiments demonstrated that LANA was localized to the 

insoluble fraction and TR and pre-RCs were also accumulated in this fraction at G1 phase. 

We tried to identify the regions of LANA involved in the localization to the insoluble 

fraction. The N-terminally deleted LANA, which maintains domain for dimerization and 

DNA binding, lost the ability to localize to the insoluble fraction. We investigated whether 

these LANA mutants could replicate the TR-containing plasmid using a transient 

replication assay. All N-terminally deleted LANA including a mutant keeping only 

dimerization and DNA binding domains dramatically decreased the efficiency of 

replication. 

The insoluble fraction is DNase I- and high salt-resistant, and contains mainly nuclear 

matrix components. It has been reported that the DNA replication associated with the 

nuclear matrix. In recent study, a real-time imaging showed that replication foci are 

stably anchored in the nucleus. Taken together, it is possible that the specific 

localization of LANA is important process for the KSHV replication in latency and the 

KSHV genome replication occurs in the nuclear matrix although further study is needed 

to determine what kinds of nuclear matrix proteins LANA specifically interacts with. 

Presenting author Email: eohsaki@hama-med.ac.jp 

98



MODULATION OF INTERFERON β GENE EXPRESSION BY KAPOSI’S SARCOMA 

ASSOCIATED HERPESVIRUS (KSHV) LATENCY PROTEINS 

Nathalie Cloutier and Louis Flamand. 

Laboratory of Virology, Rheumatology and Immunology Research Center, CHUQ Research 

Center and Faculty of Medicine, Laval University, Quebec, Canada, G1V 4G2. 

Abstract 

The interferon (IFN) system represents a potent antiviral defense mechanism. KSHV is an 

oncogenic virus associated with KS, Primary Effusion Lymphoma and Multicentric 

Castleman’s Disease. Latency proteins are important for immune evasion by KSHV from 

the host and ensuring viral persistence. Our objective is to determine the impact of v- 

FLIP, v-Cyclin and LANA expression on interferon-b synthesis. 

Considering that the ifn-b gene is regulated partly through NF-kB, we sought to 

determine whether v-FLIP could activate the ifn-b gene. By itself, v-FLIP protein has no 

effect on ifn-b gene activation but when combined with IFN-b inducers, a synergistic 

activation occurs. This effect is strictly dependent on NF-kB and is mediated through the 

positive regulatory domain II of the IFN-b promoter. v-Cyclin has no impact on ifn-b 

gene activation. Our preliminary results show that LANA inhibits ifn-b activation. 

v-FLIP activates NF-κB and is essential for the survival of infected cells. During the lytic 

cycle, dsRNA and dsDNA molecules produced by KSHV replication will activate the 

interferon pathway and v-FLIP is likely to potentiate this effect. The synergy between v- 

FLIP and the IFN-b inducers will favor IFN-b production by lytically infected cells. On the 

other hand, LANA expression dampens this effect by suppressing ifn-b expression. 

Studies with infected cells will be important to understand the interplay between latency 

proteins and IFN-b production during the replication of KSHV. A balance between 

activation and inhibition of the ifn-b gene must occur for infected cells to persist and 

avoid elimination by the host immune system defense mechanisms. 

Presenting author Email: Nathalie.Cloutier@crchul.ulaval.ca 

99



SEVERE CHANGES OF PHENOTYPE AND CYTOKINE EXPRESSION IN KSHV- 

INFECTED B-LYMPHOCYTES 

Guergana Iotzova 1 , Georg Malterer 1 , Kai Bratke 2 , Werner Luttmann 2 , Antoine Gessain 3 , 

Christine S. Falk 4 , Kevin Robertson 5 , Peter Ghazal 5 and Jürgen Haas 1,5 

1 Max-von-Pettenkofer Institute, Ludwig-Maximilians-Universität München; 2 Department 

of Pneumology, University Hospital, Rostock; 3 Unit of Epidemiology and Physiopathology 

of Oncogenic Viruses, Institut Pasteur, Paris; 4 Institute for Molecular Immunology, 

Helmholtz Zentrum München; 5 Division of Pathway Medicine, University of Edinburgh, 

Edinburgh 

Abstract 

KSHV is involved in the pathogenesis of Kaposi´s sarcoma (KS) and B-cell derived 

primary effusion lymphomas (PEL). The aim of this study was to systematically 

investigate the influence of KSHV infection on the expression of cellular genes in B 

lymphocytes. To study these changes, a microarray analysis was performed with 

persistently in vitro KSHV-infected B-cells from several donors. A considerable number of 

genes (408) were found to be modulated more than 4-fold by KSHV infection: 67.4% of 

these genes were downregulated and 32.6% upregulated. Intriguingly, many 

downregulated genes encoded for B-cell surface markers and B-cell specific transcription 

factors (PAX-5, Oct-2 and Spi-B), which was confirmed on the protein level. The massive 

loss of B-cell surface markers or “null” phenotype was similar to PEL tumor cells 

suggesting that the loss of B-cell identity is caused by KSHV rather than cellular factors. 

KSHV-infected cells could not be lysed by allo-reactive cytotoxic T-cells, indicating that 

the null phenotype is associated with immune evasion. The downregulation of the Tolllike 

receptors TLR7, TLR9 and TLR10, which was confirmed by real-time PCR, might 

contribute to the immune escape of KSHV-infected B-cells. On the other hand, we 

detected a marked upregulation of Granzyme A (GzmA) transcripts in KSHV-infected 

cells, as well as exceedingly high levels of secreted GzmA in supernatants of KSHVinfected 

B-cells and in effusion fluids of PEL patients. shRNA knockdown studies revealed 

that GzmA participitates in a parakrine cytokine by inducing cellular IL-10, indicating a 

potential role of GzmA in the pathogenesis of KSHV-related PEL. 

Presenting author Email: malterer@mvp.uni-muenchen.de 

100



STUDIES ON THE ROLE OF MICROENVIRONMENT IN THE DEVELOPMENT OF 


Calabrò ML, 1 Gasperini P, 2 Di Gangi IM, 1 Indraccolo S, 1 Amadori A, 1 Chieco-Bianchi L. 2 

1 Immunology and Diagnostic Molecular Oncology, Istituto Oncologico Veneto, IRCCS, 

Padova; 2 Department of Oncology and Surgical Sciences, Oncology Section, University of 

Padova, Italy. 

Abstract 

We used a SCID mouse model of primary effusion lymphoma (PEL) that mimics the 

aggressive course of this human KSHV-induced lymphoma to dissect the contribution of 

the host microenvironment in PEL development. The activity of a murine, i.e. hostspecific, 

interferon (IFN)-α1-expressing lentiviral vector (mIFN-α1-LV) was compared to 

that of a hIFN-α2b-LV on the in vitro and in vivo growth of PEL cells. A control EGFP-LV 

was used in parallel. Lentiviral vectors efficiently delivered the transgene to PEL cells and 

conferred long-term transgene expression both in vitro and in vivo. Treatment of PELinjected 

SCID mice with hIFN-α2b-LV was found to significantly prolong the survival in 

the majority of animals compared to control treatments, and was associated with a 

remarkable reduction in ascites formation. Interestingly, the delivery of a host-specific 

cytokine showed an anti-neoplastic activity comparable to that observed with the hIFNα2b-LV, 

thus suggesting that the specific targeting of the host microenvironment may 

impair PEL cell growth in vivo. 

Presenting author Email: lcalabro@unipd.it 

101



ENDOTHELIAL PROGENITORS FROM THE PERIPHERAL BLOOD OF PATIENTS 

WITH CLASSIC KAPOSI’S SARCOMA ARE PERSISTENTLY INFECTED BY KSHV 

Taddeo A 1 , Della Bella S, 1 Colombo E, 1 Brambilla L, 2 Bergamo E, 3 Calabrò ML. 3 

1Laboratory of Immunology, Dipartimento di Scienze e Tecnologie Biomediche, Università 

degli Studi di Milano; 2 Departiment di Dermatology, IRCCS Ospedale Maggiore, 

Milano; 3 Immunology and Diagnostic Molecular Oncology, Istituto Oncologico Veneto, 

IRCCS, Padova, Italy. 

