Cis and Trans acting factors for HHV8 lytic replication

HHV8 裂解复制的顺式和反式作用因子

• 批准号: 6942912
• 负责人: GREGORY S PARI
• 金额: $ 28.64万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-04-01 至 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6942912
• 关键词: Kaposi' s sarcoma; binding sites; gene expression; genetic promoter element; genetic regulatory element; human herpesvirus 8; intermolecular interaction; microorganism culture; nucleic acid sequence; open reading frames; recombinant virus; transcription factor; virus DNA; virus genetics; virus protein; virus replication

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.

描述（由申请人提供）：Kaposi的肉瘤相关疱疹病毒（KSHV）或人疱疹病毒8（HHV8）裂解DNA复制是由基因组中的两个顺式作用裂解起源之一指导的。 Orilyt-L位于开放式阅读帧（ORF）K4.2和K5之间，而Orilyt-R位于ORF69和VFLIP之间。瞬态测定确定两个富含A+T的DNA序列，三个AP1转录因子结合位点，一个ORF50响应元件（RE）和下游TATA共识序列有效地放大了Orilyt。使用裂解起源之一作为记者，我们建立了一种共转染复制测定法，并阐明了8个所需的蛋白质所需的蛋白质，足以扩增克隆的orilyt。 ORF为：ORF6（单链DNA结合蛋白），ORF9（DNA聚合酶），ORF40/41（Primase相关因子），ORF44（Helicase），ORF56（Primase），ORF56（Primase），ORF59（POR Processitive Fimity rivitation Fimity），ORF50/K ORF50/K -RTA（transactivator）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。现在，我们表明Orilyt内部的ORF50与K-RTA相互作用，并且该区域是瞬态测定中强大的启动子。另外，可以从该启动子区域下游的区域检测到RNA转录本。我们还构建了一个不表达ORF50基因产物K-RTA的重组HHV8 BAC。该病毒未能产生传染病，并且在用TPA治疗后（病毒裂解循环的诱导剂）治疗后不会积聚病毒DNA。该数据表明K-RTA在病毒裂解周期中具有双重作用。通过与Orilyt直接相互作用来诱导裂解复制和参与病毒DNA复制所需的病毒基因。该建议的假设是RAP通过与复制的裂解起源相互作用，而K-RTA通过在Orilyt中贡献transactivator函数来促进裂解复制。为了解决这一假设，在这一提议中，我们将：i）定义Orilyt内部的任何其他必要的顺式作用序列； ii）确定RAP和K-RTA在病毒基因组中的作用； 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。