In vitro and in vivo studies of Cytomegalovirus MIE gene regulation

巨细胞病毒 MIE 基因调控的体外和体内研究

• 批准号: 8824873
• 负责人: Qiyi Tang
• 金额: $ 33.22万
• 依托单位: HOWARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824873
• 关键词: AIDS/HIV problem; Achievement; Alternative Splicing; Area; Binding; Biological; Biological Assay; Cells; Co-Immunoprecipitations; Congenital Disorders; Cytomegalovirus; Cytomegalovirus Infections; DNA biosynthesis; Data; Development; Disease; Elements; Embryo; Exons; Fetus; Gene Expression; Gene Expression Regulation; Gene Order; Genes; Genetic Transcription; Goals; Health; Homologous Gene; Hybridization Array; Immediate-Early Genes; Immediate-Early Proteins; Immunocompromised Host; Immunofluorescence Immunologic; Impairment; In Situ Hybridization; In Vitro; Infection; Life; Molecular; Molecular Virology; Murid herpesvirus 1; Mus; Mutate; Neurodevelopmental Deficit; Neurologic; Newborn Infant; Nuclear; Nuclear Proteins; Organ; Organ Transplantation; Pathogenesis; Pathogenicity; Pattern; Positioning Attribute; Pregnancy; Preventive Intervention; Production; Protein Biosynthesis; Proteins; Publications; Publishing; RNA; RNA Splicing; Regulation; Relative (related person); Reporter; Research; Role; Spliced Genes; Staging; System; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transgenic Mice; Transgenic Model; Transgenic Organisms; Transplant Recipients; Viral; Viral Genes; Virus; Virus Diseases; Woman; Work; base; career; differential expression; fetal; gene synthesis; in vivo; in vivo Model; inhibitor/antagonist; innovation; mouse model; mutant; neurogenesis; novel; novel strategies; spatial relationship; tool; viral RNA

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.

描述（由申请人提供）：人类巨细胞病毒（HCMV）感染会导致免疫功能低下的宿主威胁生命的疾病，以及对怀孕期间感染的妇女发育的胎儿的严重神经损害。由HCMV主要IMMediate（MIE）基因编码的蛋白质对于生产性CMV感染和复制至关重要，但是CMV指挥官如何宿主细胞机械指导MIE蛋白质合成是未知的。我们的初步发现表明，MIE基因表达受到剪接水平的调节，从而导致体内器官特异性MIE基因表达，蛋白质合成和CMV感染，以及CMV与宿主细胞剪接设备的新型相互作用。这项提出的四年方法与以前的MIE基因调节研究根本不同，该研究的针对病毒基因转录，预计将与已知的信息高度互补。实际上，我们的长期目标是阐明CMV如何篡夺宿主细胞基因切割机制来指导MIE基因合成，包括：调节CMV剪接非常早期基因的机制； MIE基因剪接对体外和体内CMV复制的影响；核结构域（ND10和SC）及其相关蛋白在CMV基因调节和病毒复制中的新作用，以及这些如何影响CMV诱导的神经发育发病机理。我们的中心假设是，在早期感染期间，CMV抵消了包括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节剂PTB，U2AF和SC35，并通过与特定的CIS元素相互作用来激活早期基因。具体目的是：1）确定调节MIE基因剪接的MIE基因的主要顺式元素，并确定剪接因子对HCMV复制的重要性。 2）确定HCMV IE1/IE2与细胞基因剪接调节剂与核结构域之间相互作用的生物学重要性，并在调节HCMV基因剪接中的生物学重要性。 3）确定MIE基因在体内的组织选择性CMV致病性以及IE1/IE3的致病性影响对小鼠体外和体内胚胎神经发生的影响。这些研究将介绍新的概念和发现，以提高对分子病毒学的基本理解，并将在体外和体内模型和新型工具结合使用，以阐明MIE基因剪接调节的关键机制，这对CMV基因表达至关重要。积极的影响将包括在理解分子病毒学和CMV病理生物学方面的定性进步，还包括对预防和治疗干预的新候选目标的鉴定。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治疗策略的发展，因为MIE基因产物IE1和IE2对于CMV的早期/晚期基因表达和HCMV复制至关重要。