Mechanisms of Human Metapneumovirus Replication

人类偏肺病毒复制机制

• 批准号: 6718737
• 负责人: TIMOTHY R PETERS
• 金额: $ 11.86万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2006-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6718737
• 关键词: Paramyxovirus; SDS polyacrylamide gel electrophoresis; Vesiculovirus; biological signal transduction; cell membrane; chimeric proteins; confocal scanning microscopy; electron microscopy; glycoproteins; green fluorescent proteins; polymerase chain reaction; protein binding; virion; virus assembly; virus infection mechanism; virus replication; yeast two hybrid system

DESCRIPTION (provided by applicant): The objective of the proposed research is to determine mechanisms by which human metapneumovirus (hMPV) virions are assembled at the host cell plasma membrane. The paramyxovirus hMPV is an emerging human pathogen that is a major cause of lower respiratory tract infection in children. Genome organization and sequence confirms its close relationship to respiratory syncytial virus (RSV). The amino acid sequence of the hMPV fusion (F) protein shares significant homology with RSV F. The paramyxovirus F proteins are type I transmembrane glycoproteins that mediate viral fusion to cell membranes. Using GFP fusion proteins encoding RSV and hMPV F transmembrane and cytoplasmic domains, F regions have been identified that are sufficient for membrane localization in cells that support infection. The central hypothesis of this research is that hMPV assembly at the plasma membrane is a highly regulated process mediated by F protein sequences contained in the transmembrane and cytoplasmic domains. Three specific aims are proposed to define the role of F domains in hMPV assembly. In Specific Aim 1 the hypothesis that specific C-terminal regions within hMPV F determine its membrane localization will be tested. Fluorescence imaging techniques will be used to evaluate membrane localization of GFP-hMPV F fusion proteins and native hMPV F in transfected and infected cells. In Specific Aim 2 the minimal determinants of hMPV F membrane localization will be identified using truncation and amino acid substitution mutants. Vesicular stomatitis virus G-hMPV F chimeric proteins will be used to characterize trafficking signals within F. In Specific Aim 3 the role of hMPV F cytoplasmic tail domain in the coordination of viral assembly will be determined by identifying hMPV protein binding partners, using yeast two-hybrid and co-immunoprecipitation analysis. These experiments will contribute important information about the role of the F protein in regulated cellular protein trafficking and viral assembly. This work will be conducted in the context of a scientific environment and training program designed to establish Dr. Peters as an independent investigator focused on hMPV cellular virology.

描述（由申请人提供）：拟议的研究的目的是确定在宿主细胞质膜上组装人类元小病毒（HMPV）病毒体的机制。 Paramyxovirus HMPV是一种新兴的人类病原体，是儿童下呼吸道感染的主要原因。基因组组织和序列证实了其与呼吸综合病毒（RSV）的密切关系。 HMPV融合（F）蛋白的氨基酸序列与RSV F具有显着同源性。丙糖病毒F蛋白是I型跨膜糖蛋白，可介导病毒融合到细胞膜。使用编码RSV和HMPV F跨膜和细胞质结构域的GFP融合蛋白，已经鉴定出F区域足以在支持感染的细胞中膜定位。这项研究的中心假设是，质膜处的HMPV组件是由F蛋白序列介导的高度调节过程，该过程包含在跨膜和细胞质结构域中的F蛋白序列。提出了三个特定的目的来定义F域在HMPV组装中的作用。在特定目的1中，HMPV F内的特定C末端区域确定其膜定位将进行测试。荧光成像技术将用于评估转染和感染细胞中GFP-HMPV F融合蛋白和天然HMPV F的膜定位。在特定目标2中，将使用截短和氨基酸取代突变体确定HMPV F膜定位的最小决定因素。囊泡口腔炎病毒G-HMPV F奇物蛋白将用于表征F内的运输信号。在特定的目标3中，HMPV F细胞质尾域在病毒装配协调中的作用将通过使用HMPV蛋白质结合仪来确定，使用YEAST HMPV蛋白质结合仪，使用YEAST HMPV蛋白质结合构件来确定。这些实验将有助于有关F蛋白在调节细胞蛋白运输和病毒组装中的作用的重要信息。这项工作将在科学环境和培训计划的背景下进行，旨在将彼得斯博士建立为专注于HMPV细胞病毒学的独立研究者。