HCMV Glycoprotein B-Induced A Signal Pathway

HCMV 糖蛋白 B 诱导的 A 信号通路

• 批准号: 6547769
• 负责人: HUA ZHU
• 金额: $ 13.61万
• 依托单位: UNIV OF MED/DENT OF NJ-NJ MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2007-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6547769
• 关键词: DNA binding protein; biological signal transduction; cytomegalovirus; gene induction /repression; genetic library; host organism interaction; interferons; regulatory gene; virus protein

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) is a ubiquitous human pathogen. It is a major cause of infectious morbidity and mortality in immunocompromised individuals, especially in transplant recipients and AIDS patients. It is also a leading cause of congenital birth defects. My long-term research goal is to elucidate the interactions of HCMV with Its host cell at the molecular level, and evaluate the consequences of these interactions, especially the effects on viral replication and pathogenesis. In this proposal, I will focus on the significance of the induction of interferon-stimulated genes (ISGs) following HCMV infection. My specific aims are as follows: I. Identify HCMV-response elements (HCRE)-binding proteins. Two elements, the interferon stimulated element (ISRE) and gamma-interferon activated site (GAS), have been identified responsible for HCMV-mediated ISG induction. Gel shift assays have shown that four protein complexes interact with these elements. Identification of the consensus sequence of HCRE and HCRE-binding proteins are critical for understanding molecular basis of this signal transduction pathway. I propose different experiments to approach these questions, including: (1) Test base-pair substitutions to identify the sequence motifs necessary for HCMV-mediated ISG induction. (2) Examine known HCRE-binding proteins in the interferon regulatory factor family for their roles in HCMV-mediated signal transduction pathway. (3) Purify HCRE-binding proteins by chromatography and identify these binding proteins by 2-dimensional electrophoresis and mass spectrometric analysis. (4) Screen HCRE-binding proteins using a yeast one-hybrid system. (5) Screen HCRE-binding proteins using retrovirus cDNA expression libraries. II. To investigate biological significance of the induction of ISGs by HCMV infection. HCMV envelope glycoprotein B (gB) has been identified for initiating this signal transduction pathway. Importance of initiating this signal pathway in HCMV replication will be studied using biochemical and genetic approaches, including: (1) The roles of HCMV activated proteins in the induction of ISGs and HCMV IE genes will be studied. (2) Mutate two GAS elements in the HCMV major IE promoter and examine their roles in viral gene expression and replication.

描述（由申请方提供）：人巨细胞病毒（HCMV）是一种普遍存在的人类病原体。它是免疫功能低下个体，特别是移植受者和AIDS患者感染性发病率和死亡率的主要原因。它也是先天性出生缺陷的主要原因。本研究的长期目标是从分子水平阐明HCMV与宿主细胞的相互作用，并评估这些相互作用的后果，特别是对病毒复制和致病的影响。在这个建议中，我将集中在HCMV感染后的干扰素刺激基因（ISGs）的诱导的意义。我的具体目标如下： I. 识别HCMV反应元件（HCRE）结合蛋白。两个元件，干扰素刺激元件（ISRE）和γ-干扰素激活位点（GAS），已被确定负责HCMV介导的ISG诱导。凝胶迁移分析表明，四个蛋白质复合物与这些元素相互作用。鉴定HCRE及其结合蛋白的共有序列对于理解该信号转导途径的分子基础至关重要。我提出了不同的实验来解决这些问题，包括：（1）测试碱基对替换，以确定HCMV介导的ISG诱导所必需的序列基序。(2)检查干扰素调节因子家族中已知的HRE结合蛋白在HCMV介导的信号转导途径中的作用。(3)通过色谱法纯化HRE结合蛋白，并通过二维电泳和质谱分析鉴定这些结合蛋白。(4)利用酵母单杂交系统筛选HRE结合蛋白。(5)利用逆转录病毒cDNA表达文库筛选HRE结合蛋白。 二. 探讨HCMV感染诱导ISG的生物学意义。HCMV包膜糖蛋白B（gB）已被鉴定为启动该信号转导途径。本论文将从生物化学和遗传学的角度研究启动这一信号通路在HCMV复制中的重要性，包括：（1）研究HCMV激活蛋白在诱导ISG和HCMV IE基因中的作用。(2)突变HCMV主要IE启动子中的两个GAS元件，并检查它们在病毒基因表达和复制中的作用。