In vitro and in vivo studies of Cytomegalovirus MIE gene regulation

描述(申请人提供)：人类巨细胞病毒(HCMV)感染会在免疫功能受损的宿主中引起危及生命的疾病，并会在怀孕期间感染的妇女中对发育中的胎儿造成严重的神经损害。由巨细胞病毒主要即刻早期(MIE)基因编码的蛋白质对CMV的感染和复制是必不可少的，但CMV如何征用宿主细胞机制来指导MIE蛋白的合成尚不清楚。我们的初步发现表明，MIE基因的表达在剪接水平上被调控，导致器官特异性的MIE基因表达、蛋白质合成和体内CMV感染，以及CMV与宿主细胞剪接装置的新的相互作用。这种拟议的为期四年的方法从根本上不同于以前针对病毒基因转录的MIE基因调控研究，并有望与已知的信息高度互补。事实上，我们的长期目标是阐明CMV如何篡改宿主细胞的基因剪接机制来指导MIE基因的合成，包括：调节CMV非常早期基因剪接的机制；MIE基因剪接对CMV体外和体内复制的影响；核区(ND10和SC)及其相关蛋白在CMV基因调控和病毒复制中的新作用，以及这些如何影响CMV诱导的神经发育致病。我们的中心假设是，在感染早期，CMV通过与特定的顺式元件相互作用，抵消包括ND10及其相关蛋白在内的宿主细胞防御，篡夺宿主细胞剪接调控因子PTB、U2AF和SC35，并激活早期基因。其具体目的是：1)确定调控MIE基因剪接的主要顺式元件，并确定剪接因子对HCMV复制的重要性。2)确定HCMV IE1/IE2与细胞基因剪接调控因子的相互作用以及核区在调控HCMV基因剪接中的生物学意义。3)研究MIE基因组织特异性选择性剪接对CMV体内器官选择性致病力的影响，以及IE1/IE3对小鼠胚胎神经发生的影响。这些研究将引入新的概念和发现，促进对分子病毒学的基本理解，并将结合创新的体外和体内模型和新工具来阐明MIE基因剪接调控的关键机制，这些机制对CMV基因表达和高效宿主感染至关重要。积极影响将包括在理解分子病毒学和巨细胞病毒病理生物学方面取得的质的进展，以及为预防和治疗干预确定新的候选目标。了解CMV调节MIE基因剪接的机制可能因此推动新的、选择性的抗CMV治疗策略的发展，因为MIE基因产物IE1和IE2对CMV早期/晚期基因表达和HCMV复制至关重要。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) infection causes life-threatening disease in immunocompromised hosts and serious neurological impairment to developing fetuses in women infected during pregnancy. Proteins encoded by the HCMV major-immediate early (MIE) gene are essential to productive CMV infection and replication, but it is unknown how CMV commandeers host cell machinery to direct MIE protein synthesis. Our preliminary findings reveal that MIE gene expression is regulated at the splicing level, causing organ-specific MIE gene expression, protein synthesis and CMV infection in vivo, and novel interactions of CMV with host cell splicing apparatus. This proposed four-year approach is fundamentally different from previous MIE gene regulation studies that target the viral gene transcription, and is expected to be highly complementary with already-known information. Indeed, our long-term goal is to elucidate how CMV usurps host cell gene-splicing machinery to direct MIE gene synthesis, including: mechanisms that regulate splicing of CMV very early genes; the influence of MIE gene splicing on CMV replication in vitro and in vivo; the novel roles of nuclear domains (ND10 and SC) and their associated proteins in CMV gene regulation and viral replication, and how these influence CMV-induced neurodevelopmental pathogenesis. Our central hypothesis is that during early-stage infection, CMV counteracts host cell defenses including ND10 and its associated proteins, usurps the host cell splicing regulators PTB, U2AF and SC35, and activates early genes by interacting with specific cis-elements. The specific aims are: 1) To identify major cis-elements of the MIE gene that regulate MIE gene splicing, and determine the importance of splicing factors for HCMV replication. 2) Determine the biological importance of interactions between HCMV IE1/IE2 and cellular gene splicing regulators and of nuclear domains in regulating HCMV gene splicing. 3) Determine the influence of tissue- specific alternative splicing of the MIE gene upon organ-selective CMV pathogenicity in vivo, and the pathogenic influence of IE1/IE3 on embryonic neurogenesis in vitro and in vivo in mice. These studies will introduce novel concepts and findings that advance basic understanding of molecular virology, and will combine innovative in vitro and in vivo models and novel tools to elucidate key mechanisms of MIE gene splicing regulation that are critical to CMV gene expression and productive host infection. Positive impacts will include qualitative advances in understanding of molecular virology and CMV pathobiology in particular and also identification of new candidate targets for preventive and therapeutic intervention. Understanding the mechanisms CMV uses to regulate MIE gene splicing may thus drive development of new, selective anti-CMV therapeutic strategies, as the MIE gene products IE1 and IE2 are essential to CMV early/late gene expression and HCMV replication.