The Cell Biology of Coronavirus Infection

描述(申请人提供)：冠状病毒已经证明了宿主-物种转换的能力，无论是在SARS等自然流行病中，还是在不同物种的细胞中，在传代、重组尖峰蛋白交换或基因组RNA电穿孔之后都是如此。像其他正链RNA病毒一样，冠状病毒在宿主细胞细胞质中结合修饰的细胞膜进行复制。冠状病毒引起细胞膜的深刻改变，诱导含有双膜小泡的网状泡状网络，这些小泡可能是病毒RNA合成的场所，也称为复制复合体。然而，病毒和细胞复制复合体的形成和功能的决定因素尚不清楚。此外，感染冠状病毒小鼠肝炎病毒(MHV)与持续的膜褶皱和内化有关，但膜褶皱在冠状病毒复制中的机制和作用尚不清楚。该研究计划的总体目标是阐明介导冠状病毒诱导的细胞膜修饰和复制复合体形成的保守而独特的病毒和细胞蛋白、膜和途径，并建立冠状病毒复制中膜修饰的要求。这项建议的三个综合目标是利用MHV和SARS-CoV在复制、细胞成像、蛋白质组学和生化实验中确定复制复合体的细胞和病毒成分，并建立膜褶皱在冠状病毒复制中的机制和作用。这些研究的结果将确定新的病毒-宿主相互作用，这可能对冠状病毒在多种细胞类型中建立复制和在物种之间移动的能力至关重要。此外，这些研究可能会为研究病毒宿主范围和干扰病毒复制确定新的病毒靶点。最后，这些实验可能阐明宿主细胞生物学以及与细胞内病原体相互作用的新途径。 与公共卫生相关：冠状病毒是一种RNA病毒，它在受感染宿主细胞的细胞质中复制，并诱导宿主细胞膜的修饰，作为病毒RNA合成的场所。该研究计划的目标是明确冠状病毒感染过程中冠状病毒复制酶蛋白靶向和病毒RNA合成的过程，识别细胞相互作用蛋白，并测试成功复制冠状病毒对细胞蛋白和途径的要求。这些研究的结果将确定冠状病毒复制的常见途径，确定病毒和细胞靶标，用于研究病毒的毒力、减毒和对冠状病毒感染的干扰。

DESCRIPTION (provided by applicant): Coronaviruses have demonstrated the capacity for host-species switching, both in natural epidemics, such as SARS, and in the laboratory in cells from different species following passage, recombinant swapping of the spike protein, or following electroporation of genome RNA. Like other plus-strand RNA viruses, coronaviruses replicate in the host cell cytoplasm in association with modified cellular membranes. Coronaviruses cause profound modifications of cytoplasmic membranes, inducing a reticulovesicular network containing double- membrane vesicles as putative sites of viral RNA synthesis, also known as replication complexes. However, the viral and cellular determinants of replication complex formation and function are not known. In addition, infection with the coronavirus mouse hepatitis virus (MHV) is associated with continuous membrane ruffling and internalization, but the mechanisms and role of membrane ruffling in coronavirus replication is not known. The overall goals of the proposed research program is to elucidate the conserved and unique viral and cellular proteins, membranes, and pathways that mediate coronavirus induced cell membrane modifications and replication complex formation and establish the requirements for the membrane modifications in coronavirus replication. The three integrated aims of this proposal will use MHV and SARS-CoV in replication, cell imaging, proteomic and biochemical experiments to define the cellular and viral components of replication complexes, and establish the mechanisms and role of membrane ruffling in coronavirus replication. The results of these studies will identify new virus-host interactions that may be critical in the ability of coronaviruses to establish replication in multiple cell types and move between species. In addition the studies will likely define new viral targets for studies of virus host range and interference with virus replication. Finally the experiments may elucidate new pathways in host cell biology and interaction with intracellular pathogens. PUBLIC HEALTH RELEVANCE: Coronaviruses are RNA viruses that replicate in the cytoplasm of the infected host cell and induce modifications to host cell membranes that serve as sites for viral RNA synthesis. The goal of the proposed research program is to define the process of coronavirus replicase protein targeting and viral RNA synthesis during coronavirus infection, identify cellular interacting proteins, and test the requirements for cellular proteins and pathways in successful coronavirus replication. The results of these studies will define common pathways of coronavirus replication, identify viral and cellular targets for studies of viral virulence, attenuation, and interference with coronavirus infection.