Molecular Analysis of Coronavirus Assembly

描述（由申请方提供）：冠状病毒（CV）是人类和许多家畜的广泛分布的、医学上重要的呼吸道和肠道病原体，引起很大一部分人类上呼吸道感染。这些包膜病毒含有正（+）义单链RNA基因组，其是所有RNA病毒中最大的（约30 kb）。关于有包膜RNA病毒组装的分子细节还有许多问题有待回答。本文提出的研究重点是小鼠肝炎病毒（MHV）的组装机制，获得其包膜的核衣壳的非依赖性方式在内质网高尔基中间室（ERGIC）的膜。病毒样颗粒（VLP）组装时，只有小包膜（E）和膜（M）蛋白共表达。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。以前的研究解决E和M在病毒组装中的作用依赖于病毒感染的细胞，VLP和靶向RNA重组。随着一个新的MHV感染性克隆的可用性，我们处于独特的地位，直接操纵病毒基因组研究的机制，其中E和M功能的病毒粒子组装。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。我们将精确地确定E.将鉴定E中的保留信号和参与与M的潜在共定位的结构域。将研究E、M和宿主蛋白的作用，以确定它们在细胞内膜组装/出芽中的作用。目的2和3是理解E和M蛋白在使用VLP、MHV感染性克隆和复制子的病毒体形成中的作用。我们将使用生物化学和显微镜分析的嵌合体和位点特异性突变体的E和M表达从各种载体和MHV感染性克隆，研究野生型和改变VLP和病毒的蛋白质，亚细胞组分和装配。从本研究中获得的信息可用于（i）增加我们对医学重要性病毒在细胞内膜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标，（iii）协助开发用于疫苗的CV异源基因表达载体，以及（iv）有助于理解蛋白质-蛋白质和蛋白质-膜相互作用和转运。

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.