Elements mediating maintenance of latent Kaposi's sarcoma -associated herpesvirus genomes.

Abstract

Latent infection by members of the gammaherpesvirus family is typically characterized by stable episomal maintenance of genomic viral plasmids. In the case of the prototype gammaherpesvirus Epstein-Barr virus (EBV), this is dependent upon binding of the Epstein-Barr nuclear antigen 1 (EBNA1) to sites in the origin of plasmid replication (oriP). The recently discovered related agent, Kaposi's sarcoma-associated herpesvirus (KSHV) encodes the latency-associated nuclear antigen (LANA), which is one of few viral proteins expressed during latent infection. In this dissertation, we find that LANA colocalizes with viral episomes at mitotic host chromosomes and likely mediates a tethering mechanism, whereby LANA attaches viral episomes to host chromosomes through simultaneous interaction with chromosomal proteins such as histone H1 and specific sequences located within the left end of the viral genome. Additional characterization describes site-specific binding of the LANA protein to three different <italic>cis</italic>-acting sequences within this region which appear to be critical for tethering and maintenance of KSHV episomes. Of the three sites, terminal LANA binding region 4 (TLBR4) binds LANA with the highest affinity. Mutagenesis studies indicate the minimal TLBR4 binding sequence to be 5<super>'</super> CGCCCGGGCATGG, which is bound by the very distal C-terminus of the LANA protein. Finally, studies of latent replication indicate that LANA may enhance but not be strictly necessary for maintenance of KSHV-based plasmids. Taken together, these data support the hypothesis that latent KSHV episomes are stabilized and segregated to daughter cells through tethering to host chromosomes by LANA, however, LANA's role in recruitment of the replicative machinery remains somewhat obscure.