Abstract 

Accumulating evidence indicates that tumor angiogenesis is supported by the 

mobilization and incorporation of endothelial progenitor cells (EPCs), highly proliferative 

precursors of bone marrow origin. Our recent demonstration that EPCs are increased in 

the peripheral blood of patients with Kaposi’s sarcoma (KS), together with the intrinsic 

biologic properties of these cells, strongly suggests that EPCs could be involved in the 

pathogenesis of KS. The fact that the characteristic spindle cells share many markers 

with vascular endothelial cells and are thought to be of endothelial origin further supports 

this hypothesis. A possible scenario may be that EPCs may act as preferential KSHV 

reservoirs and, whether infected, may home to permissive sites and propagate to 

produce KS lesions. Novel insights into the state of KSHV infection of EPCs could greatly 

improve the comprehension of KS pathogenesis. Therefore, we investigated KSHV 

infection of ex-vivo cultured late-EPCs that, among other cell populations with endothelial 

features, contribute more directly to neovascularization and might represent a major 

source of endothelial progenitors in vivo. We found that late-EPCs from KS patients 

harbor KSHV DNA and retain the virus after multiple passages. Lytic phase induction or 

hypoxia could amplify the virus in cells and supernatants, indicating that late-EPCs 

support KSHV productive replication. EPCs appear therefore to represent potential virus 

reservoirs and putative precursors of KS spindle cells. The biological mechanisms that 

govern the infection of EPC by KSHV are currently under investigation. 


102



KSHV infection of human placental histocultures 

Di Stefano M, 1 Fiore JR 2 , Di Gangi IM, 3 Chieco-Bianchi L, 4 Greco P, 5 Gesualdo L, 1 Menu E, 6 

Calabrò ML. 3 

1 Laboratory of Molecular Medicine, University of Foggia, Foggia, Italy; 2 Department of 

Clinical and Occupational Health, University of Foggia, Foggia, Italy; 3 Immunology and 

Diagnostic Molecular Oncology, Istituto Oncologico Veneto, IRCCS, Padova, Italy; 

4 Department of Oncology and Surgical Sciences, Oncology Section, University of Padova, 

Padova, Italy; 5 Department of Surgical Sciences, University of Foggia, Foggia, Italy; 

6 Unité de Régulation des Infections Rétrovirales, Institut Pasteur, Paris, France. 

Abstract 

A placenta histoculture system was used to analyse the susceptibility of placental cells to 

in vitro KSHV infection. KSHV quantitative detection was performed by real-time PCR in 

cultured villi and supernatants, and virus expression was determined by 

immunohistochemistry for latent and lytic KSHV antigens. Increasing amounts of KSHV 

DNA were detected in placental tissues and culture supernatants. Immunohistochemistry 

analyses showed that both cyto- and syncitiotrophoblasts, as well as endothelial cells, 

expressed latent and lytic antigens. Moreover, relevant apoptotic phenomena were 

observed in infected histocultures. 

These data indicate that placental cells may be productively infected in vitro by KSHV, 

and suggest that this phenomenon might influence vertical transmission and pregnancy 

outcome in KSHV-infected women. 


103



SUMO-MODIFICATION MODULATES THE SUBCELLULAR LOCALIZATION AND 

ACTIVITY OF THE KSHV LATENT PROTEIN LANA2 

1 2 3 3 2 

Laura Marcos-Villar, Pedro Gallego, Fernando Lopitz, Manuel S Rodriguez & Carmen 

Rivas 

1Dpt Microbiologia II, Fac Farmacia, Universidad Complutense de Madrid, Madrid, Spain; 

2 Dpt Biología Molecular y Celular, Centro Nacional de Biotecnología, CSIC, Madrid, Spain; 

3 Ubiquitin-like proteins & Cancer Group, Proteomics Unit. CIC-BioGUNE, Derio, Spain. 

Abstract 

Small ubiquitin-related modifier (SUMO) family proteins function by becoming covalently 

attached to other proteins as post-translational modifications. SUMO modifies many 

proteins that participate in diverse processes, including transcriptional regulation, nuclear 

transport, maintenance of genome integrity, and signal transduction. The functional 

consequences of SUMO attachment vary greatly from substrate to substrate; however, 

two general modes of action have been suggested. Sumoylation may alter protein 

stability or may alter protein-protein interactions that could result in a variety of 

consequences, including changes in cellular localization. 

LANA2 is a multifunctional protein exclusively expressed in KSHV infected B cells that is 

required for the survival of KSHV-infected primary effusion lymphoma cells. Studies 

carried out in our laboratory demonstrate that LANA2 is covalently modified by SUMO. 

Sumoylation of LANA2 does not affect protein stability but modulates its subcellular 

localization and protein activity. 

Presenting author Email: lmarcos@cnb.csic.es 

104



HHV8-ASSOCIATED K5/MIR2 EXPRESSION IN VIVO CORRELATES WITH 

REDUCED COEXPRESSION OF CD31, MHC-1, AND ®-CATENIN IN KAPOSI’S 

SARCOMA 

Liron Pantanowitz1, Mandana Mansouri2, Sharon Marconi1, Eric Bartee2, Ashlee V. 

Moses2, Klaus Früh2 

1 Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, 

Springfield, MA; 2 Vaccine and Gene Therapy Institute, Oregon Health & Science 

University, 505 NW185th Ave, Beaverton, OR, 97006, USA 

Abstract 

K5/MIR2 eliminates MHC-I, CD31 and the VE-Cadherin/β-catenin complex from KSHVinfected 

endothelial cells in vitro, thereby reducing their immune recognition, cell 

adhesion and migration. Expression of K5 in vivo and downregulation of K5-target 

proteins in Kaposi’s sarcoma (KS) has not been studied. Therefore, we investigated K5 in 

patient procured KS lesions, and sought to determine whether this expression correlated 

with loss of CD31, MHC-1, and β-Catenin. Immunohistochemistry was performed on 

biopsied KS lesions of various stages (patch 2, plaque 10, tumor 8) using novel 

antibodies to K5, as well as antibodies to CD31, CD34, MHC-I, β-Catenin, LNA-1, and D2- 

40. All cases were immunoreactive with CD34, D2-40 and LNA-1. K5-specific staining 

was observed in 100% of tumor stage lesions and 40% of KS plaques. While only a few 

spindle cells were K5 positive (3+) in plaque lesions, up to 90% of lesional cells were K5 

positive in KS tumors. In only 10% of cases with K5 staining was concomitant expression 

of K5-targeted molecules (CD31, MHC-1, and β-Catenin) noted, whereas 50% of K5negative 

cases stained for K5-targeted molecules. These data suggest that K5 expression 

in vivo is more wide-spread than currently assumed, particularly in advanced KS lesions. 

Moreover, K5 expression in KS tumors appears to be associated with an absence of the 

K5-targeted molecules CD31, MHC-1, and β-Catenin in KSHV-infected lymphatic 

endothelial lesional cells. This striking inverse correlation between K5 and CD31/MHC- 

1/β-Catenin expression supports a crucial role for K5 in modulating tumor cell function in 

proliferating KS lesions. 

