Modulation of Kaposi's Sarcoma-associated Herpesvirus (KSHV/HHV8) origin-dependent DNA replication by K-bZIP, K-Rta, and LANA
AuthorRossetto, Cyprian Constance
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There are eight Kaposi Sarcoma-associated Herpesvirus (KSHV) viral-encoded proteins that are necessary for lytic origin-dependent DNA replication. Six of the eight proteins have a high level of homology to other herpesvirus proteins necessary for oriLyt-dependent DNA replication. There are two proteins involved in oriLyt DNA replication that are unique to KSHV, the major transactivator K-Rta and the relatively functionally unknown protein K-bZIP. During the course of our investigation on the role of K-Rta and K-bZIP in DNA replication we discovered that the major Latency Associated Protein (LANA) also plays a role in oriLyt-dependent DNA replication. To determine which K-bZIP protein domains contribute to oriLyt-dependent DNA replication and facilitate suppression of K-Rta-mediated transcriptional activation, we generated a series of deletion constructs and site-directed mutations in the K-bZIP ORF. Mutation of key leucine residues within the putative leucine zipper (LZ) motif eliminated the ability of the protein to homodimerize and complement oriLyt-dependent DNA replication. Deletion of the basic amino acid region (BR) or LZ domain did not affect the ability of K-bZIP to bind to K-Rta indicating that either region contributes to heterodimerization with K-Rta. However, deletion or mutations introduced into both the LZ and BR resulted in elimination of the suppressive activity of K-bZIP even in the presence of a K-bZIP--K-Rta interaction. Interestingly, mutants that lacked the ability to suppress K-Rta transactivation were still capable of complementing oriLyt-dependent DNA replication, indicating that this activity does not contribute to the DNA synthesis-related activity of K-bZIP.To determine the role of K-bZIP in the context of the viral genome, we generated a recombinant KSHV bacterial artificial chromosome (BAC) with a deletion in the K-bZIP open reading frame. This BACmid, BAC36ΔK8, displayed an enhanced growth phenotype with respect to virus production and accumulation of virus-encoded mRNAs measured by real-time PCR under conditions where K-Rta was used to induce the virus lytic cycle. Induction of the virus lytic cycle using tetradecanoyl phorbol acetate/n-butyrate (TPA/NaBut) resulted in no virus production and an aberrant gene expression pattern from BAC36ΔK8-containing cells compared to wild-type (wt) BAC. Immunofluorescence staining revealed that subcellular localization of K-Rta was unchanged but there was a disruption of LANA subcellular localization in cells harboring BAC36ΔK8, suggesting that K-bZIP influences LANA localization. Coimmunoprecipitation experiments confirmed the K-bZIP interacts with LANA in BCBL-1 cells and in contransfection assays. In an effort to understand the consequences of a K-bZIP--LANA interaction we developed a modification of the transient cotransfection replication assay where both lytic (oriLyt) and latent (terminel repeats-TR) DNA replication are evaluated simultaneously. LANA repressed oriLyt-dependent DNA replication in a dose dependent manner when added to the cotransfection replication assay. This repression of oriLyt-dependent DNA replication was overcome by increasing amounts of a K-bZIP expression plasmid in the contransfection mixture of by dominant-negative inhibition of the interaction of LANA with K-bZIP by the over expression of the K-bZIP--LANA interaction domain. Using the chromatin immunoprecipitation assay (ChIP) we show that LANA interacts with oriLyt in the region associated with K-bZIP binding suggesting suppression of lytic replication by LANA is mediated by direct binding. These data suggest that the interaction of LANA with K-bZIP modulates lytic and latent replication.