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Interaction of Kaposi's sarcoma-associated herpesvirus ORF59 with oriLyt is dependent upon binding with K-Rta
AuthorSUSILARINI, NI KETUT
AdvisorPari, Gregory S.
Biochemistry and Molecular Biology
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ABSTRACT There are six Kaposi Sarcoma-associated Herpesvirus (KSHV/HHV-8) viral-encoded core proteins required for lytic origin-dependent DNA replication. They are: ORF9 (DNA polymerase), ORF6 (single-stranded DNA binding protein), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase) and ORF59 (DNA polymerase processivity factor). These six core proteins have a high level of homology to other herpesvirus proteins necessary for oriLyt-dependent DNA replication. In addition to these core proteins, the major transactivator for KSHV, K-Rta, is also required for lytic origin-dependent DNA replication.KSHV/HHV-8 displays two distinct life stages, latency and lytic reactivation. Progression through the lytic cycle and replication of the viral genome is an essential step towards the production of infectious virus and human disease. KSHV K-Rta has been shown to be the major transactivator required for the initiation of lytic reactivation. In the transient cotransfection replication assay, K-Rta is the only non-core protein required for DNA synthesis. K-Rta was shown to interact with both C/EBPα binding motifs and the R response elements (49) within oriLyt. It is postulated that K-Rta acts in part, to facilitate the recruitment of replication factors to oriLyt. In order to define the role of K-Rta in the initiation of lytic DNA synthesis we show an interaction with ORF59, the DNA polymerase processivity factor (PF), one of the eight virally encoded proteins necessary for origin-dependent DNA replication. Using the chromatin immunoprecipitation assay (ChIP), both K-Rta and ORF59 interact with the RRE and C/EBPα binding motifs within oriLyt in cells harboring the KSHV BAC. A transient transfection ChIP assay demonstrated that the interaction of ORF59 with oriLyt is dependent upon binding with K-Rta and ORF59 fails to bind to oriLyt in the absence of K-Rta. Also, using the cotransfection replication assay, over-expression of the interaction domain of K-Rta has a dominant-negative effect on oriLyt amplification, suggesting that the interaction of K-Rta with ORF59 is essential for DNA synthesis and supports the hypothesis that K-Rta facilitates the formation of a replication complex at oriLyt.