Molecular Analysis of Coronavirus Assembly

冠状病毒组装的分子分析

• 批准号: 6679931
• 负责人: BRENDA G. HOGUE
• 金额: $ 19万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-15 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6679931
• 关键词: Coronaviridae; immunofluorescence technique; laboratory rabbit; membrane proteins; molecular cloning; murine hepatitis virus; protein localization; protein protein interaction; protein structure function; replicon; transfection /expression vector; virion; virus assembly; virus envelope; virus protein; viruslike particle

DESCRIPTION (provided by applicant): Coronaviruses (CVs) are widespread, medically important respiratory and enteric pathogens of humans and many domestic animals, causing a significant portion of human upper respiratory infections. These enveloped viruses contain a positive(+)-sense, single-stranded RNA genome that is the largest (approximately 30 kb) of all the RNA viruses. Many questions remain to be answered about the molecular details of assembly of enveloped RNA viruses. The studies proposed herein focus on the mechanism of assembly of mouse hepatitis virus (MHV) that acquire their envelopes by a nucleocapsid independent manner at membranes of the endoplasmic reticulum Golgi intermediate compartment (ERGIC). Virus-like-particles (VLPs) assemble when only the small envelope (E) and membrane (M) proteins are coexpressed. We hypothesize that E performs its role through its interplay with M and the ERGIC lipid membranes and possibly host proteins. Previous studies addressing the role of E and M in virus assembly relied on virus-infected cells, VLPs, and targeted RNA recombination. With the availability of a new MHV infectious clone we are uniquely positioned to directly manipulate the virus genome to study the mechanism by which E and M function in virion assembly. In Aim 1 we will focus on understanding the role of the E and M proteins in targeting assembly to intracellular membranes. We will precisely identify the site of intracellular localization for E. The retention signal in E and domains involved in potential co-localization with M will be identified. The role of E, M and host proteins will be studied to determine their roles in assembly/budding at intracellular membranes. Aims 2 and 3 are to understand the roles of the E and M proteins in virion formation using VLPs, the MHV infectious clone and replicons. We will use biochemical and microscopic analyses of chimerics and site-specific mutants of E and M expressed from various vectors and the MHV infectious clone to study the proteins, subcellular fractions and assembly of wild-type and altered VLPs and viruses. Information gained from this study can be used to (i) increase our understanding of fundamental mechanisms by which viruses of medical importance acquire their envelopes at intracellular membranes, (ii) identify major targets for antiviral drug development, (iii) assist in development of CV heterologous gene expression vectors for vaccine use, and (iv) contribute to understanding of protein-protein and protein-membrane interactions and transport.

描述（由申请人提供）：冠状病毒（CVs）是广泛存在的，医学上重要的人类和许多家畜呼吸道和肠道病原体，导致人类上呼吸道感染的很大一部分。这些包膜病毒含有一个正（+）感的单链RNA基因组，是所有RNA病毒中最大的（约30 kb）。包膜RNA病毒组装的分子细节仍有许多问题有待解答。本文的研究重点是小鼠肝炎病毒（MHV）在内质网高尔基中间室（ERGIC）膜上以核衣壳独立的方式获得包膜的组装机制。当只有小包膜蛋白(E)和膜蛋白(M)共表达时，病毒样颗粒（vlp）组装。我们假设E通过与M和ERGIC脂膜以及可能的宿主蛋白的相互作用来发挥其作用。先前研究E和M在病毒组装中的作用依赖于病毒感染的细胞、VLPs和靶向RNA重组。随着一个新的MHV感染性克隆的可用性，我们处于独特的位置，可以直接操纵病毒基因组来研究E和M在病毒粒子组装中的作用机制。在目标1中，我们将重点了解E和M蛋白在靶向细胞膜组装中的作用。我们将精确地确定E的细胞内定位位点。E中的保留信号和与M潜在共定位的结构域将被确定。E， M和宿主蛋白的作用将被研究，以确定它们在细胞膜组装/出芽中的作用。目的2和3是了解E和M蛋白在利用vlp、MHV感染克隆和复制子形成病毒粒子中的作用。我们将使用生化和显微分析的嵌合和位点特异性突变的E和M表达从各种载体和MHV感染克隆，以研究蛋白质，亚细胞组分和组装野生型和改变的VLPs和病毒。从这项研究中获得的信息可用于(i)增加我们对具有医学意义的病毒在细胞膜上获得包膜的基本机制的理解，（ii）确定抗病毒药物开发的主要靶点，（iii）协助开发用于疫苗的CV异种基因表达载体，以及（iv）有助于理解蛋白质-蛋白质和蛋白质-膜相互作用和运输。