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