Presenting author Email: fruehk@ohsu.edu 

105



KBZIP SERVES AS A GLOBAL TRANSCRIPTIONAL REPRESSOR VIA 

SUMOYLATION AND DIRECT INHIBITION OF JMJD2A, A HISTONE DEMETHYLASE 

Latricia, Fitzgerald, D 1 , Yoshihiro, Izumiya 1 ; Cliff, Tepper, G. 1 ; Datsun, Hsia 1 ; Chang, 

Pei-Ching 1 ; Ellison, Tom 1 ; Luciw, Paul A. 2 ; Hsing-Jien, Kung 1 

UC Davis Cancer Center, Sacramento, CA, United States 1 ; University of California at 

Davis, Davis, California, United States 2 

Abstract 

Sumoylation and histone H3K9 methylation are hallmarks of heterochromatin and 

silenced genes. Polycomb complex, a global gene silencer, contains both SUMO ligase 

and histone methylase. KbZIP is an early gene expressed in the KSHV genome upon lytic 

activation. It is a strong transcriptional repressor and a moderate transcriptional 

activator. In the absence of other viral proteins, K-bZIP is a general repressor of host 

genes and an effective inducer of H3K9 trimethylation. K-bZIP is sumoylated at lysine 

158, when this site is mutated K-bZIP no longer serves as a global repressor. K-bZIP 

also represses transcription through its interaction with transcriptional machinery, such 

as JMJD2a, a histone demethylase. We were able to demonstrate that K-bZIP interacts 

with JMJD2a and this interaction leads to inhibition of JMJD2A activity. As K-bZIP is a 

SUMO-binding protein and behaves like a SUMO-ligase/adaptor, we asked whether 

JMJD2A is sumoylated. The results showed that JMJD2A is sumoylated and colocalized 

with SUMO1 foci. In addition, JMJD2A is transciptionally downmodulated by K-bZIP. Thus, 

K-bZIP utilizes multiple ways to shut off JMJD2A which was observed that overexpression 

of JMJD2A in BCBL-1 increases the level of reactivation of latent genome. consistent with 

the notion that chromosomal remodeling plays a pivotal part of latency switch. These 

data implicates K-bZIP in the establishment of latency by K-Rta inhibition and modulating 

the chromatin structure of the infecting genome. 

Presenting author Email: lfitzgerald@ucdavis.edu 

106



ANALYSIS METHODS FOR THE RELIABLE PREDICTION OF VIRUS AND HOST 

MIRNA EXPRESSION FROM THE AGILENT MIRNA MICROARRAY 

Eve Coulter, Dan Frampton, Paul Kellam 

MRC Centre for Medical Molecular Virology, Department of Infection, UCL, 46 Cleveland 

Street, London, W1T 4JF 

Abstract 

MicroRNAs play an important role in the down-regulation of gene expression by blocking 

translation of their mRNA targets. Hundreds of miRNAs have been identified within the 

human genome, many of which are predicted to be involved in the regulation of major 

cellular processes such as cell development and apoptosis. It is thought their abnormal 

expression may contribute to cancer development and proliferation. In addition, miRNAs 

have been shown to be present in every herpesvirus examined to date suggesting they 

play a similar role in viral gene regulation. 

The development of suitable miRNA array technology allows us to detect the presence or 

absence of both human and viral miRNAs and to use miRNA profiles to differentiate 

between virus-infected and non-infected cell types. However, there is some uncertainty 

over the design of reliable probes for viral miRNAs, with several different probes being 

used to measure the level expression of a given miRNA. We have used the Agilent miR 

array platform and a panel of herpesvirus positive B-cell lymphomas to develop an 

accurate data analysis method which discards unreliable probes and show it is better able 

to discriminate between KSHV- and EBV-infected B-cell tumour cell lines than the 

standard Agilent method. Statistical validation was performed using ROC analysis of a 

large dataset of B cell tumour cell line miRNA arrays and predicted differentially 

expressed miRNAs confirmed by real time PCR. 

Presenting author Email: d.frampton@ucl.ac.uk 

107



KAPOSI’S SARCOMA-ASSOCIATED HERPESVIRUS (KSHV) INFECTED PRIMARY 

EFFUSION LYMPHOMA CELLS SHOWS A DISTINCT GENE EXPRESSION PROFILE 

FROM BURKITT LYMPHOMA CELLS EITHER WITH OR WITHOUT EPSTEIN-BARR 

VIRUS AND THE OTHER T CELL LYMPHOBLASTIC LEUKEMIA CELLS 

Keiji Ueda, Eriko Ohsaki, Tohru Suzuki and Masato Karayama 

Dept. of Infectious Disease, Hamamatsu University School of Medicine, 

1-20-1 Handayama, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan 

Abstract 

Analysis on a gene expression profile using various kinds of normal or abnormal tissues, 

including tumors, and cell lines etc., is very informative to know the nature of the cells, 

which leads to further analyses and exploring new methods to treat diseased cells. 

Though many reports have already published on gene expression profiles of KSHV 

infected tissues, either in vitro or in vivo, we will report here gene expression profiles of 

cultured cell lines; KSHV infected primary effusion lymphoma cell lines such as BC1, BC3, 

BCBL1 and TY1, Burkitt cell lines such as BJAB, Ramos, which are not infected with 

Epstein-Barr virus (EBV) and Daudi, Raji, Akaka, which are infected with EBV, and T 

lymphoblastic cell lines as Sup T1, Molt 3 and Jurkat, and HIV-1 infected MT4. 

What we found is that each categorized group of cell lines showed distinct gene 

expression profiles and Burkitt cell lines infected with or without EBV showed a similar 

profile. Since all PEL cell lines are infected with KSHV and BC1 is infected with both EBV 

and KSHV, we can not conclude that KSHV infection governs the gene expression profile 

but it is likely that EBV status does not affect the profile. 

We are further studying how a typical gene is activated in the PEL cell lines and will 

discuss about it. 

Presenting author Email: kueda@hama-med.ac.jp 

108



THE M TYPE K15 PROTEIN OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS 

INDUCES CELL MIGRATION, INVASION AND REGULATES MICRORNA 

EXPRESSION VIA ITS SH2-BINDING MOTIF 

Yu-Hsuan Wu 1 , Min-Fen Wu 1 , Yuan-Hau Tsai 1 , Su-Fang Lin 3 , Tyson V. Sharp 4 and Hsei- 

Wei Wang 1,2,5 

1 Institute of Microbiology and Immunology, 2 Institute of Clinical Medicine, National Yang- 

Ming University; 3 Division of Clinical Research, National Health Research Institute, 

Taiwan; 4 School of Biomedical Sciences, University of Nottingham Medical School, 

Queen’s Medical Centre, Nottingham, UK; 5 Department of Teaching and Research,Taipei 

City Hospital, Taipei, Taiwan 

Abstract 

Kaposi’s sarcoma (KS) associated herpesvirus (KSHV) is the etiological agent of KS. In 

vivo KS is a tumor capable of spreading throughout the body, and pulmonary metastasis 

is observed clinically. In vitro KSHV induces the invasiveness of endothelial cells. 

However, no viral gene has yet been implicated in cell invasion. The KSHV ORF K15 is a 

KSHV-specific gene encoding a transmembrane protein. Two highly divergent forms of 

K15, the predominant (P) and minor (M) forms (K15P and K15M, respectively) have been 

identified in different KSHV strains. K15 resembles the LMP2A gene of Epstein-Barr virus 

(EBV) in their genomic locations and protein topology. Also, both K15 proteins have 

similar motifs to those found in EBV LMP1 protein. K15 therefore appears to be a hybrid 

of a distant evolutionary relative of both EBV LMP1 and 2A. Since both LMP1 and LMP2A 

proteins are capable of inducing cell motility and have been linked to NPC metastasis, we 

questioned whether K15 also possesses similar abilities. In this study, we show by the 

use of a K15M-specific mAb and PCR, that K15M is latently expressed in KSHV positive 

PEL cells. K15M localizes on lysosomal membrane and is capable of activating the NF-kB 

transcription factor via its SH2-binding motif. K15M induces cell migration, invasion and 

the expression of microRNAs miR-21 and miR-31 via this conserved motif. K15M 

therefore may contribute to KSHV-mediated tumor metastasis and angiogenesis. 

Targeting of K15 proteins or their downstream micorRNAs for therapy may represent a 

novel avenue of treatment for KSHV-associated neoplasia. 

