Characterization of the gamma HV68 v-cyclin

描述(由申请人提供)：这是研究由？-疱疹病毒(v-Cyclins)编码的周期蛋白同源基因(v-Cyclins)对？-疱疹病毒的致病机制和潜伏期做出贡献的第一次拨款续期。人类疱疹病毒KSHV和EBV是癌症的重要原因，特别是在免疫功能低下的人中。由于这些病毒的物种特异性，对其发病机制的体内研究一直很有限。我们和其他人一直在开发一种小型动物模型系统，即近交系小鼠感染小鼠伽马疱疹病毒68(YHV68)，用于分析γ-疱疹病毒感染的发病机制。本应用重点介绍了？hv68v-细胞周期蛋白在疾病发病机制中的作用。值得注意的是，2-疱疹病毒(HVS、KSHV和yHV68)都编码D型细胞周期蛋白的同源物，而EBV感染则上调宿主D型细胞周期蛋白的表达。因此，我们期望对？hv68v-细胞周期蛋白的分析将为保守的致病机制提供重要的见解。我们已经证明？hv68v-细胞周期蛋白是促进原代淋巴细胞细胞周期进程的癌基因，并且hv68v-细胞周期蛋白突变体对潜伏感染的M？和/或B细胞(进度报告)。这些观察结果导致了以下4个目标。 目的1.v-Cyclin在慢性感染中的作用[评估感染剂量和途径对急性病毒复制、潜伏期和重新激活的影响；与wt和v-Cyclin缺失yHV68的体内感染竞争分析；进一步表征v-Cyclin缺失yHV68重新激活缺陷；含有KSHV v-Cyclin的嵌合病毒的特征]。 目的2.v-Cyclin基因表达的转录调控[裂解周期相关的v-Cyclin基因转录的作用；潜伏期/再激活相关的v-Cyclin基因剪接转录本的分析；P1和P2启动的LANA/v-Cyclin剪接转录本的特征]。 目的3.建立和鉴定组织培养模型以研究v-Cyclin的功能[急性病毒复制时依赖于CDK的功能；潜伏期病毒重新激活时依赖于CDK的功能]。 目的4.鉴定CDK非依赖的v-Cyclin功能[绘制病毒重新激活所需的结构域；比较wt和CDK结合突变体v-Cyclin的基因表达谱；鉴定和鉴定与v-Cyclin相互作用的细胞和病毒蛋白]。

DESCRIPTION (provided by applicant): This is the first renewal of a grant to investigate the mechanisms by which cyclin homologs encoded by ?-herpesviruses (v-cyclins) contribute to ?-herpesvirus pathogenesis and latency. The human ?-herpes viruses KSHV and EBV are important causes of cancer, especially in immunocompromised individuals. Because of the species specificity of these viruses, in vivo studies of their pathogenesis have been limited. We and others have been developing a small animal model system, infection of inbred mice with murine gammaherpesvirus 68 (yHV68), for analysis of the pathogenesis of ?-herpesvirus infection. This application focuses on the role of the ?HV68 v-cyclin in disease pathogenesis. Notably, the ?2-herpesviruses (HVS, KSHV and yHV68) all encode homologs of D-type cyclins, while EBV infection upregulates expression of host D-type cyclins. Thus, we expect analysis of the ?HV68 v-cyclin will provide important insights into a conserved pathogenic mechanism. We have shown that the ?HV68 v-cyclin is an oncogene that promotes cell cycle progression in primary lymphocytes and that a ?HV68 v-cyclin mutant reactivates inefficiently from latently infected M? and/or B cells (Progress Report). These observations lead to the following 4 aims. Aim 1. Role of v-cyclin during chronic infection [assess impact of dose and route of infection on acute virus replication, latency and reactivation; in vivo infection competition analysis with wt and v-cyclin null yHV68; further characterize v-cyclin null yHV68 reactivation defect; characterization of chimeric virus harboring KSHV v-cyclin]. Aim 2. Transcriptional regulation of v-cyclin gene expression [role of lytic cycle-associated v-cyclin gene transcription; analysis of latency/reactivation-associated spliced v-cyclin gene transcripts; characterization of P1- and P2-initiated LANA/v-cyclin spliced transcripts]. Aim 3. Develop and characterize tissue culture models to investigate v-cyclin functions [CDK-dependent function during acute virus replication; CDK-independent function during virus reactivation from latency]. Aim 4. Characterize CDK-independent v-cyclin function [map domains required for virus reactivation; gene expression profiling comparing wt and CDK-binding mutant v-cyclin; identify and characterize cellular and viral proteins interacting with v-cyclin].