Chromosome Binding Site of Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Is Essential for Persistent Episome Maintenance and Is Functionally Replaced by Histone H1

Abstract

Latency-associated nuclear antigen 1 (LANA1) of Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) persistently maintains a plasmid containing the KSHV latent origin of replication (oriP) as a closed circular episome in dividing cells. In this study, we investigated the involvement of chromosome binding activity of LANA1 in persistent episome maintenance. Deletion of the N-terminal 22 amino acids of LANA1 (⌬N-LANA) inhibited the interaction with mitotic chromosomes in a human cell line, and the mutant concomitantly lost activity for the long-term episome maintenance of a plasmid containing viral oriP in a human B-cell line. However, a chimera of ⌬N-LANA with histone H1, a cellular chromosome component protein, rescued the association with mitotic chromosomes as well as the long-term episome maintenance of the oriP-containing plasmid. Our results suggest that tethering of KSHV episomes to mitotic chromosomes by LANA1 is crucial in mediating the long-term maintenance of viral episomes in dividing cells.Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is a gamma 2 herpesvirus and is associated with the development of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease (5,6,10,17,21,24). KSHV can establish long-term persistent infections in tumor cells and lymphoma-derived cell lines. In such cells, the double-stranded KSHV DNA genome can persist as multiple copies of closed circular episomes (5, 9), like the genome of its close relative, Epstein-Barr virus (EBV).Two viral components can mediate the long-term episome maintenance of KSHV in infected cells. One is latency-associated nuclear antigen 1 (LANA1), encoded by open reading frame 73, and the other is a cis-acting DNA sequence (latent origin of replication [oriP]) in the 5Ј end of the KSHV genome (terminal repeat [TR] sequence) (1, 2, 7). LANA1 persistently maintains a plasmid containing multiple TRs as an episome in a human B-cell line (1, 2).Previous studies showed that LANA1 was directly bound to the KSHV TR sequence (2,8,11). Furthermore, an imperfect 20-bp palindrome in the TR sequence exhibited binding activity for LANA1, and a plasmid containing three TRs was persistently maintained as an episome in a human cell line (2, 11). Domain map analysis revealed that the C-terminal region of LANA1 bound to this 20-bp palindrome in the TR sequence (11). Since this region contains the LANA1 dimerization domain (22), it seems likely that LANA1 binds to the TR sequence as a dimer, like EBNA1, the episome maintenance protein of EBV.About 40 to 80 copies of episomes are stably maintained in KSHV-infected lymphoma cells in vivo (1, 4). The constant KSHV copy number in dividing cells indicates that KSHV has an efficient system for segregating viral episomes into two daughter cells in every cell division. In the present study, we examined the involvement of the chromosome interaction activity of LANA1 in long-term episome maintenance. A 22-amino-acid deletion from the N terminus of LANA1 (⌬N-LANA) inhibit...

Symmetrical localization of extrachromosomally replicating viral genomes on sister chromatids

“…At least two other viruses, Kaposi's sarcomaassociated herpesvirus (KSHV, human herpesvirus 8) and bovine papillomavirus-1, are known to attach to host mitotic chromosomes (reviewed by Botchan, 2004). Among these, KSHV encodes latency-associated nuclear antigen (LANA), which is a functional counterpart of EBNA1 in EBV (Ballestas et al, 1999;Barbera et al, 2006;Shinohara et al, 2002). Previous studies demonstrated that the LANA colocalizes with KSHV genomes on mitotic chromosomes (Ballestas et al, 1999) and occasionally exhibits symmetrical localization on sister chromatids of mitotic chromosomes (Szekely et al, 1999).…”

Mitotic Chromosome-Binding Activity of Latency-Associated Nuclear Antigen 1 Is Required for DNA Replication from Terminal Repeat Sequence of Kaposi's Sarcoma-Associated Herpesvirus

“…As in the case of papillomavirus E2, EBNA-1 associates with host chromosomes (45), and it has been suggested that the action of the FR in retaining the plasmid involves the viral genome hitchhiking on the host chromosome via EBNA-1. It seems likely that KSHV has evolved a similar chromosome-tethering mechanism for its persistence in host cells (5,14,49,59) and that LANA1 is responsible not only for the stable maintenance but also for the replication of KSHV TRcontaining plasmids (26,28,42). In a transient-replication assay using a panel of LANA1 deletion mutants, the minimal domain of LANA1 able to support the replication of a TRcontaining plasmid was identified as LANA1 ⌬23-950, which contains the N-terminal CBS in addition to the C-terminal DNA-binding and dimerization domain (Lim et al, submitted).…”

“…Its C terminus binds to sequences within the TRs located at both ends of the KSHV genome and represses TR-dependent transcription (6,15,24,42). As expected from the chromosome-tethering model, the LANA1 CBS is necessary for long-term replication of a KSHV TR-containing plasmid (59). In addition, we and others have shown that LANA1 is required for the transient replication of KSHV TR-containing plasmids, indicating that it may play an essential role not only in plasmid maintenance but also in DNA replication from the KSHV TR (26,28,42).…”

Latency-Associated Nuclear Antigen of Kaposi's Sarcoma-Associated Herpesvirus Recruits Uracil DNA Glycosylase 2 at the Terminal Repeats and Is Important for Latent Persistence of the Virus

“…Recombinant KSHV with LANA deleted as well as LANA transcript depletion by shRNA reduced the number of KSHV genomic copies, demonstrating that tethering is important for long-term persistence (26,74). LANA has been shown to bind to cellular proteins to support tethering and replication of the KSHV genome (7,16,40,63,71). In order to identify cellular proteins associated with LANA when LANA is bound to its cognate sequence, we performed an LBS affinity binding assay (71).…”

“…Later studies mapped the domains of LANA important for tethering to host chromosomes (5,6,17). LANA associates with human chromatin through the amino-terminal domain and remains attached during all of the phases of the cell cycle (7,54,63). LANA tethers the KSHV genome to host chromosomes through binding at the terminal repeats (TRs) of the KSHV genome (62).…”