Presenting author Email: b881625@life.nthu.edu.tw 

109



IDENTIFICATION OF NOVEL CONSERVED GAMMAHERPESVIRUS MICRORNAS 

Nicole Walz , Thomas Christalla and Adam Grundhoff 

Heinrich-Pette Institute for Experimental Virology and Immunology, D-20251 Hamburg, 

Germany 

Abstract 

microRNAs (miRNAs) are small (~22 nt.), non-coding RNA molecules which posttranscriptionally 

regulate mRNA expression and play important roles in the regulation of 

diverse cellular processes. The current release of the miRNA database (miRBase) lists a 

total of 132 known viral miRNAs, the vast majority of which are encoded by members of 

the herpesvirus family. While the propensity to encode miRNAs appears to be a 

conserved feature of many herpesviruses, the miRNAs themselves show little 

conservation: with the exception of 7 miRNAs known to be shared between Epstein-Barr 

Virus (EBV) and its close relative Rhesus Lymphocryptovirus (rLCV), all other herpesvirus 

miRNAs appear unrelated in sequence. We have recently developped VMir, a 

computational ab initio prediction method to identify viral miRNAs, and have employed 

the program to identify miRNAs in SV40, KSHV and EBV. We have modified the algorithm 

to allow consideration of evolutionary conservation and conducted an extended analysis 

of the herpesvirus family. We were able to identify and experimentally verify 2 novel 

EBV- and 17 novel rLCV-encoded miRNAs; with a total of 33 miRNAs rLCV is now the 

virus with the highest number of known miRNAs. In contrast, we have found little 

evidence for the existence of hitherto unknown conserved miRNAs shared by other 

herpesviruses, including KSHV and the closely related Rhesus Rhadinovirus (RRV). While 

EBV and rLCV thus share even more miRNAs than previously thought, our results 

underline the notion that, in contrast to their cellular counterparts, viral miRNAs show an 

almost total lack of sequence conservation. 

Presenting author Email: Adam.Grundhoff@hpi.uni-hamburg.de 

110



LYTIC INDUCTION OF KAPOSI'S SARCOMA-ASSOCIATED HERPESVIRUS IN 

PRIMARY EFFUSION LYMPHOMA CELLS WITH NON-TOXIC NATURAL PRODUCTS 

Hye-Jeong Cho 1 , Fuqu Yu 2 , Ren Sun 2 , Dongho Lee 1 and Moon Jung Song 1 

1 Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, 

Seoul 136-713, Republic of Korea, and 2 Department of Molecular and Medical 

Pharmacology, University of California at Los Angeles, Los Angeles, CA90095, U.S.A. 

Abstract 

Kaposi’s sarcoma-associated herpesvirus (KSHV) has been linked to Kaposi’s sarcoma, 

primary effusion lymphoma (PEL), and multicentric Castleman’s disease. Intentional lytic 

induction of gammaherpesviruses in the presence of antiviral drugs is thought to be an 

effective treatment option for gammaherpesvirus-related tumors. In this study, we used 

a cell-based fluorescence bioassay system in which a KSHV-infected PEL cell line was 

stably transfected with a potent viral promoter-driven reporter gene to identify effective 

non-toxic reagents capable of inducing latent KSHV. Among 400 plant extracts screened, 

three extracts increased reporter gene expression in a dose-dependent manner. The 

three extracts activated the RTA promoter and induced expressions of lytic genes in the 

endogenous viral genomes of KSHV-infected tumor cells. Furthermore, these extracts 

were also capable of inducing lytic replication of Epstein-Barr virus in B95.8 cells, 

suggesting a conserved reactivation mechanism(s) among gammaherpesviruses. 

Together, our results demonstrate the effectiveness of the screening system to identify 

natural products capable of inducing KSHV reactivation, thereby facilitating the 

development of novel therapeutic agents for KSHV-associated malignancies. 

Presenting author Email: chohyejeong@korea.ac.kr 

111



KSHV DNA QUANTIFICATION IN MATCHED PLASMA AND PBMCs SAMPLES OF 

HIV+ PATIENTS WITH KSHV-RELATED LYMPHOPROLIFERATIVE DISEASES 

Rosamaria Tedeschi 1 , Alessia Marus 1 , Ettore Bidoli 2 , Cecilia Simonelli 3 , and Paolo De 

Paoli 1 

1 Microbiology-Immunology and Virology Unit; 2 Epidemiology Unit; 3 Medical Oncology 

Dept.; Centro di Riferimento Oncologico, IRCCS, Aviano, Italy 

Abstract 

Background: The quantitative evaluation of Kaposi’s sarcoma Herpesvirus (KSHV) viral 

load is not well described in the clinical management of KSHV-related lymphoproliferative 

diseases. 

Aim: To evaluate and to compare KSHV viral load in different blood compartments from 

HIV+ patients with Multicentric Castleman’s disease (MCD), Primary Effusion Lymphoma 

(PEL) and virus-associated solid lymphoma (SLY) and to establish which clinical sample 

would be preferable for KSHV DNA testing. 

Methods: We assessed KSHV DNA in plasma and PBMCs paired samples from 7 PEL, 8 

MCD, 2 SLY HIV+ patients at the diagnosis and during the course of the illness by using a 

real time PCR assay. EBV DNA, HIV RNA and CD4 cell counts were also measured. 

Results: KSHV viremia was always detectable at diagnosis among the three groups of 

patients. KSHV DNA levels were correlated in matched pairs of samples at diagnosis and 

also during follow-up (Spearman correlation coefficient: r=0.73; p



PATHOLOGY OF RITUXIMAB-INDUCED KAPOSI SARCOMA FLARE 

Liron Pantanowitz1, Bruce J. Dezube1, Sharon Marconi1, Ashlee V. Moses2, Klaus Früh2 

Department of Pathology, Baystate Medical Center, Tufts University School of Medicine, 

Springfield, MA; 2 Vaccine and Gene Therapy Institute, Oregon Health & Science 

University, 505 NW185th Ave, Beaverton, OR, 97006, USA 

Abstract 

Background: Kaposi sarcoma (KS) flare may occur following therapy with 

corticosteroids, as part of the immune reconstitution inflammatory syndrome seen with 

highly active antiretroviral therapy (HAART), and after rituximab therapy. The exact 

mechanism responsible for iatrogenic KS flare is unclear. 

Methods: A case of AIDS-associated cutaneous KS flare following rituximab therapy was 

compared to similar controls by means of immunohistochemistry using vascular makers 

(CD34, CD31), monoclonal antibodies to Human Herpesvirus 8 (HHV8) gene products 

(LNA-1, K5), as well as B-lymphocyte (CD20) and T-lymphocyte (CD3, CD4, CD8) 

markers. 

Results: CD20+ B-cell depletion with rituximab in KS flare occurred concomitantly with 

activation of the HHV8 immediate early gene protein K5. KS flare in this patient was 

successfully treated with liposomal doxorubicin and valganciclovir. 

Conclusion: Rituximab-induced KS flare appears to be related to HHV8 activation. 

Effective management of iatrogenic KS flare therefore depends upon the control of HHV8 

viremia in conjunction with specific chemotherapy for KS. 

Presenting author Email: fruehk@ohsu.edu 

113



C-MAF: A KSHV MICRORNA TARGET 

Amy Hansen 1 , Dimitris Lagos 1 , Stephen Henderson 1 , Vicky Emuss 1 Fiona Gratrix 1 , Rolf 

Renne 2 and Chris Boshoff 1 

1 Cancer Research UK Viral Oncology Group, UCL Cancer Institute, Paul O’Gorman 

Building, Huntley Street, University College London, WC1E 6BT, London, U.K. 

Abstract 


gene expression by either blocking translation or inducing mRNA degradation. KSHV 

encodes 12 microRNAs located within the latency-associated region of the genome; ten 

microRNAs are clustered and co-expressed. Despite being identified 3 years ago, few 

KSHV microRNA targets have been identified. We present microRNA profiling data which 

confirms the expression of viral microRNAs in our in vitro endothelial cell model of 

primary infection and in vivo within KS lesions. We sought to identify cellular targets of 

the KSHV microRNA cluster in human lymphatic endothelial cells (LECs). Gene expression 

microarray profiling of LECs infected with lentivirus expressing either microRNA cluster or 

empty vector identified cellular targets silenced by microRNA induced mRNA degradation. 

Several genes were significantly deregulated; amongst these was the transcription factor 

c-Maf, an oncogenic avian retrovirus homologue. C-maf overexpression transforms B and 

T cells, however its function in endothelial cells is poorly characterised. In silico 

prediction analysis identified several potential KSHV microRNA target sites within the c- 

Maf 3’UTR. We have subsequently identified miR-K12-11 and miR-K12-6 as the principle 

silencers of c-Maf. In addition we show that silencing is mediated by direct microRNA 

interaction with the c-Maf 3’UTR. This work identifies and experimentally validates c-Maf 

as the first endothelial-specific KSHV microRNA target. 

