Host factors in the alphavirus replication complex

甲病毒复制复合物中的宿主因子

基本信息

批准号：
7185849
负责人：
MARGARET R MACDONALD
金额：
$ 32.05万
依托单位：
ROCKEFELLER UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-02-01 至 2010-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7185849
关键词：
Alphavirus Antibodies Antibody Affinity Arthritis Binding Binding Proteins Biochemical Biological Assay Bioterrorism Categories Cells Centers for Disease Control and Prevention (U.S.)Cessation of life Complex Cytolysis Cytoplasm Development Disease Encephalitis Encephalitis Viruses Endoribonucleases Environment Equus caballus Exanthema Family Family member Fever Fluorescence Microscopy Future Genome Genomics Goals Green Fluorescent Proteins Human Immunologic Techniques Infection Integration Host Factors Intervention Mammals Maps Membrane Modeling Molecular Molecular Genetics National Institute of Allergy and Infectious Disease Polyproteins Process Production Protein Overexpression Proteins Proteolytic Processing Proteomics RNA RNA Interference RNA Viruses RNA chemical synthesis RNA-Protein Interaction Ras Signaling Pathway Replicon Research Role Ross river virus SH3 Domains Sindbis Virus Switching Complex Techniques Testing Therapeutic Agents Togaviridae Venezuelan Equine Encephalomyelitis Viral Viral Nonstructural Proteins Viral Structural Proteins Virus Virus Replication domain mapping endoribonuclease genome sequencing inhibitor/antagonist knock-down magnetic beads member mutant pathogen ras GTPase-Activating Proteins replicase research study therapeutic target viral RNA

项目摘要

DESCRIPTION (provided by applicant): Infection of humans and other mammals by members of the Alphavirus genus within the Togaviridae can result in fever, rash, arthritis, encephalitis and death. Several alphaviruses have been designated by the Centers for Disease Control and Prevention as Category B agents of concern for possible use in bioterrorism, and as Priority Pathogens by the NIAID. There is currently no specific treatment available for diseases caused by these pathogens. We are studying Alphavirus replication with the long-term goal of identifying potential therapeutic targets. Replication of incoming positive-sense genomic RNA, which occurs in membrane-associated complexes containing the viral nonstructural proteins (nsPs), first generates a minus strand copy, followed by new progeny plus strand RNAs. We hypothesize that in addition to known differences in the viral nsP components of the minus and plus strand replicases, that recruitment of different host factors facilitates their disparate functions. We also hypothesize that alphaviruses utilize a common strategy of host factor recruitment to replicate their respective genomes. Using mutants expressing a tagged nsP3 protein, we propose to use immunological techniques to isolate the plus and minus strand replicases from three representative alphaviruses, Sindbis virus, Ross River virus, and Venezuelan equine encephalitis virus. The host factors present in the replication complexes will be identified by proteomic techniques. Focusing on factors commonly present in the Alphavirus plus and/or minus strand replicase complexes, we will verify each factor's association with the replicase in intact cells using confocal fluorescence microscopy. Using molecular and biochemical approaches, we will assess the effects of associated host components on viral replication and map the protein-protein (or protein-RNA) interactions of the host factors. Understanding the components of and molecular interactions within the Alphavirus minus and plus strand replicases will facilitate future exploration for inhibitors of these important pathogens.

描述（由申请人提供）：togaviridae内α病毒属成员对人类和其他哺乳动物的感染可能导致发烧，皮疹，关节炎，脑炎和死亡。疾病控制和预防中心已将几种α病毒指定为B类关注生物恐怖主义的关注者，而NIAID的优先病原体被指定为使用。目前尚无针对这些病原体引起的疾病的特定治疗方法。我们正在研究α病毒复制，其长期目标是确定潜在的治疗靶标。传入的阳性基因组RNA的复制，发生在包含病毒非结构蛋白（NSP）的膜相关复合物中，首先生成负链拷贝，然后再产生新的后代加上链链RNA。我们假设，除了负和链复制酶的病毒NSP成分的已知差异外，募集不同宿主因素还有助于其不同的功能。我们还假设α病毒利用宿主因子募集的共同策略来复制其各自的基因组。使用表达标记为NSP3蛋白的突变体，我们建议使用免疫学技术将来自三种代表性α的加分和负链复制酶分离出来，Sindbis病毒，Ross River病毒和委内瑞拉人马肺炎病毒。复制络合物中存在的宿主因子将通过蛋白质组学技术识别。侧重于α病毒Plus和/或减去链复制酶复合物中常见的因素，我们将使用共焦荧光显微镜验证每个因素与完整细胞中复制酶的关联。使用分子和生化方法，我们将评估相关宿主成分对病毒复制的影响，并绘制宿主因子的蛋白质蛋白质（或蛋白-RNA）相互作用。了解α病毒减去和加上链复制中的成分和分子相互作用的成分和分子相互作用，将促进对这些重要病原体的抑制剂的未来探索。