Molecular Analysis of Coronavirus Assembly

描述(申请人提供)：冠状病毒(CV)是人类和许多家畜的广泛的、医学上重要的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。这些被包裹的病毒包含一个阳性(+)意义的单链RNA基因组，是所有RNA病毒中最大的(约30kb)。关于被包裹的RNA病毒组装的分子细节，还有许多问题有待回答。本文主要研究小鼠肝炎病毒(MHV)在内质网高尔基体中间隔膜(ERGIC)上以不依赖于核衣壳的方式获得包膜的组装机制。只有小包膜蛋白(E)和膜蛋白(M)共表达时，病毒样颗粒(VLP)才会聚集在一起。我们假设E通过与M和ERGIC类脂膜以及可能的宿主蛋白的相互作用来发挥作用。以前关于E和M在病毒组装中的作用的研究依赖于病毒感染细胞、VLP和靶向RNA重组。随着一种新的MHV感染性克隆的出现，我们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蛋白在靶向组装到细胞内膜中的作用。我们将准确地确定E的细胞内定位位置。E中的保留信号以及与M可能共同定位的结构域将被确定。将研究E、M和宿主蛋白的作用，以确定它们在细胞膜组装/出芽中的作用。目标2和3是了解E和M蛋白在使用VLP、MHV感染性克隆和复制子形成病毒粒子中的作用。我们将利用不同载体和MHV感染性克隆表达的E和M嵌合体和定点突变的生化和显微分析来研究野生型和变异型VLP和病毒的蛋白质、亚细胞组分和组装。从这项研究中获得的信息可以用来(I)加深我们对具有重要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)帮助开发用于疫苗的CV异源基因表达载体，以及(Iv)有助于了解蛋白质-蛋白质和蛋白质-膜的相互作用和运输。

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.