Role of Coronavirus Membrane Protein Carboxy Tail Virus Assembly

描述（由申请方提供）：冠状病毒是一组具有医学意义的包膜、正链RNA病毒，主要引起人类和许多动物的呼吸道和肠道感染。该提案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。病毒体包膜含有至少两种主要的结构蛋白，即膜蛋白（M）和刺突蛋白（S）。此外，一些包膜（E）蛋白分子也存在于包膜中。核衣壳（N）磷蛋白包封包装在包膜内的基因组RNA。N蛋白与基因组RNA结合形成螺旋状核衣壳。病毒体包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关键作用。M蛋白含有病毒体外的短N-末端结构域、三个跨膜结构域和病毒体内的长C-末端结构域。C-末端含有长的两亲性结构域和高度带电的亲水性尾部。两亲性结构域已经被暗示在各种病毒蛋白质-蛋白质相互作用中发挥作用，包括M-S和M-E缔合。此外，M的末端C-末端~ 25个氨基酸已被证明在通过与核衣壳相互作用的组装中具有重要的功能。参与介导这些蛋白质-蛋白质相互作用的特定残基和要求尚未完全定义和理解。拟议研究的具体目标是确定在冠状病毒组装中重要的M蛋白胞质尾内保守结构域和带电残基的作用。为了实现这一目标，将产生一组突变体病毒，其在带电残基和M尾内的保守结构域中具有各种变化。将在全长小鼠肝炎病毒（MHV-CoV）-A59感染性克隆的背景下分析所有突变体。与野生型病毒相比，将研究突变体的空斑形态和生长动力学。生化分析，共免疫沉淀（co-IP）和共聚焦显微镜将用于阐明M在组装中的机械作用，在特定的残基和结构要求。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（包括最近出现的严重急性呼吸道综合征冠状病毒（SARS-CoV））的抗病毒开发将为医疗保健治疗方案提供重大改进。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.