Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus8 (HHV-8), is a human tumor virus which is associated with Kaposi' s sarcoma and several lymphoproliferative diseases. Phylogenetic analysis of KSHV genomic sequence revealed that KSHV belongs to the gamma-herpesvirus family, which characteristically establishes latent infection in lymphoid cells. When latency is disrupted, viruses can switch to lytic life cycle. In general, herpesviruses utilize different origins of replication during latent versus lytic infection. Latent DNA replication depends on the cellular DNA replication machinery, whereas lytic cycle DNA replication requires virally encoded replication proteins. In lytic DNA replication, the lytic origin (ori-Lyt) is bound by a virus-specified origin-binding protein (OBP) that recruits the core replication machinery. Although six core replication proteins have been identified in KSHV, an OBP and the ori-Lyt of KSHV were unidentified. Recently, some progresses were made in our laboratory. (i) two regions in the KSHV genome, between K4.2 and K5 and between K12 and ORF71, were found to be able to serve as origins for lytic cycle-specific DNA replication; (ii) a KSHV-encoded bZip protein K8 was found to bind to a sequence within KSHV ori-Lyt by an in vitro binding site selection assay. These two discoveries opened an avenue to study the mechanism of KSHV lytic DNA replication. In the proposed studies, we will investigate the mechanism of KSHV lytic DNA replication. Emphasis will be placed on the role of K8 bZip protein in the lytic-origin dependent DNA replication. (1) KSHV ori-Zyt domain will be dissected using site-directed mutagenesis to define cis-acting requirements for viral lytic replication and map K8 binding element within the ori-Lyt. (2 ) Since the function of K8 in viral lytic DNA replication appears to be mediated through interacting with the core viral replication proteins, revelation of interaction of K8 with other proteins becomes of paramount importance in understanding the role of K8 in KSHV DNA replication. We will attempt to identify the viral or cellular proteins that are associated with K8. (3) The functional role(s) of K8 in KSHV lytic DNA replication and other events in lytic cycle will be determined by using transient DNA replication assay and by blocking K8 gene expression using the external guide sequence (EGS) technique. The proposed studies will provide an insight into KSHV lytic cycle replication.
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