HHV-8 ORIGIN-DEPENDENT DNA REPLICATION

卡波西氏肉瘤疱疹病毒（KSHV）或人类疱疹病毒8型（HHV-8）是卡波西氏肉瘤（KS）的可能病因。迄今为止收集的大部分数据涉及流行病学证据，证明HHV-8 DNA序列存在于各种淋巴细胞衍生的癌症中。例如，HHV-8与多中心Castleman病和体腔大细胞淋巴瘤有关。尽管进行了大量的研究，但人们对HHV-8的裂解复制知之甚少。对HHV-8核苷酸序列的检查确实确定了与解旋酶引物酶、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，这些同源物都没有被功能分析证明是裂解复制所必需的。现在整个HHV-8基因组已经测序，推测的orf已经确定，下一步是确定HHV-8的裂解性复制起源（oriLyt），并阐明裂解性DNA复制所需的病毒因子。为此，在BC3细胞中使用瞬时测定法确定了一个假定的复制裂解起源。该提案将：i)表征HHV-8的推测的裂解复制起源，ii)鉴定起源依赖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表征HHV-8启动蛋白。

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.