Presenting author Email: amy.hansen@wibr.ucl.ac.uk 

114



KSHV KAPOSIN B SUPPRESSES THE HOST INTERFERON RESPONSE BY 

PREVENTING STAT1 PHOSPHORYLATION 

Christine A King, Jennifer A Corcoran and Craig McCormick 

Dalhousie University, Department of Microbiology and Immunology, Halifax, Nova Scotia, 

B3H 1X5, Canada. 

Abstract 

A central component of host antiviral defence is the interferon (IFN)-mediated antiviral 

pathway. Detection of virus by infected cells triggers the secretion of type I IFN (IFN-α or 

IFN-β) that engages specific receptors on target cells, resulting in activation of two 

receptor-associated tyrosine kinases, Jak1 and Tyk2. This is followed by tyrosine 

phosphorylation and heterodimerization of the STAT1 and STAT2 proteins. 

Phosphorylated STAT1 and STAT2 combine with IRF-9 to form the ISGF-3 complex, 

which, upon translocation to the nucleus, binds to the cis element ISRE (IFN-stimulated 

response element), to drive transcription of IFN responsive genes. To ensure viral 

persistence and productive infection, KSHV has evolved a variety of countermeasures to 

disrupt IFN signal transduction. Here we show that the latent KSHV kaposin B protein 

potently inhibits transcription from the interferon stimulated response element (ISRE) in 

response to IFN-α. Furthermore, primary human umbilical vein endothelial cells 

(HUVECs) expressing kaposin B fail to accumulate phosphorylated STAT1 in response to 

IFN-α and harbor decreased levels of total STAT1. Our data suggest that kaposin B 

interferes with the host IFN response thereby contributing to successful immune evasion 

by KSHV. 

