Kaposi's sarcoma-associated herpesvirus LANA recruits the DNA polymerase clamp loader to mediate efficient replication and virus persistence

Kaposi''s sarcoma-associated herpesvirus (KSHV) latently infects tumor cells and persists as a multiple-copy, extrachromosomal, circular episome. To persist, the viral genome must replicate with each cell cycle. The KSHV latency-associated nuclear antigen (LANA) mediates viral DNA replication and persistence, but little is known regarding the underlying mechanisms. We find that LANA recruits replication factor C (RFC), the DNA polymerase clamp proliferating cell nuclear antigen (PCNA)] loader, to drive DNA replication efficiently. Mutated LANA lacking RFC interaction was deficient for LANA-mediated DNA replication and episome persistence. RFC depletion had a negative impact on LANA’s ability to replicate and maintain viral DNA in cells containing artificial KSHV episomes or in infected cells, leading to loss of virus. LANA substantially increased PCNA loading onto DNA in vitro and recruited RFC and PCNA to KSHV DNA in cells. These findings suggest that PCNA loading is a rate-limiting step in DNA replication that is incompatible with viral survival. LANA enhancement of PCNA loading permits efficient virus replication and persistence, revealing a previously unidentified mechanism for KSHV latency.Kaposi''s sarcoma-associated herpesvirus (KSHV or human herpesvirus 8) has a causative role in Kaposi''s sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman disease (1–4). KSHV infection of tumor cells is predominantly latent. During latent infection, the viral genome persists as a multicopy, circular, extrachromosomal episome (plasmid) (5, 6). To persist, the genome must replicate and segregate to progeny nuclei with each cell division. Latency-associated nuclear antigen (LANA), a 1,162-residue protein, is one of a few KSHV genes expressed in latency. LANA is necessary and sufficient for episome persistence in the absence of other viral genes (7, 8). Both N-terminal LANA (N-LANA) and C-terminal LANA (C-LANA) are essential for function. N-LANA associates with mitotic chromosomes via binding histones H2A/H2B, and C-LANA simultaneously binds KSHV terminal repeat (TR) DNA (7, 9–20). Thus, LANA tethers the viral genome to host chromosomes and distributes viral DNA to daughter nuclei during mitosis. Importantly, LANA, which lacks enzymatic function, also mediates KSHV TR DNA replication (10, 21–23) through recruitment of host cell machinery, but little is known regarding the details of this process.We recently identified an internal 59-aa LANA region critical for efficient DNA replication and persistence (24, 25). Here, we find that LANA recruits replication factor C (RFC), the DNA polymerase clamp proliferating cell nuclear antigen (PCNA)] loader (26), through this sequence to mediate viral DNA replication and episome persistence.