Characterization of the Minimal Replicator of Kaposi's Sarcoma-Associated Herpesvirus Latent Origin

Abstract

The latency-associated nuclear antigen (LANA) of Kaposi's sarcoma-associated herpesvirus (KSHV) binds to two sites within the 801-bp-long terminal repeat (TR) and is the only viral protein required for episomal maintenance. While two or more copies of TR are required for long-term maintenance, a single TR confers LANA-dependent origin activity on plasmid DNA. Deletion mapping revealed a 71-bp-long minimal replicator containing two distinctive sequence elements: LANA binding sites (LBS1/2) and an adjacent 29-to 32-bp-long GC-rich sequence which we termed the replication element. Furthermore, the transcription factor Sp1 can bind to TR outside the minimal replicator and contributes to TR's previously reported enhancer activity.Kaposi's sarcoma-associated herpesvirus (KSHV) is associated with Kaposi's sarcoma (KS) and two lymphoproliferative diseases: primary effusion lymphoma and multicentric Castleman's disease (5,6,30). The 140-kbp KSHV long unique region encodes approximately 90 genes, which are flanked on both sides with 20 to 40 copies of 801-bp-long GC-rich terminal repeats (TR) (20,24,29). During latency, only a small subset of viral genes, including the latency-associated nuclear antigen (LANA), is expressed and the viral genome is maintained as multiple copies of episomal DNA (10,11,28,32). LANA is a functional homologue of origin binding proteins EBNA-1 from Epstein-Barr virus (EBV) and E2 from human papillomavirus (15,22). LANA is necessary and sufficient to support stable long-term episomal maintenance in dividing cells by (i) tethering viral episomes to cellular chromosomes (1,2,7,8,12,13,19,26) and (ii) supporting the initiation of DNA replication of TR-containing plasmids (15,17,23), presumably by bridging the viral origin of replication (ori) and the host cellular replication machinery. Using short-term replication assays, we have previously demonstrated that all cis-regulatory sequences required for LANA-dependent ori activity are located within a single copy of TR (17). By performing a detailed deletion analysis, we determined the minimal replicator within TR and showed that the transcription factor Sp1 can bind to at least two sites within TR and contributes to its previously described enhancer activity (13).Mapping the minimal replicator within TR. The TR unit is 801 bp long and 89% GC rich (20,29). LANA binds in a cooperative fashion to two sites (LBS1/2) in TR located between nucleotides (nt) 571 and 610, and both binding sites contribute to ori activity as determined by short-term replication assays (12, 17). Located directly upstream of LBS1/2 is an 89-bp highly GC-rich element adjacent to a 101-bp AT-rich region (Fig. 1A). AT-rich stretches are often found in origins of replication and are believed to function in DNA unwinding (4). We have previously reported that deletion of TR sequences from nt 1 to 482, including the AT-rich region, and nt 610 to 801 downstream of LBS1/2 does not decrease replication efficiency. In contrast, a deletion of the GC-rich region (up to nt 551) completely abo...

Kaposi sarcoma–associated herpesvirus: immunobiology, oncogenesis, and therapy

“…Update on LANA function and structure LANA binds the viral terminal repeats, specifically two sequenceconserved, high-affinity binding sites (LBS1 and LBS2) and a more divergent third, low-affinity site (114)(115)(116). LANA can be thought of as a dumbbell-like structure in which a stalk of internal repeats separates the two globular terminal regions.…”

Identification of Kaposi's Sarcoma-Associated Herpesvirus LANA Regions Important for Episome Segregation, Replication, and Persistence

“…Each contains two copies of the TR and also a GFP expression cassette. p2TR-⌬RE-GFP lacks the 32-bp replication element (RE), which is adjacent to the two LANA binding sites present in each TR (32); this RE is required for LANA-mediated replication but not for the binding of LANA to the TR. Therefore, the plasmid can be used to investigate the ability of LANA to segregate TR-containing DNA in the absence of replication.…”

Kaposi’s Sarcoma-Associated Virus Governs Gene Expression Profiles Toward B Cell Transformation

“…LANA has two binding sites called LANA binding sequences (LBS) in TR and replication origin of the KSHV genome in latency (ori-P) consists of the LBS and the following 32bp GCrich segment (32GC) (Garber, Hu, and Renne, 2002;Garber et al, 2001). One of two LBS is required for the viral replication at least but it is not enough, i.e., 32GC is also required (Hu and Renne, 2005). Though it remains to be solved how the viral ori-P is determined among repeated TR sequences and how 32GC is functioning, LANA has been reported to interact with components of cellular replication machinery; origin recognition complex 1 to 6 (ORC1~ORC6) (Verma et al, 2006).…”