Presenting author Email: caking@dal.ca 

115


Delegate Contact Details 

116

Adam Grundhhoff 

Friedensallee 63 

Hamburg 

22763 

Germany 

Adam.Grundhoff@hpi.uni-hamburg.de 

Akira Shimizu, 

Division of Infection & Immunity, 

UCL, 

46 Cleveland Street, 

London, 

W1T 4JF 

ashimizu@med.gunma-u.ac.jp 

Amy Hansen 

Viral Oncology Team 

UCL Cancer Institute 

Paul O'Gorman Building 


72 Huntley Street 

WC1E 6BT 


a.hansen@ucl.ac.uk 

Andrea Suarez 

12800 E 19th Ave MS 8333 

PO Box 6511 

Aurora Colorado 

80045 

United States 

thomas.shallow@uchsc.edu 

Angus Wilson 

NYU School of Medicine 

Dept. Microbiology 

550 First Avenue 

New York 

NY 10016 

United States 

wilsoa02@med.nyu.edu 

Bahram Ebrahimi 

Division of Medical Microbiology 

University of Liverpool 

Daulby Street 

Liverpool 

L69 3GA 


ebrahimi@liv.ac.uk 


Adrian Whitehouse 

Institute of Molecular and Cellular Biology 


Leeds 

LS2 9JT 


a.whitehouse@leeds.ac.uk 

Alexis Madrid 

Ganem Lab 

HSW1501 

513 Parnassus Ave 

San Francisco 

94143-0552 

United States 

alexis.madrid@ucsf.edu 

Andrea O’Hara 

3 Georgetown Ct 

Durham 

NC 

27705 

United States 

ohara@unc.edu 

Andrew Hislop 


Institute for Cancer Studies 

Edgbaston 

Birmingham 

West Midlands 

B15 2TT 


a.d.hislop@bham.ac.uk 

Anika Haevemeier 


Institute of Virology OE5230 

Carl-Neuberg-Str.1 

Hannover 

30625 

Germany 

anikahaevemeier@web.de 

Bala Chandran 

Department of Microbiology and Immunology 

RFUMS 

3333 Green Bay Road 

North Chicago IL 

60064 

United States 

bala.chandran@rosalindfranklin.edu 

117

Bettina Kempkes 

Helmholtz Center Munich 

Institute of Clinical Molecular Biology 

Marchioninistr. 25 

Munich 

81377 

Germany 

kempkes@helmholtz-muenchen.de 

Brad Spiller 

Department of Child Health 

Cardiff University School of Medicine 

5th Floor University Hospital of Wales 

Cardiff 

South Glamorgan 

CF144XN 


spillerb@cf.ac.uk 

Charles Rinaldo 

A419C Crabtree Hall- GSPH 

130 DeSoto Street 

University of Pittsburgh 

Pittsburgh 

15261 

United States 

rinaldo@pitt.edu 

Chris Parsons 

86 Jonathan Lucas St 

Hollings Cancer Center- Room HO506 

Charleston South Carolina 

29425 

United States 

parsonch@musc.edu 

Craig McCormick 

15 Silverwood Terrace 

Halifax 

Nova Scotia 

B3M2Y9 

Canada 

craig.mccormick@dal.ca 


Blossom Damania 

Lineberger Cancer Ctr RM 31-353 

UNC-Chapel Hill CB7295 

Chapel Hill 

27599 

United States 

damania@med.unc.edu 

Bridget Robinson 

831 SW Vista Ave 

Apt 201 

Portland 

USA 

97205 

United States 

robinsob@ohsu.edu 

Charles Wood 

University of Nebraska - Lincoln 

102C Morrison Center 

Lincoln 

Nebraska 

68583-0900 

United States 

cwood1@unl.edu 

Christine A King 

121 John Stewart Dr 

Dartmouth 

B2W5W8 

Canada 

caking@dal.ca 

Cristina Areste 



Edgbaston 

Birmingham 


B15 2TT 


m.c.arestecalero@bham.ac.uk 

118

Dan Frampton 

Department of Infection 



London 

W1T 4JF 


dan.frampton@gmail.com 

Dean Kedes 

1300 Jefferson Park Ave. 

Dept. of Microbiology 

University of Virginia 

Charlottesville 

22908 

United States 

kedes@virginia.edu 

Denise Whitby 

SAIC-Frederick, Inc 

National Cancer Institute 

P.O. Box B, Bldg. 535 

Frederick 

MD 

21702 

United States 

whitbyd@ncifcrf.gov 

Dirk Dittmer 

72005 Wilkinson 

Chapel Hill 

NC 

27517 

United States 

ddittmer@med.unc.edu 

Dr Bernd Hillenbrand 

Engelhardstr. 7 

Hannover 

30173 

Germany 

hillenbrand.bernd@mh-hannover.de 


David Blackbourn 

Cancer Research UK Institute for Cancer Studies 


Vincent Drive 

Edgbaston 

Birmingham 

B15 2TT 


d.j.blackbourn@bham.ac.uk 

Debasmita Roy 

140 BPW Club Road Apt B18 

Carrboro 

Orange 

NC27510 

United States 

debasmita_roy@med.unc.edu 

Denys Khaperskyy 

12-6059 Shirley St 

Halifax 

B3H 2M9 

Canada 

D.Khaperskyy@dal.ca 

Donald Alcendor 

1005 Dr. D.B. Todd Jr. Blvd. 

Hubbard Hospital 5th Floor Rm 5025 

Nashville TN 

Davidson 

37208 

United States 

dalcendor@mmc.edu 

Dr Dimitris Lagos 

Cancer Research UK Viral Oncology Group 


Paul O'Gorman Building 

72 Huntley Street 

WC1E 6BT 


d.lagos@ucl.ac.uk 

119

Dr Janet Douglas 

505 NW 185th Ave 

Beaverton 

97006 

United States 

dougljan@ohsu.edu 

Edward Tsao 




London 

W1T 4JF 


e.tsao@ucl.ac.uk 

Emilee Knowlton 

312 Lehigh Avenue 

Apt 3 

Pittsburgh 

15232 

United States 

erk21@pitt.edu 

Eriko Osaki 

Handayama Higashi-ku Hamamatsu-shi 

1-20-1 

Hamamatsu 

Shizuoka 

431-3129 

Japan 

eohsaki@hama-med.ac.jp 

Ethel Cesarman 

820 Park Ave 

Hoboken NJ 

7030 

United States 

ecesarm@med.cornell.edu 


Dr Lynn Butler 


Edgbaston 

Birmingham 

B15 2TT 

butlerlm@adf.bham.ac.uk 

Elizabeth Read-Connole 

6130 Executive Blvd. 

Rockville MD 20852 

Rockville 

Montgomery 

20852 

United States 

econnole@mail.nih.gov 

Enrique Mesri 

University of Miami Sylvester Cancer Center 

Microbiology and Immunology 

1550 NW 10th Ave Pap Bldg Room 109 

Miami 

33136 

United States 

emesri@med.miami.edu 

Erle Robertson 

3610 Hamilton Walk 

201E Johnson Pavilion 

Philadelphia 

Pennsylvania 

19104 

United States 

amyho2@mail.med.upenn.edu 

Eve Coulter 

Department of Infection 



London 

W1T 4JF 


evecoulter@hotmail.com 

120

Fang Cheng 

Vuolukiventie 1b g145 

Helsinki 

710 

Finland 

cheng.fang@helsinki.fi 

Gary Hayward 

1026 Cowpens Ave 

Baltimore 

MD 

21286 

United States 

ghayward@jhmi.edu 

Gianna Ballon 

21-59 45th Street 

Astoria 

New York 

11105 

United States 

gib2004@med.cornell.edu 

Grzegorz Sarek 

Ahventie 5A11 

Espoo 

2170 


grzegorz.sarek@helsinki.fi 

Hye-Jeong Cho 

Anam-dong Seongbuk-Gu 

Division of Biotechnology College of Life 

Sciences and Biotechnology 

Korea University 

Seoul 

136-713 

Republic of Korea 

chohyejeong@korea.ac.kr 


Gaby Sander 

Molecular and Experimental Surgery 

Schwabachanlage 10 

Erlangen 

Bavaria 

91054 

Germany 

gaby.sander@uk-erlangen.de 

Georg Malterer 

Max von Pettenkofer Institut 

Dept. of Virology 

Pettenkofer Strasse 9a 

Muenchen 

80336 

Germany 

malterer@mvp.uni-muenchen.de 

Greg Bruce 

9756 Lakeshore Blvd NE 

Seattle 

Washington 

98115 

United States 

greg.bruce@seattlechildrens.org 

Imogen Lai 



London 

W1T 4JF 


imogen.lai@ucl.ac.uk 

121

James Boyne 

Institute of Molecular and Cellular Biology 

Garstang Building 


Leeds 

LS2 9JT 


j.r.boyne@leeds.ac.uk 

Jennifer Corcoran 

Room 7F Tupper 5850 College Street 

Dept. Microbiology and Immunology 

Dalhousie University 

Halifax NS 

B3H 1X5 

Canada 

jcorcora@dal.ca 

Jinjong Myoung 

1322 3rd Ave 

Rm1 

San Francisco 

94122 

United States 

jinjong.myoung@ucsf.edu 

John Nicholas 

Johns Hopkins Oncology Center 

1650 Orleans Street 

CRB-I Room 309 

Baltimore 

Maryland 

21231 

United States 

nichojo@jhmi.edu 

John West 

5803 Sentinel Dr. 

Raleigh 

27609 

United States 

john_west@med.unc.edu 

Karen Misstear 


Division of Cancer Studies 

Birmingham 


B30 1TR 


kxm694@bham.ac.uk 


Jean Gustin 

VGTI 

505 NW 185th Avenue 

Beaverton 

Washington 

97006 

United States 

gustinj@ohsu.edu 

Jiang-Mei Qin 

12 Blenheim Crescent 

Leeds 

LS2 9AY 


qinjiangmei@yahoo.com.cn 

Johanna Viiliäinen 

Genome-Scale Biology Research Program 

Institute of Biomedicine, Biomedicum Helsinki 

A530b1 

P.O Box 63 (Haartmaninkatu 8) 

FIN-00014 University of Helsinki 


johanna.viiliainen@helsinki.fi 

Johnan Kaleeba 

4301 Jones Bridge Road 

Bethesda MD 

20814 

United States 

jkaleeba@usuhs.mil 

Karlie Plaisance 

1376 Mowry Rd 

Renne Lab Room 375E 

Gainesville 

Florida 

32610 

United States 

kplais4@ufl.edu 

122

Keiji Ueda 

Dept.of Infectious Disease 

Hamamatsu Univ. School of Medicine 

1-20-1 Handayama Higashi-ku Hamamatsu 

Shizuoka 

431-3192 

Japan 

kueda@hama-med.ac.jp 

Kwun Wah Wen 

126 Marlowe Ct 

Carrboro 

27510 

United States 

kenwen@med.unc.edu 

Lauren Lepone 

721 Copeland St 

Apt 2 

Pittsburgh 

PA 

15232 

United States 

lml33@pitt.edu 

Lucy Dalton-Griffin 


Windeyer Institute of Medical Sciences 


London 

W1T 4JF 


ucbclda@ucl.ac.uk 


Maria Luisa Calabro' 

Department of Oncology and Surgical Sciences 

Oncology Section 

via Gattamelata 64 

Padova 

35128 

Italy 

lcalabro@unipd.it 

Klaus Frueh 


Beaverton 

Oregon 

97006 

United States 

fruehk@ohsu.edu 

Laura Marcos 

Centro Nacional de Biotecnologia CSIC 

Darwin 3 Campus Universidad Autonoma 

Madrid 

28049 

Spain 

lmarcos@farm.ucm.es 

Lei Yang 

Key Laboratory of Xinjiang Endemic & Ethnic 

Disease 

Shihezi University 

Shihezi 

XinJiang 

832002 

China 

Lyang@shzu.edu.cn 

Magdalena Weidner-Glunde 


Institute of Virology 

Carl-Neuberg.-Str.1 Bldg.I6 

Hannover 

30625 

Germany 

weidnerg@allmail.mh-hannover.de 

Maria Nazzarena Labo 




Frederick 

MD 

21702 

United States 

labon@mail.nih.gov 

123

Mark Cannon 

2016 Fremont Ave S 

Minneapolis 

55405 

United States 

canno101@umn.edu 

Michael Stuerzl 

Schwabachanlage 10 

Erlangen 

91054 

Germany 

Michael.Stuerzl@uk-erlangen.de 


Nathalie Cloutier 

Rhumatology and Immunology Research Center 

2705 Laurier blvd 

Room T1-49 

Sainte-Foy 

Quebec 

G1V 4G2 

Canada 

nathalie.cloutier@crchul.ulaval.ca 

Paul Kellam 




London 

W1T 4JF 


p.kellam@ucl.ac.uk 

Michael Lagunoff 

8816 8th ave N.E. 

