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），因为它将在本提案中提到，是卡波西肉瘤（KS）的可能原因。 迄今为止收集的大部分数据涉及流行病学证据，表明HHV-8 DNA序列存在于各种淋巴源性癌症中。 例如，HHV-8与多中心Castleman病和体腔大细胞淋巴瘤有关。尽管进行了大量的研究，但对HHV-8裂解复制知之甚少。 对HHV-8核苷酸序列的检查确实鉴定了与解旋酶-引发酶、聚合酶及其持续合成因子以及单链DNA结合蛋白的候选复制基因同源物。 然而，这些同源物中没有一个已经通过功能测定显示为裂解复制所需的。 现在整个HHV-8基因组已经测序，并已确定推定的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，已经使用BC 3细胞中的瞬时测定法鉴定了推定的裂解复制起点。这项建议将：i）表征HHV-8的推定裂解复制起点，ii）鉴定起点依赖性DNA复制所需的HHV-8基因，和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起始蛋白。

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.