Primary effusion lymphoma (PEL) cells harbor Kaposi's sarcoma-associated herpesvirus (KSHV) episomes and express a KSHV-encoded latency-associated nuclear antigen (LANA). In PEL cells, LANA and KSHV DNA colocalized in dots in interphase nuclei and along mitotic chromosomes. In the absence of KSHV DNA, LANA was diffusely distributed in the nucleus or on mitotic chromosomes. In lymphoblasts, LANA was necessary and sufficient for the persistence of episomes containing a specific KSHV DNA fragment. Furthermore, LANA colocalized with the artificial KSHV DNA episomes in nuclei and along mitotic chromosomes. These results support a model in which LANA tethers KSHV DNA to chromosomes during mitosis to enable the efficient segregation of KSHV episomes to progeny cells.

Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) (also known as human herpesvirus 8) latently infects KS tumors, primary effusion lymphomas (PELs), and PEL cell lines. In latently infected cells, KSHV DNA is maintained as circularized, extrachromosomal episomes. To persist in proliferating cells, KSHV episomes must replicate and efficiently segregate to progeny nuclei. In uninfected B-lymphoblastoid cells, KSHV latency-associated nuclear antigen (LANA1) is necessary and sufficient for persistence of artificial episomes containing specific KSHV DNA. In previous work, the cis-acting sequence required for episome persistence contained KSHV terminal-repeat (TR) DNA and unique KSHV sequence. We now show that cis-acting KSHV TR DNA is necessary and sufficient for LANA1-mediated episome persistence. Furthermore, LANA1 binds TR DNA in mobility shift assays and a 20-nucleotide LANA1 binding sequence has been identified. Since LANA1 colocalizes with KSHV episomes along metaphase chromosomes, these results are consistent with a model in which LANA1 may bridge TR DNA to chromosomes during mitosis to efficiently segregate KSHV episomes to progeny nuclei.Kaposi's sarcoma (KS)-associated herpesvirus (KSHV) or human herpesvirus 8 is a gamma-2 herpesvirus tightly linked to KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease, an aggressive lymphoproliferative disorder (8,9,35,47). KSHV infection in tumors and PEL cell lines is predominantly latent. Latently infected cells have multiple copies of extrachromosomal, circularized KSHV DNA (episomes) (8, 13). To persist in proliferating cells, viral episomes must first replicate and then segregate to progeny cells.The latency-associated nuclear antigen (LANA1), encoded by KSHV open reading frame 73, is one of a limited number of KSHV genes expressed during latent infection (23,24,40). Confocal microscopy using immunofluorescence and fluorescent in situ hybridization demonstrated that LANA1 colocalized with KSHV episomes in PEL cell nuclei in interphase and along mitotic chromosomes (4,12,(22)(23)(24)48). Furthermore, in KSHV-uninfected lymphoblastoid cells, LANA1 mediates extrachromosomal persistence of artificial KSHV episomes containing specific KSHV DNA (4). The KSHV Z6 cosmid, which includes KSHV terminal-repeat (TR) elements and the left end of the KSHV genome, persisted as an episome in LANA1-expressing cells. In contrast, the Z8 cosmid, which contains sequence from near the center of the KSHV genome, did not persist as an episome in LANA1-expressing cells. Further, DNA containing the left end of Z6, but not other Z6 segments, persisted as episomes in LANA1-expressing cells (4,43). Here

To persist in latently infected, proliferating cells, Kaposi's sarcoma-associated herpesvirus (KSHV) episomes must replicate and efficiently segregate to progeny nuclei. Episome persistence in uninfected cells requires latency-associated nuclear antigen 1 (LANA1) in trans and cis-acting KSHV terminal repeat (TR) DNA. The LANA1 C terminus binds TR DNA, and LANA1 mediates TR-associated DNA replication in transient assays. LANA1 also concentrates at sites of KSHV TR DNA episomes along mitotic chromosomes, consistent with a tethering role to efficiently segregate episomes to progeny nuclei. LANA1 amino acids 5 to 22 constitute a chromosome association region (Piolot et al., J. Virol. 75:3948-3959, 2001). We now investigate LANA1 residues 5 to 22 with scanning alanine substitutions. Mutations targeting LANA1 5 GMR 7 , 8 LRS 10 , and 11 GRS 13 eliminated chromosome association, DNA replication, and episome persistence. LANA1 mutated at 14 TG 15 retained the ability to associate with chromosomes but was partially deficient in DNA replication and episome persistence. These results provide genetic support for a key role of the LANA1 N terminus in chromosome association, LANA1-mediated DNA replication, and episome persistence.