Seattle 

Washington 

98115 

United States 

lagunoff@u.washington.edu 

Moon Jung Song 

1 Anam-dong 

Divison of Biotechnology College of Life Sciences 

and Biotechnology 

Korea Univeristy 

Seoul 

Seongbuk-gu 

136-713 

Republic of Korea 

moonsong@korea.ac.kr 

Paivi Ojala 

Haartmaninkatu 8 

University of Helsinki 

14 


Paivi.Ojala@helsinki.fi 

Priya Bellare 

355 Crestmont Drive 

San Francisco 

California 

94131 

United States 

Priya.Bellare@ucsf.edu 

124

Prof Juergen Haas 

Max-von-Pettenkofer Institute 

Pettenkoferstrasse 9a 

Muenchen 

80336 

Germany 

haas@lmb.uni-muenchen.de 

Rachel Wheat 

Univeristy of Birmingham 

Division of Cancer Studies 

Birmingham 


B30 1TR 


r.l.wheat@bham.ac.uk 

Rosamaria Tedeschi 

Centro di Riferimento Oncologico 

via F Gallini 2 

Aviano 

33081 

Italy 

rtedeschi@cro.it 

Scott Wong 

Vaccine & Gene Therapy Institute 

505 NW 185th Avenue 

Beaverton Oregon 

97006 

United States 

wongs@ohsu.edu 

Sally Watterson 

Division of Infection & Immunity, 

UCL, 

46 Cleveland Street, 

London, 

W1T 4JF 

sally_watterson@hotmail.com 

Sheila Dollard 

1600 Clifton Rd 

Atlanta 

30333 

United States 

sgd5@cdc.gov 


Rachel Colman 


Institute of Cancer Studies 

Edgbaston 

Birmingham 


B15 2TT 


r.colman@bham.ac.uk 

Rolf Renne 

1376 Mowry Road 

PO BOX 103633 

Gainesville 

Florida 

32610 

United States 

rrenne@ufl.edu 

Ryan Estep 


Beaverton OR 

97006 

United States 

estepr@ohsu.edu 

Sean Gregory 

140 BPW Club Rd 

Apt E7 

Carrboro 

27510 

United States 

sgregory@med.unc.edu 

Sherren Sabbah 



Edgbaston 

Birmingham 


B15 2TT 


SXS463@bham.ac.uk 

125

Simon Chanas 



Edgbaston 

Birmingham 


B15 2TT 


s.a.chanas@bham.ac.uk 

Terri DiMaio 

4038 Stone Way N. 303 

Seattle 

King 

98103 

United States 

tdimaio@u.washington.edu 

Timothy Rose 

9514 Lake Shore Blvd NE 

Seattle 

Washington 

98115 

United States 

timothy.rose@seattlechildrens.org 

Utthara Nayar 

1233 York Ave Apt 12B 

New York 

NY 

10065 

United States 

utn2001@med.cornell.edu 

Vickie Marshall 




Frederick 

MD 

21702 

United States 

marshall@ncifcrf.gov 


Subhash Verma 

201E Johnson Pavilion 

3610 Hamilton Walk 

Philadelphia 

PA 

19104 

United States 

vermas@mail.med.upenn.edu 

Thomas Schulz 

Institute of Virology 


Carl-Neuberg-Str. 1 

Hannover 

30625 

Germany 

schulz.thomas@mh-hannover.de 

Tracey Barrett 

School of Crystallography 

Birkbeck College 

Malet Street 

London 

WC1E 7HX 


t.barrett@mail.cryst.bbk.ac.uk 

Veenu Minhas 

UNL Nebraska Center for Virology 

102A Morrison Ctr 

4240 Fair Street 

Lincoln 

Nebraska 

68583 

United States 

veenu9@yahoo.com 

Vicky Emuss 

UCL Cancer Institute 

74 Huntley Sreett 

Paul O’Gorman Building 

London 

WC1E 6BT 


v.emuss@ucl.ac.uk 

126

Vladimir Majerciak 

4817 36th Street NW 

Apt. 208 

Washington 

DC 

20008 

United States 

majerciv@mail.nih.gov 

Wiebke Albrecht 

Medical School Hanover 

Institute of Virology OE5230 

Carl-Neuberg-Str.1 

Hannover 

30625 

Germany 

albrecht.wiebke@mh-hannover.de 

Yan Yuan 

Department of Microbiology 

University of Pennsylvania Dental School 

240 S. 40th Street 

Philadelphia 

Pennsylvania 

19104 

United States 

yuan2@pobox.upenn.edu 

Yoshihiro Izumiya 

UCDMC Research III Rm2400 

4645 2nd Ave. 

Sacramento 

95817 

United States 

yizumiya@ucdavis.edu 

Zhi-Ming Zheng 

14507 Cartwright Way 

North Potomac MD 

Montgomery 

20878 

United States 

zhengt@exchange.nih.gov 


Wendall Miley 




Frederick 

MD 

21702 

United States 

miley@ncifcrf.gov 

Xuefeng Zhang 

264 Grove Street Apt. 1 

Newton 

2466 

United States 

xzhang3@bidmc.harvard.edu 

Yan Zeng 

Key Laboratory of Xinjiang Endemic & Ethnic 

Disease 

Shihezi University 

Shihezi 

XinJiang 

832002 

China 

zengyan910@yahoo.com.cn 

Yu-Hsuan Wu 

No.155- Sec.2- Linong Street- Taipei- 112 

Taiwan (ROC) 

