Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

The Department of Molecular BiologyViral Pathogenicity Examined inRNA VirusesOur laboratory’s long-term goal is tounderstand the molecular basis of thepathogenicity of viruses of the negativestrandRNA (nsRNA) family, using vesicularstomatitis virus (VSV) and human parainfluenzavirus type 3 (HPIV-3) as the prototype viruses. Athorough understanding of the mode of viraltranscription and replication is fundamental todeveloping reagents to combat them. Ouremphasis is on establishing the functions of keyviral proteins, such as L (the RNA polymerase),P (the transcription factor), and N (the nucleocapsidprotein), encapsidating the genome RNA.The virus ribonucleoprotein (RNP) complexcontaining these polypeptides transcribes thegenome RNA in vitro as well as in vivo, by which itinitiates infection within the infected cells. Wealso study host-virus interaction and havediscovered several host proteins that play criticalroles in the gene expression of these viruses.Vesicular Stomatitis VirusUsing recombinant expression vectors, wehave expressed, in biologically active form, viralpolypeptides that constitute the transcribingRNP (i.e., L, N and P proteins) in prokaryoticand eukaryotic cells. Several important discoverieshave been made, especially with respect to thesubunit composition of the L protein and theputative replicase complex. We have shown thatL protein expressed in insect cells associates withthe cellular translation elongation factor EF-1 a/b/g subunits for its activity. In this respect, VSVRNA polymerase bears a striking similarity tobacteriophage Qb replicase, which requires thebacterial homologue of the translation elongationfactors Ts and Tu. We have shown that thehost cell capping enzyme specifically interactswith the viral RNA polymerase (L) and mediatesthe capping of viral mRNAs, whereas themethyltransferase activity that methylates thecapped structure is encoded by the L protein.These unique findings may help unravel the rolesof these cellular proteins in VSV RNA polymerasefunction. Using a reverse-genetics systemto study transcription and replication of speciallyconstructed mini-genome or defective interfering(DI) particles using cDNAs encoding P proteinsand wild-type L and N proteins, we havedemonstrated that although the transcriptioncomplex is composed of L-P 2-3, the replicaseappears to be a tripartite complex between L (N-P) that initiates the replication reaction. Understandingthe structure and function of thereplicase complex is key to gaining insight into thereplicative pathways in the VSV life cycle.Human parainfluenza virusThe transcription complex of HPIV-3consists of L, P, and N proteins in which the Nprotein encapsidates the genome RNA. We haveshown that specific interaction and polymerizationof actin on the RNP complex leads to theactivation of transcription in vitro, and that thevirus replicates in the cytoskeletal network, whereactin plays a critical role in the replication process.Continuing studies have led to discovery of twoadditional cellular proteins that specificallyinteract with cis-acting viral RNAs, including thekey glycolytic enzyme glyceraldehyde 3-phosphatedehydrogenase (GAPDH) and the nuclearautoantigen La protein. Understanding, in detail,the molecular basis of the interplay of viruses andcellular proteins will further our knowledge ofthe host’s role in promoting virus replication. Wehave shown that HPIV-3 entry and budding is bidirectional,although the apical pole is greatlypreferred. We are identifying and characterizingthe receptor(s) involved in the entry process ofHPIV-3. We have further shown that HPIV-3upregulates MHC class I and II expression onrespiratory epithelial cells without involvementof the STAT1 and class II transactivator (CIITA)pathways. These results suggest that HPIV-3 playsan important role in infection-related immunityand pathogenesis.A full-length cDNA clone of the HPIV-3genome (called pHPIV-3) was constructed, andrecombinant, infectious HPIV-3 was generated bytransfecting pHPIV-3 and support plasmidsencoding the HPIV-3 NP, P, and L proteins intoHeLa cells infected with a recombinant vacciniavirus expressing T7 RNA polymerase. We havemapped the replication and transcriptionpromoters on the genome RNA using a uniqueHPIV-3 mini-genome system. Availability of aninfectious clone for HPIV-3 and the mini-genomesystem will enhance our understanding of themolecular biology of HPIV-3 gene expression andmay help develop an effective vaccine against thisimportant human pathogen.VIROLOGYTHE A.K. BANERJEELABORATORYPROJECT STAFFSantanu Bose, Ph.D.Manjula Mathur, Ph.D.RESEARCH ASSOCIATESSantanu Bose, Ph.D.Achut Malur, Ph.D.POSTDOCTORAL FELLOWSMausumi BasuPatricia Bates, Ph.D.Shaji Daniel, Ph.D.Yuwen Huo, Ph.D.Jared LeMaster, Ph.D.Kaustubha Qanungo, Ph.D.TECHNOLOGISTDouglas Younger, III, B.S.COLLABORATORSAsit K. Pattnaik, Ph.D. 1Richard Ransohoff, M.D. 2Focco van den Akker, Ph.D. 31Dept. of Microbiology andImmunology, Univ. of Miami, FL2Dept. of Neurosciences, CCF3Case Western ReserveUniversity, Cleveland, OHMathur, M., and A.K. Banerjee (2002) Novel binding of GTP to the phosphoprotein (P) of vesicular stomatitisvirus. Gene Exp. 10:193-200.Gupta, A.K., Mathur, M., and A.K. Banerjee (2003) Unique capping activity of the recombinant RNApolymerase (L) of vesicular stomatitis virus: association of cellular capping enzyme with the L protein.Biochem. Biophys. Res. Commun. 293:264-268.Bose, S., and A.K. Banerjee (2002) Role of heparan sulfate in human parainfluenza virus type 3 infection.Virology 298:73-83.Gupta, A.K., Shaji, D., Banerjee AK. Identification of a novel tripartite complex involved in replication ofvesicular stomatitis virus genome RNA. J. Virol. 77:732-738.Amiya K. Banerjee, Ph.D.Head, Section of Virology113