title: The minimal replicator element of the Kaposi's sarcoma-associated herpesvirus terminal repeat supports replication in a semiconservative and cell-cycle-dependent manner.
abstract: Kaposi's sarcoma-associated herpesvirus (KSHV) persists as episomes in infected cells by circularizing at the terminal repeats (TRs). The KSHV episome carries multiple reiterated copies of the terminal repeat, and each copy is capable of supporting replication. Expression of the latency-associated nuclear antigen (LANA) is critical for the replication of TR-containing plasmids. A 32-bp sequence upstream of LANA binding site 1 (LBS1), referred to as RE (replication element), along with LANA binding sites 1 and 2 (RE-LBS1/2), is sufficient to support replication (J. Hu and R. Renne, J. Virol. 79:2637-2642, 2005). In this report we demonstrate that the minimal replicator element (RE-LBS1/2) replicates in synchrony with the host cellular DNA, and only once, in a cell-cycle-dependent manner. Overexpression of the mammalian replication inhibitor geminin blocked replication of the plasmid containing the minimal replicator element, confirming the involvement of the host cellular replication control mechanism, and prevented rereplication of the plasmid in the same cell cycle. Overexpression of Cdt1 also rescued the replicative ability of the RE-LBS1/2-containing plasmids. A chromatin immunoprecipitation assay performed using anti-origin recognition complex 2 (alpha-ORC2) and alpha-LANA antibodies from cells transfected with RE-LBS1/2, RE-LBS1, LBS1, or RE showed the association of ORC2 with the RE region. Expression of LANA increased the number of copies of chromatin-bound DNA of replication elements, suggesting that LANA is important for the recruitment of ORCs and may contribute to the stabilization of the replication protein complexes at the RE site.
authors.affiliation: Department of Microbiology and Tumor Virology Program of the Abramson Comprehensive Cancer Center, University of Pennsylvania, School of Medicine, 201E Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104, USA.
authors.forename: Subhash C
authors.initials: SC
authors.lastname: Verma
authors.forename: Tathagata
authors.initials: T
authors.lastname: Choudhuri
authors.forename: Erle S
authors.initials: ES
authors.lastname: Robertson
mesh_terms: D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002453:Cell Cycle; D002460:Cell Line; D004261:DNA Replication / Q000235:genetics*; D004279:DNA, Viral / Q000235:genetics; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D020079:Terminal Repeat Sequences / Q000235:genetics*; D014779:Virus Replication / Q000187:drug effects
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; D009687:Nuclear Proteins; C109540:latency-associated nuclear antigen