Functional analysis of the latent origin for DNA replication of Kaposi's sarcoma-associated herpesvirus

Kaposi's sarcoma-associated herpesvirus (KSHV), also called human herpesvirus 8 (HHV8), is linked to the development of Kaposi's sarcoma, primary effusion lymphomas and multicentric Castleman's disease. The viral genome is maintained as circular episomes in KSHV-associated tumor cells. During latency, the latency-associated nuclear antigen (LANA) is highly expressed. LANA associates with host chromosomes and specifically binds to two adjacent LANA binding sites (LBS1/2) within the terminal repeat (TR), thereby allowing the faithful segregation of viral episomes or TR-containing plasmids during mitosis. In addition to maintaining viral genomes, LANA modulates viral and cellular gene expression by interacting with host cellular proteins including p53, Rb, and GSK-3beta.; The research work in this dissertation focuses on LANA's role in latent DNA replication and a detailed characterization of the putative latent origin of replication (ori) of KSHV. By performing short-term DNA replication assays, I demonstrated that LANA acts in trans to support DNA replication of TR-containing plasmids. The LANA-C domain by itself provides partial function for DNA replication. A detailed mapping analysis revealed that two cis-regulatory elements located within a single unit of the TR constitute the putative minimal replicator of KSHV: LBS1/2 and an adjacent 29 bp long GC-rich sequence which we have termed the replication element (RE). In addition, we found that the transcription factor Sp1 can bind to TR outside of the minimal replicator and contributes to the previously reported transcriptional enhancer activity of TR.; LANA is thought to recruit cellular replication factors for DNA replication due to the lack of any enzymatic activity. To investigate the mechanisms by which LANA functions in DNA replication, we have been attempting to identify those cellular proteins. We were unable to show LANA association with the eukaryotic origin recognition complex (ORC) proteins by immunoprecipitation, or the interaction of ORC proteins with TR by chromatin immunoprecipitation assays. However, we successfully isolated 12 cellular proteins that associated with epitope-tagged LANA-C by preparative mass spectrometry. These novel LANA-interacting proteins may be involved in LANA-dependent DNA replication and other functions. Further studies will be conducted to reveal the in vivo binding of these proteins with LANA and their potential involvement in KSHV latent DNA replication.; These data contribute to the understanding of the molecular biology of KSHV and the evolution of gamma-herpesvirus, and might aid the development of novel strategies to develop antiviral therapeutics. Our data also indicate that the KSHV latent replication origin is a good model for studies on cellular DNA replication.