Mechanisms of Human Metapneumovirus Replication

人类偏肺病毒复制机制

• 批准号: 6874526
• 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/6874526
• 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）：拟议研究的目的是确定人偏肺病毒（hMPV）病毒粒子在宿主细胞质膜上组装的机制。副粘病毒 hMPV 是一种新出现的人类病原体，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道合胞病毒（RSV）的密切关系。 hMPV 融合 (F) 蛋白的氨基酸序列与 RSV F 具有显着的同源性。副粘病毒 F 蛋白是介导病毒与细胞膜融合的 I 型跨膜糖蛋白。使用编码 RSV 和 hMPV F 跨膜和细胞质结构域的 GFP 融合蛋白，已鉴定出 F 区足以在支持感染的细胞中进行膜定位。这项研究的中心假设是 hMPV 在质膜上的组装是一个由跨膜和细胞质结构域中包含的 F 蛋白序列介导的高度调控的过程。提出了三个具体目标来定义 F 结构域在 hMPV 组装中的作用。在具体目标 1 中，将测试 hMPV F 内的特定 C 末端区域决定其膜定位的假设。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。在具体目标 2 中，将使用截短和氨基酸取代突变体来鉴定 hMPV F 膜定位的最小决定因素。水疱性口炎病毒 G-hMPV F 嵌合蛋白将用于表征 F 内的运输信号。在特定目标 3 中，hMPV F 胞质尾结构域在病毒组装协调中的作用将通过使用酵母二鉴定 hMPV 蛋白结合伴侣来确定。 -混合和免疫共沉淀分析。这些实验将提供有关 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，旨在使 Peters 博士成为专注于 hMPV 细胞病毒学的独立研究者。