Cis and Trans acting factors for HHV8 lytic replication

描述（由申请方提供）：卡波西肉瘤相关疱疹病毒（KSHV）或人疱疹病毒8型（HHV 8）裂解DNA复制由基因组内两个顺式作用裂解起点之一指导。OriLyt-L位于开放阅读框（ORF）K4.2和K5之间，OriLyt-R位于ORF 69和vFLIP之间。瞬时测定确定了两个富含A+T的DNA序列、三个AP 1转录因子结合位点、一个ORF 50应答元件（RE）和一个下游TATA共有序列都是高效扩增oriLyt所需的。使用一个裂解的起点作为报告，我们建立了共转染复制试验，并阐明了8个所需的蛋白质的必要和足够的扩增克隆oriLyt。ORF是：ORF 6（单链DNA结合蛋白）、ORF 9（DNA聚合酶）、ORF 40/41（引发酶相关因子）、ORF 44（解旋酶）、ORF 56（引发酶）、ORF 59（聚合持续合成因子）、ORF 50/K-Rta（反式激活因子）和K8（未知功能）。先前的研究表明，K-Rta和RAP在感染细胞中相互作用。我们现在表明oriLyt内的ORF 50 RE与K-Rta相互作用，并且该区域在瞬时测定中充当强有力的启动子。此外，可以从该启动子区域的下游区域检测RNA转录物。我们还构建了不表达ORF 50基因产物K-Rta的重组HHV 8 BAC。该病毒不能产生感染性病毒，并且在用TPA（病毒裂解周期的诱导剂）处理时不积累病毒DNA。该数据表明，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直接相互作用参与病毒裂解DNA复制。该提议的假设是RAP通过与裂解性复制起点相互作用来执行基本复制功能，并且K-Rta通过在oriLyt内贡献反式激活因子功能来促进裂解性复制。为了解决这一假设，在本提案中，我们将：i）定义oriLyt内任何额外的必需顺式作用序列; ii）确定RAP和K-Rta在病毒基因组背景下的作用;以及iii）确定oriLyt内的RAP和K-Rta结合位点，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.