In vitro and in vivo studies of Cytomegalovirus MIE gene regulation

描述（由申请方提供）：人巨细胞病毒（HCMV）感染在免疫功能低下的宿主中引起危及生命的疾病，并在妊娠期间感染的女性中对发育中的胎儿造成严重的神经损伤。由HCMV主要立即早期（MIE）基因编码的蛋白质对于生产性CMV感染和复制是必不可少的，但CMV如何征用宿主细胞机器来指导MIE蛋白质合成尚不清楚。我们的初步研究结果表明，MIE基因的表达调控剪接水平，导致器官特异性MIE基因的表达，蛋白质合成和CMV感染体内，和新的相互作用的CMV与宿主细胞剪接装置。这种为期四年的方法与以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;核结构域的新作用（ND 10和SC）及其相关蛋白在CMV基因调控和病毒复制中的作用，以及这些蛋白如何影响CMV诱导的神经发育发病机制。我们的中心假设是，在早期感染，CMV抵消宿主细胞防御，包括ND 10及其相关蛋白，篡夺宿主细胞剪接调节PTB，U2 AF和SC 35，并激活早期基因通过与特定的顺式元件相互作用。具体目标是：1）鉴定调节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 IE 1/IE 2和细胞基因剪接调节因子之间的相互作用以及核结构域在调节HCMV基因剪接中的生物学重要性。3)确定MIE基因的组织特异性选择性剪接对体内器官选择性CMV致病性的影响，以及IE 1/IE 3对小鼠体外和体内胚胎神经发生的致病性影响。这些研究将引入新的概念和发现，推进分子病毒学的基本理解，并将结合联合收割机创新的体外和体内模型和新的工具来阐明MIE基因剪接调控的关键机制，这对CMV基因表达和生产性宿主感染至关重要。积极的影响将包括在分子病毒学和CMV病理学的理解方面取得质的进步，特别是还将确定预防和治疗干预的新候选目标。因此，了解CMV用于调节MIE基因剪接的机制可能会推动新的选择性抗CMV治疗策略的开发，因为MIE基因产物IE 1和IE 2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.