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The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Latency Abstract 4 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
The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Latency Abstract 5 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]: ?
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Dan Frampton Department of Infectio
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Keiji Ueda Dept.of Infectious Disea
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Prof Juergen Haas Max-von-Pettenkof
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Vladimir Majerciak 4817 36th Street
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