Department of Microbiology & Immunology, University of Nevada, Reno, School of Medicine, Center for Molecular Medicine, Reno, Nevada, USA.
J Virol. 2012 Sep;86(18):9983-94. doi: 10.1128/JVI.00839-12. Epub 2012 Jul 3.
The latency-associated nuclear antigen (LANA) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) plays a major role in maintaining latency and is critical for the perpetual segregation of viral episomes to the progeny nuclei of newly divided cells. LANA binds to KSHV terminal repeat (TR) DNA and tethers the viral episomes to host chromosomes through the association of chromatin-bound cellular proteins. TR elements serve as potential origin sites of KSHV replication and have been shown to play important roles in latent DNA replication and transcription of adjacent genes. Affinity chromatography and proteomics analysis using KSHV TR DNA and the LANA binding site as the affinity column identified topoisomerase IIβ (TopoIIβ) as a LANA-interacting protein. Here, we show that TopoIIβ forms complexes with LANA that colocalize as punctuate bodies in the nucleus of KSHV-infected cells. The specific TopoIIβ binding region of LANA has been identified to its N terminus and the first 32 amino acid residues containing the nucleosome-binding region crucial for binding. Moreover, this region could also act as a dominant negative to disrupt association of TopoIIβ with LANA. TopoIIβ plays an important role in LANA-dependent latent DNA replication, as addition of ellipticine, a selective inhibitor of TopoII, negatively regulated replication mediated by the TR. DNA break labeling and chromatin immunoprecipitation assay using biotin-16-dUTP and terminal deoxynucleotide transferase showed that TopoIIβ mediates a transient DNA break on viral DNA. These studies confirm that LANA recruits TopoIIβ at the origins of latent replication to unwind the DNA for replication.
卡波氏肉瘤相关疱疹病毒（KSHV）编码的潜伏相关核抗原（LANA）在维持潜伏状态方面发挥着重要作用，对于病毒游离体向新分裂细胞的子细胞核的永久分离至关重要。LANA 与 KSHV 末端重复（TR）DNA 结合，并通过与染色质结合的细胞蛋白的关联将病毒游离体固定在宿主染色体上。TR 元件可作为 KSHV 复制的潜在起点，并且已被证明在潜伏 DNA 复制和相邻基因的转录中发挥重要作用。使用 KSHV TR DNA 和 LANA 结合位点作为亲和柱的亲和层析和蛋白质组学分析鉴定拓扑异构酶 IIβ（TopoIIβ）为 LANA 相互作用蛋白。在这里，我们表明 TopoIIβ 与 LANA 形成复合物，这些复合物在 KSHV 感染细胞的核中作为点状体共定位。已经确定 LANA 与 TopoIIβ 结合的特定区域位于其 N 端和包含对于结合至关重要的核小体结合区域的前 32 个氨基酸残基。此外，该区域还可以作为显性负突变体来破坏 TopoIIβ 与 LANA 的结合。TopoIIβ 在 LANA 依赖的潜伏 DNA 复制中起着重要作用，因为加入 Ellipticine，一种拓扑异构酶 II 的选择性抑制剂，会负调控由 TR 介导的复制。使用生物素-16-dUTP 和末端脱氧核苷酸转移酶进行 DNA 断裂标记和染色质免疫沉淀测定表明，TopoIIβ 在病毒 DNA 上介导瞬时 DNA 断裂。这些研究证实，LANA 将 TopoIIβ 募集到潜伏复制的起点处，以解开 DNA 进行复制。
