Role of Coronavirus Membrane Protein Carboxy Tail Virus Assembly

描述(申请人提供)：冠状病毒是一组具有重要医学意义的包膜、阳性缠绕的RNA病毒，主要在人类和许多动物中引起呼吸道和肠道感染。这项建议的总体重点是了解冠状病毒蛋白在病毒组装中的机制和功能。病毒粒子被膜至少含有两种主要结构蛋白，膜蛋白(M)和刺突蛋白(S)。此外，包膜(E)蛋白的几个分子也存在于包膜中。核衣壳(N)磷酸蛋白将包装在包膜内的基因组RNA包裹起来。N蛋白与基因组RNA结合形成螺旋核衣壳。病毒粒子被膜中最丰富的结构蛋白是M蛋白。它与自身、其他结构蛋白和核衣壳相互作用，因此在病毒组装中起着关键作用。M蛋白在病毒粒子外含有一个短的N-末端结构域，在病毒粒子内部含有三个跨膜区和一个长的C-末端结构域。C-末端含有一个长的两亲性结构域和一个高电荷的亲水性尾巴。两亲性结构域已被认为在病毒蛋白-蛋白相互作用中发挥作用，包括M-S和M-E结合。此外，M的极端C-末端-25个氨基酸已被证明通过与核衣壳相互作用在组装中具有重要的功能。参与调节这些蛋白质-蛋白质相互作用的特定残基和要求还没有完全定义和理解。这项研究的具体目的是确定在冠状病毒组装中重要的M蛋白细胞质尾部的保守结构域和带电残基的作用。为了实现这一目标，将产生一组突变病毒，其带电残基和M尾巴内的保守结构域发生各种变化。所有突变体都将在全长小鼠肝炎病毒(MHV-CoV)-A59感染性克隆的背景下进行分析。与野生型病毒相比，将研究突变型病毒的空斑形态和生长动力学。生化分析、免疫共沉淀(co-IP)和共聚焦显微镜将被用来从特定残基和结构要求的角度阐明M在组装中的机制作用。拟议的研究将提供适用于确定潜在抗病毒靶点的信息。针对冠状病毒的抗病毒发展，包括最近出现的严重急性呼吸综合征冠状病毒(SARS-CoV)，将显著改善卫生保健治疗方案。针对冠状病毒的抗病毒试剂和疫苗将有助于治疗和预防呼吸道和肠道感染，从而有助于全球改善人类健康。

DESCRIPTION (provided by applicant): Coronaviruses are a medically significant group of enveloped, positive stranded RNA viruses that cause primarily respiratory and enteric infections in humans and many animals. The overall focus of this proposal is to understand the mechanism and function of coronavirus proteins in virus assembly. The virion envelope contains at least two major structural proteins, the membrane (M) and spike (S) proteins. Additionally, a few molecules of the envelope (E) protein are also present in the envelope. The nucleocapsid (N) phosphoprotein encapsidates the genomic RNA packaged inside the envelope. The N protein binds the genomic RNA to form a helical nucleocapsid. The most abundant structural protein in the virion envelope is the M protein. It interacts with itself, other structural proteins and the nucleocapsid and therefore plays a key role in viral assembly. The M protein contains a short N-terminal domain outside the virion, three transmembrane domains and a long C-terminus domain inside the virion. The C-terminus contains a long amphipathic domain and a highly charged hydrophilic tail. The amphipathic domain has been implicated to play a role in various viral protein-protein interactions, including M-S and M-E associations. Additionally, the extreme C-terminal -25 amino acids of M have been shown to be functionally important in assembly through interactions with the nucleocapsid. The specific residues and requirements that are involved in mediating these protein-protein interactions are not fully defined and understood. The specific aims of the proposed research are to determine the role of a conserved domain and charged residues within the M protein cytoplasmic tail that are important in coronavirus assembly. To achieve this goal, a panel of mutant viruses will be generated with various changes in charged residues and the conserved domain within the M tail. All of the mutants will be analyzed in the context of the full-length mouse hepatitis virus (MHV-CoV)-A59 infectious clone. Mutants will be studied for plaque morphology and growth kinetics compared to wild-type virus. Biochemical analysis, coimmunoprecipitation (co-IP) and confocal microscopy will be used to elucidate the mechanistic roles of M in assembly, in terms of specific residue and structure requirements. The proposed study will provide information which is applicable to identifying potential antiviral targets. Antiviral development against coronaviruses, including the recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV), will provide a significant improvement in health care treatment options. Antiviral reagents and vaccines directed towards coronaviruses will aid in the treatment and prevention of respiratory and enteric infections, thus contributing to global improvement of human health.