In vitro and in vivo studies of Cytomegalovirus MIE gene regulation

描述（由申请人提供）：人巨细胞病毒（HCMV）感染在免疫功能低下的宿主中导致危及生命的疾病，并对怀孕期间感染的妇女发育中的胎儿造成严重的神经损伤。由HCMV主要-即时早期（MIE）基因编码的蛋白质对巨细胞病毒感染和复制至关重要，但目前尚不清楚巨细胞病毒如何利用宿主细胞机制来指导MIE蛋白的合成。我们的初步研究结果表明，MIE基因的表达在剪接水平受到调控，在体内引起器官特异性的MIE基因表达、蛋白合成和CMV感染，以及CMV与宿主细胞剪接装置的新型相互作用。这一提议的为期四年的方法与之前针对病毒基因转录的MIE基因调控研究有根本不同，预计将与已知信息高度互补。事实上，我们的长期目标是阐明CMV如何篡夺宿主细胞基因剪接机制来指导MIE基因合成，包括：调节CMV早期基因剪接的机制；MIE基因剪接对巨细胞病毒体外和体内复制的影响；核结构域（ND10和SC）及其相关蛋白在巨细胞病毒基因调控和病毒复制中的新作用，以及它们如何影响巨细胞病毒诱导的神经发育发病机制。我们的中心假设是，在感染早期，巨细胞病毒抵消宿主细胞防御，包括ND10及其相关蛋白，篡位宿主细胞剪接调节因子PTB， U2AF和SC35，并通过与特定的顺式元件相互作用激活早期基因。具体目的是：1)鉴定MIE基因中调控MIE基因剪接的主要顺式元件，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结构域在HCMV基因剪接调控中的生物学重要性。3)确定MIE基因的组织特异性选择性剪接对体内CMV器官选择性致病性的影响，以及IE1/IE3对小鼠体外和体内胚胎神经发生的致病影响。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，这些机制对巨细胞病毒基因表达和宿主感染至关重要。积极的影响将包括对分子病毒学和巨细胞病毒病理生物学的定性理解，以及对预防和治疗干预的新候选靶点的确定。了解CMV用于调节MIE基因剪接的机制可能因此推动新的、选择性抗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.