Institute of Microbiology & Immunology 

Room 411 

Taipei 

112 

Taiwan 

b881625@life.nthu.edu.tw 

127


Author index 

128


Author Abstract # Author Abstract # 

Ageichik, Alexander V. 23 Chahal, Hema 44 

Albrecht, W. 5 Chandran, Bala 19, 28, 60 

Alcendor, Donald J. 21 Chang, Pei-Ching 37, P9 

Alkharsah, Khaled R. 20, 24 Cheng, Fang 36 

Amadori, A. P4 Chieco-Bianchi, L. P4, P6 

Amin, Minal M. 48 Cho, Hye-Jeong P14 

Anh, Pham Thi Hoang 51 Christalla, Thomas P13 

Arico, E. 67 Cloutier, Nathalie P2 

Bagnéris, Claire 23 Collins, Mary 23 

Bagni, Rachel 26, 27 Colman, Rachel 13, 44 

Bakken, Thomas 63 Colombo, E. P5 

Ballon, Gianna 15 Corcoran, Jennifer A. P18 

Barrett, Tracey 23 Coulter, Eve M. 30, P10 

Bartee, Eric P8 Crabtree, Kay L. 53 

Bellare, Priya 33 Cronin, Nora 23 

Bergamo, E. P5 Csernus, Balasz 49 

Bidoli, Ettore P15 Dalton-Griffin, Lucy 58 

Blackbourn, David J. 13, 44 Damania, Blossom A. 1, 11, 31, 32, 50 

Blom, Anna M. 43 De Paoli, Paolo P15 

Boshoff, Chris 12, 23, 63, P17 De Sanjose, Silvia 51 

Bottero, Virginie 19 Della Bella, S. P5 

Bower, Mark 12 Deng, Merlyn 65 

Boyne, James R. 39, 41 Desai, Prashant 21 

Brambilla, L. P5 Dezube, Bruce J. 32, P16 

Bratke, Kai P3 Di Gangi, I.M. P4, P6 

Brayfield, Brad P. 55 Di Stefano, M. P6 

Brown, Cedric 48 DiBartolo, Daniel 25 

Brown, Elizabeth 54 Dillner, Joakim 43 

Bruce, A. Gregory 16 Dinkel, Holger 56 

Bubman, Darya 49 DiPersio, C. Michael 59 

Bürger, Antje 20 Dittmer, Dirk P. 1, 31, 32, 50 

Burnside, Kellie L. 2 Dollard, Sheila C. 48 

Cai, Qiliang 6 Douglas, Janet 10 

Calabrò, M.L. P4, P5, P6 Downing, Robert 48 

Cannon, Mark 63 Dufresne, Andrew T. 33 

Cardenas, Elisa 14 Duran, E. Margarita 22 

Casabona, Jordi 54 Dutia, B. 34 

Cavallin, Lucas 22 Ebrahimi, B. 34, 67 

Cesarman, Ethel 15, 25, 49 Ellison, Thomas J. 37, 66, P9 

Chadburn, Amy 15, 25 Emuss, Vicky P17 

129


Estep, Ryan D. 14 Hensler, Heather 8 

Falk, Christine S. P3 Herrero, Rolando 51 

Felber, Barbara K. 65 Hieu, Nguyen Trong 51 

Fiore, J.R. P6 Hillenbrand, Bernd 20 

Fischer, Irina 20 Hilscher, Chelsey 1, 31 

Fitzgerald, Latricia D. 37, P9 Hislop, Andrew D. 13, 45 

Flamand, Louis P2 Hladik, Wolfgang 48 

Fossum, Even 57 Hollow, Charles 35 

Fowlkes, Ashley L. 48 Hsia, Datsun P9 

Frampton, Dan 30, P10 Hu, Soo-Jin Han Jianhong 26, 27 

Franceschi, Silvia 51 Huang, Jin 29, 52 

Frank, Ronald 4 Hyjek, Elizabeth 25 

Früh, Klaus 10, P8, P16 Imami, Nesrina 12 

Gallego, Pedro P7 Indraccolo, S. P4 

Ganem, Don 33, 42, 47 Iotzova, Guergana P3 

Garrigues, H. Jacques 59 Isaacs, Jennifer 62 

Gasperini, P. P4 Izumiya, Chie 66 

Gellermann, E. 5 Izumiya, Yoshihiro 37, 66, P9 

Gessain, Antoine P3 Jais, Mariel 8, 46 

Gesualdo, L. P6 Järviluoma, Annika 3 

Ghazal, Peter P3 Jenkins, Frank J. 8, 46 

Goldschmidt-Clermont, Pascal J. 22 Jochmann, Ramona 24 

Gonin-Laurent, Nathalie 24 Kankasa, Chipepo 53, 55 

Gotch, Frances 12 Karayama, Masato P11 

Gratrix, Fiona 12, P17 Kellam, Paul 9, 30, 38, 58, P10 

Gravett, Courtney 16 Keller, Shannon 25 

Greco, P. P6 Kempkes, Bettina 57 

Gregory, Sean 11, 31 King, Christine A. P18 

Groopman, Jerome E. 17 Kim, Chang Hee 26, 27 

Grundhoff, Adam P13 Knowles, Daniel M. 25 

Guasparri, Ilaria 25, 49 Knowlton, Emilee 8, 46 

Guo, Shu-Xia 52 Konrad, Andreas 56 

Gustin, Jean 10 Korodi, Zoltan 43 

Haas, Jürgen 57, P3 Koskinen, Päivi J. 36 

Hansen, Amy P17 Kremmer, Elisabeth 56, 57 

Harrington Jr., William 32 Kung, Hsing-Jien 37, 66, P9 

Hävemeier, Anika 18 Labo, Maria Nazzarena 54 

Hayward, Gary S. 21 Lagos, Dimitris 12, P17 

Heinzelmann, Katharina 57 Lai, Imogen Yi-Chun 9, 38 

Henderson, Stephen P17 Laiho, Marikki 3 

130


Lampson, Michael 6 Millman, Scott 64 

Lane, B. 34, 67 Minhas, Veenu 53, 55 

Lazcano, Eduardo 51 Misstear, Karen 13, 44 

Lee, Dongho P14 Mitchell, Charles D. 53, 55 

Lehtonen, Anne 36 Moses, Ashlee V. 10, P8, P16 

Lepone, Lauren 46 Muñoz, Nubia 51 

Leubert, Rene 56 Myoung, Jinjong 47 

Levine, Rita 19 Naschberger, Elisabeth 24 

Li, Dong-Mei 29, 52 Nasu, Ryo 7 

Li, Feng 29, 52 Nayar, Utthara 49 

Lin, Su-Fang P12 Neil, Stuart 9 

Liu, Yi-Fang 15 Neipel, Frank 24, 56 

Long, Heather 13 Nzaro, Esau 48 

Lopitz, Fernando P7 O’Hara, Andrea J. 32, P11 

Lord, Janet 44 Ohsaki, Eriko P1 

Luciw, Paul A. 37, 66, P9 Ojala, Päivi M 3, 36 

Luo, Xian-Dao 29 Okroj, Marcin 43 

Luttmann, Werner P3 Ottinger, Matthias 4 

M’soka, Tendai J. 53, 55 Pantanowitz, Liron P8, P16 

Ma, Qi 22 Pardieu, Claire 9 

Madrid, Alexis 42 Parsons, Chris 62 

Majerciak, Vladimir 40, 65 Perez, Sussana 51 

Malterer, Georg P3 Persson, Linda 64 

Manso, B. 67 Piazza, Paolo 8, 46 

Mansouri, Mandana 10, P8 Pietrek, Macel 18 

Maor, Yehoshua 17 Plaisance, Karlie 27 

Marconi, Sharon P8, P16 Pliquet, Daniel 4 

Marcos-Villar, Laura P7 Qin, Zhiqiang 62 

Mark, Linda 43 Qing, Jiang-Mei 52 

Marshall, Vickie 54 Quereshi, Omar 13 

Martró, Eliza 54 Raghu, Hari 28, 60 

Marus, Alessia P15 Rajewsky, Klaus 15 

Matos, Elena 51 Rappocciolo, Giovanna 8, 46 

Matsumura, Satoko 7 Ray, Alex 54 

Mbisa, Georgina 51 Renne, Rolf 26, 27, 54, P17 

McCormick, Craig P18 Rinaldo, Charles R. 8, 46 

Menu, E. P6 Rivas, Carmen P7 

Mermin, Jonathan 48 Roaden, L. 34, 67 

Mesri, Enrique A. 22 Roback, John 48 

Miley, Wendell 26, 27 Robertson, Erle S. 6 

131


Robertson, Kevin P3 Thurau, Mathias 24, 56 

Rodriguez, Manuel S. P7 Tojkander, Sari 3 

Rose, Timothy M. 2, 16, 59 Towers, Greg 9 

Roy, Debasmita 50 Tsai, Yuan-Hau P12 

Rubinchikova, Yelena E. 59 Tsao, Edward 9, 30, 58 

Ryan, Jonathan T. 16 Ueda, Keiji P1, P11 

Sabbah, Shereen 45 van Dyk, L.F. 61 

Sadagopan, Sathish 19, 28, 60 Varga, Laszlo 60 

Sander, Gaby 24, 56 Varjosalo, Markku 36 

Sansom, David 13 Vart, Richard James 12 

Sarek, Grzegorz 3 Vartia, Salla 3 

Sasaki, Yoshiteru 15 Veettil, Mohanan Valiya 19, 28, 60 

Scholz, Barbara 57 Verma, Subhash C. 6 

Schulz, Thomas F. 4, 5, 18, 20, 24, Viejo-Borbolla, A. 

5 

36, 56 

Viswanathan, Kasinath 10 

Sharma-Walia, Neelam 19, 28, 60 Waksman, Gabriel 23 

Sharp, Tyson V P12 Wallace, Bonnie 23 

Sheridan, V. 34 Walz, Nicole P13 

Shin, Hai-Rim 51 Wang, Dian 54 

Si, Huaxin 6 Wang, Fu-Zhang 60 

Simonelli, Cecilia P15 Wang, Hsei-Wei P12 

Sin, Sang-Hoon 50 Wang, Ling 31, 50 

Skalsky, Rebecca L. 26 Wang, Sheng 29 

Song, Moon Jung P14 Wang, Yan 35 

Spiller, O. Brad 43 Webb, Ben 9 

Stürzl, Michael 

24, 56 

Weidner-Glunde, 

Suárez, A.L. 

61 

Magdalena 

4, 36 

Sukvirach, Sukhon 51 Weinländer, Kristina 24 

Sun, Ren 25, 34, P14 Wen, Kwun Wah 1 

Suzuki, Tohru P1, P11 West, John 11, 31 

Syrjäkari, Henna 3 Westrop, Samantha Jane 12 

Taddeo, A. P5 Whitby, Denise 26, 27, 51, 54 

Taipale, Jussi 36 Whitehouse, Adrian 39, 41 

Tan, Xiao-Hua 29, 52 Wies, Effi 56 

Tanese, Naoko 7 Wilson, Angus 7, 64 

Taylor, Adam 41 Wilson, Sam 9 

Tedeschi, Rosamaria 43, P15 Wojcicki, Janet 53 

Thome, Margot 24 Wong, Ping-Pui 12 

Tepper, Cliff G. P9 Wong, Scott W. 14 

Thomas, Jaiye O. 51 Wood, Charles 53, 55 

132


Wu, Min-Fen P12 Zeng, Yan 29, 52 

Wu, Yu-Hsuan P12 Zhang, Hui 29 

Xian, Ling-ling 29 Zhang, Rui 22 

Xie, Jian-Xin 29, 52 Zhang, Xuefeng 17 

Yang, Lei 29, 52 Zheng, Zhi-Ming 40, 65 

Yarchoan, Robert 54 Zhou, Xiao–Fei 29 

Yu, Fuqu P14 Zhu, Wen Qui 21 

Yuan, Yan 35 

133

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