HHV-8 ORIGIN-DEPENDENT DNA REPLICATION

卡波西肉瘤疱疹病毒(KSHV)或人类疱疹病毒8型(HHV-8)可能是卡波西肉瘤(KS)的病因。到目前为止收集的大部分数据涉及流行病学证据，表明HHV-8DNA序列存在于各种淋巴源性癌症中。例如，HHV-8与多中心Castleman病和以体腔为基础的大细胞淋巴瘤有关。尽管进行了密集的研究，但人们对HHV-8裂解复制知之甚少。对HHV-8核苷酸序列的检查确实确定了与解旋酶-启动酶、聚合酶及其加工因子和单链DNA结合蛋白同源的候选复制基因。然而，功能分析表明，这些同源物都不是裂解复制所必需的。现在已经对HHV-8全基因组进行了测序，并确定了可能的ORF，下一步是确定HHV-8裂解复制起点(OriLyt)，并阐明裂解DNA复制所需的病毒因子。为此，已在BC3细胞中使用瞬时试验确定了假定的复制裂解来源。这一建议将：i)确定HHV-8的假定裂解复制来源，ii)确定依赖于来源的DNA复制所需的HHV-8基因，以及iii)确定哪些基因直接参与DNA复制，哪些基因起辅助作用，并确定HHV-8启动子蛋白的特征。

Kaposi's sarcoma herpesvirus (KSHV) or human herpesvirus type 8 (HHV-8) as it will be referred to in this proposal, is the likely cause of Kaposi's sarcoma (KS). Much of the data collected to date involves epidemiological evidence demonstrating that HHV-8 DNA sequences are present in a variety of lymphoid-derived cancers. For example, HHV-8 is implicated in multicentric Castleman's disease and body cavity-based large cell lymphomas. Despite intense research, little is known about HHV-8 lytic replication. Inspection of the HHV-8 nucleotide sequence does identify the candidate replication gene homologs to helicase- primase, polymerase and its processivity factor, and a single- stranded DNA binding protein. However none of these homologs have been shown by functional assay to be required for lytic replication. Now that the entire HHV-8 genome has been sequenced and putative ORFs have been identified, the next step is to identify the HHV-8 lytic origin of replication (oriLyt) and to elucidate those viral factors required for lytic DNA replication. To this end, a putative lytic origin of replication has been identified using a transient assay in BC3 cells. This proposal will: i) Characterize the putative lytic origin of replication for HHV-8, ii) identify the HHV-8 genes required for origin-dependent DNA replication, and iii) determine which genes participate directly in DNA replication and which play an ancillary role and characterize the HHV-8 initiator protein.