Chan S R, Chandran B
Department of Microbiology, Molecular Genetics, and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160-7700, USA.
J Virol. 2000 Dec;74(23):10920-9. doi: 10.1128/jvi.74.23.10920-10929.2000.
Human herpesvirus 8 (HHV-8) or Kaposi's sarcoma-associated herpesvirus (KSHV) ORF59 protein (PF-8) is a processivity factor for HHV-8 DNA polymerase (Pol-8) and is homologous to processivity factors expressed by other herpesviruses, such as herpes simplex virus type 1 UL42 and Epstein-Barr virus BMRF1. The interaction of UL42 and BMRF1 with their corresponding DNA polymerases is essential for viral DNA replication and the subsequent production of infectious virus. Using HHV-8-specific monoclonal antibody 11D1, we have previously identified the cDNA encoding PF-8 and showed that it is an early-late gene product localized to HHV-8-infected cell nuclei (S. R. Chan, C. Bloomer, and B. Chandran, Virology 240:118-126, 1998). Here, we have further characterized PF-8. This viral protein was phosphorylated both in vitro and in vivo. PF-8 bound double-stranded DNA (dsDNA) and single-stranded DNA independent of DNA sequence; however, the affinity for dsDNA was approximately fivefold higher. In coimmunoprecipitation reactions, PF-8 also interacted with Pol-8. In in vitro processivity assays with excess poly(dA):oligo(dT) as a template, PF-8 stimulated the production of elongated DNA products by Pol-8 in a dose-dependent manner. Functional domains of PF-8 were determined using PF-8 truncation mutants. The carboxyl-terminal 95 amino acids (aa) of PF-8 were dispensable for all three functions of PF-8: enhancing processivity of Pol-8, binding dsDNA, and binding Pol-8. Residues 10 to 27 and 279 to 301 were identified as regions critical for the processivity function of PF-8. Interestingly, aa 10 to 27 were also essential for binding Pol-8, whereas aa 1 to 62 and aa 279 to 301 were involved in binding dsDNA, suggesting that the processivity function of PF-8 is correlated with both the Pol-8-binding and the dsDNA-binding activities of PF-8.
人类疱疹病毒8型（HHV-8）或卡波西肉瘤相关疱疹病毒（KSHV）的ORF59蛋白（PF-8）是HHV-8 DNA聚合酶（Pol-8）的持续性因子，与其他疱疹病毒表达的持续性因子同源，如单纯疱疹病毒1型UL42和爱泼斯坦-巴尔病毒BMRF1。UL42和BMRF1与其相应的DNA聚合酶的相互作用对于病毒DNA复制及随后感染性病毒的产生至关重要。利用HHV-8特异性单克隆抗体11D1，我们先前已鉴定出编码PF-8的cDNA，并表明它是定位于HHV-8感染细胞核的早期-晚期基因产物（S. R. 陈、C. 布卢默和B. 钱德兰，《病毒学》240:118 - 126，1998）。在此，我们进一步对PF-8进行了特性分析。这种病毒蛋白在体外和体内均被磷酸化。PF-8与双链DNA（dsDNA）和单链DNA结合，与DNA序列无关；然而，它对dsDNA的亲和力大约高五倍。在共免疫沉淀反应中，PF-8也与Pol-8相互作用。在以过量聚（dA）:寡聚（dT）作为模板的体外持续性分析中，PF-8以剂量依赖的方式刺激Pol-8产生延长的DNA产物。使用PF-8截短突变体确定了PF-8的功能结构域。PF-8的羧基末端95个氨基酸（aa）对于PF-8的三种功能均非必需：增强Pol-8的持续性、结合dsDNA和结合Pol-8。残基10至27和279至301被确定为对PF-8的持续性功能至关重要的区域。有趣的是，氨基酸10至27对于结合Pol-8也是必需的，而氨基酸1至62和氨基酸279至301参与结合dsDNA，这表明PF-8的持续性功能与PF-8的Pol-8结合和dsDNA结合活性均相关。
