Cis and Trans acting factors for HHV8 lytic replication

描述(申请人提供)：卡波西肉瘤相关疱疹病毒(KSHV)或人类疱疹病毒8型(HHV8)裂解DNA复制是由基因组中两个顺式作用裂解起源之一引导的。OriLyt-L位于开放阅读框K4.2和K5之间，OriLyt-R位于ORF69和vFlIP之间。瞬时分析表明，两个富含A+T的DNA序列、三个AP1转录因子结合位点、一个ORF50反应元件(RE)和一个下游的TATA共同序列都是有效扩增oriLyt所必需的。以其中一个裂解来源为报告基因，我们建立了共转染-复制实验，并阐明了扩增oriLyt克隆所需的8个必需蛋白质。它们分别是：ORF6(单链DNA结合蛋白)、ORF9(DNA聚合酶)、ORF40/41(启动酶相关因子)、ORF44(解旋酶)、ORF56(启动酶)、ORF59(Poll处理因子)、ORF50/K-RTA(反式激活因子)和K8(功能未知)。以往的研究表明，K-RTA和RAP在感染细胞中相互作用。我们现在证明了oriLyt内的ORF50 RE与K-RTA相互作用，并且该区域在瞬时分析中起到了强有力的启动子的作用。此外，可以从该启动子区域下游的区域检测到RNA转录本。我们还构建了不表达ORF50基因产物K-RTA的重组HHV8 BAC。这种病毒不能产生传染性病毒，在用TPA治疗时也不积累病毒DNA，TPA是病毒裂解周期的诱导剂。这一数据表明，K-RTA在病毒裂解周期中具有双重作用：诱导裂解复制所需的病毒基因，并通过与oriLyt的直接相互作用参与病毒裂解DNA复制。这一提议的假设是，RAP通过与复制的裂解起始点相互作用来执行基本的复制功能，而K-RTA通过在oriLyt内贡献反式激活功能来促进裂解复制。为了解决这一假设，在这个建议中，我们将：i)在oriLyt中定义任何额外的基本顺式作用序列；ii)确定RAP和K-RTA在病毒基因组中的作用；以及iii)确定oriLyt中的RAP和K-RTA结合部位，并阐明负责反式激活和/或DNA复制的蛋白质结构域。

DESCRIPTION (provided by applicant): Kaposi's sarcoma-associated herpesvirus (KSHV) or human herpesvirus 8 (HHV8) lytic DNA replication is directed by one of two cis acting lytic origins within the genome. OriLyt-L is located between open reading frames (ORFs) K4.2 and K5 and OriLyt-R is located between ORF69 and vFLIP. Transient assays determined that two A+T rich DNA sequences, three AP1 transcription factor binding sites, an ORF50 response element (RE) and a downstream TATA consensus sequence are all required for efficient amplification of oriLyt. Using one of the lytic origins as a reporter we established a cotransfection-replication assay and elucidated 8 required proteins necessary and sufficient to amplify cloned oriLyt. The ORFs are: ORF6 (single-stranded DNA binding protein), ORF9 (DNA polymerase), ORF40/41 (primase-associated factor), ORF44 (helicase), ORF56 (primase), ORF59 (pol processivity factor), ORF50/K-Rta (transactivator) and K8 (unknown function). Previous studies have demonstrated that K-Rta and RAP interact in infected cells. We now show that the ORF50 RE within oriLyt interacts with K-Rta and this region acts as a powerful promoter in transient assays. In addition, RNA transcripts can be detected from a region just downstream of this promoter region. We have also constructed a recombinant HHV8 BAC that does not express the ORF50 gene product, K-Rta. This virus fails to produce infectious virus and does not accumulate viral DNA upon treatment with TPA, an inducer of the viral lytic cycle. This data suggests that K-Rta has a dual role in the viral lytic cycle; induction of viral genes required for lytic replication and participation in viral lytic DNA replication by direct interaction with oriLyt. The hypothesis for this proposal is that RAP performs an essential replication function by interacting with the lytic origin of replication and K-Rta facilitates lytic replication by contributing a transactivator function within oriLyt. To address this hypothesis, in this proposal we will: i) define any additional essential cis acting sequences within oriLyt; ii) determine the role of RAP and K-Rta in the context of the viral genome; and iii) determine RAP and K-Rta binding sites within oriLyt and elucidate protein domains responsible for transactivation and/or DNA replication.