Paramyxovirus Assembly

副粘病毒组装

• 批准号: 8225168
• 负责人: TORU TAKIMOTO
• 金额: $ 38.63万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-02-15 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8225168
• 关键词: A549; Actins; Antiviral Agents; Apical; Attenuated Vaccines; Biochemical; Biological Assay; Cell membrane; Cells; Child; Confocal Microscopy; Coupled; Cytoplasm; Data; Dominant-Negative Mutation; Electrons; Endosomes; Epithelial; Epithelial Cells; Exhibits; Gene Proteins; Genes; Genetic Transcription; Genome; Grant; Growth; Human; Human Virus; Infant; Knowledge; Life; Light; Lower respiratory tract structure; Lung; Mediating; Methods; Microscopic; Microtubules; Molecular; Monitor; Motor; Movement; Nucleocapsid; Para-Influenza Virus Type 1; Parainfluenza; Paramyxovirus; Pathogenicity; Pathway interactions; Pharmaceutical Preparations; Process; Proteins; Public Health; RNA Viruses; Reagent; Recombinants; Recycling; Research; Respiratory syncytial virus; Role; Sendai virus; Site; Specificity; Surface; System; Tertiary Protein Structure; Testing; Therapeutic; Time; Vaccines; Vesicle; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus Diseases; Virus Replication; base; cellular imaging; citrate carrier; design; enhanced green fluorescent protein; genetic regulatory protein; membrane assembly; mutant; novel vaccines; polarized cell; positional cloning; public health relevance; rab GTP-Binding Proteins; recombinant virus; research study; respiratory; respiratory infection virus; respiratory virus; small hairpin RNA; trafficking; transmission process; virus host interaction

DESCRIPTION (provided by applicant): Respiratory virus infection caused by enveloped RNA viruses, such as parainfluenza and respiratory syncytial viruses, is a major public health concern. These viruses infect and replicate in the upper and/or lower respiratory tracts and transmit through airways. These respiratory viruses bud from the apical surface of polarized epithelial cells. Obviously, efficient virus assembly in respiratory epithelial cells is a key factor for virus transmission and pathogenicity. However, the molecular mechanism of virus assembly, especially how viral structural components are transported to the plasma membrane assembly/budding sites is poorly understood, partially due to lack of a method to monitor trafficking of viral components in live infected cells. In this proposal, we will use Sendai virus (SeV), one of the best-characterized paramyxoviruses, to reveal how viral nucleocapsids (vRNPs) are transported to assembly sites. Using the reverse genetics system, we successfully rescued a recombinant SeV (rSeV-LeGFP) whose L protein is tagged with enhanced green fluorescent protein (eGFP). This virus allows us to monitor real time trafficking of the vRNP in live infected cells. Using the virus, we successfully recorded vRNP movement through microtubules in infected cells. Additional data strongly suggest that the intracellular vesicular trafficking pathway regulated by Rab11a is involved in vRNP translocation. Taking full advantage of rSeV-LeGFP and our ability to rescue various SeV mutants, we propose to unveil the cellular and viral factors involved in the process of vRNP trafficking and virus assembly in respiratory epithelial cells. We will identify the cellular machinery and proteins responsible for vRNP transport, as well as the viral proteins that mediate the process. In this project, we will uncover the process of assembly of SeV and related human respiratory paramyxoviruses, especially the role of the intracellular vesicular trafficking pathway in vRNP transport and assembly of virions at the apical surface budding sites of epithelial cells. This research will shed light on the unknown critical process of respiratory virus assembly, which is expected to provide valuable data to develop safe attenuated vaccines or antiviral agents. PUBLIC HEALTH RELEVANCE: Although parainfluenza and respiratory syncytial viruses are the major causes of respiratory viral disease in infants and young children, no vaccines or therapeutic drugs are available. Studies proposed in this grant will unveil the virus-host interactions required for virus assembly and formation, which will provide important implications for the design of novel vaccines and antiviral reagents.

描述（由申请方提供）：由包膜RNA病毒（如副流感病毒和呼吸道合胞病毒）引起的呼吸道病毒感染是一个主要的公共卫生问题。这些病毒在上呼吸道和/或下呼吸道中感染和复制，并通过气道传播。这些呼吸道病毒从极化上皮细胞的顶端表面出芽。显然，病毒在呼吸道上皮细胞中的高效组装是病毒传播和致病的关键因素。然而，病毒组装的分子机制，特别是病毒结构组分如何被运输到质膜组装/出芽位点的了解甚少，部分原因是缺乏一种方法来监测活感染细胞中病毒组分的运输。在这项提案中，我们将使用仙台病毒（SeV），最好的特点之一，副粘病毒，揭示病毒核衣壳（vRNP）是如何被运送到装配网站。利用反向遗传学系统，我们成功地拯救了一株重组SeV（rSeV-LeGFP），其L蛋白被增强型绿色荧光蛋白（eGFP）标记。这种病毒使我们能够监测活的感染细胞中vRNP的真实的运输。使用该病毒，我们成功地记录了vRNP通过感染细胞中微管的运动。额外的数据强烈表明，细胞内的囊泡运输途径的Rab 11 a的调节参与vRNP易位。充分利用rSeV-LeGFP和我们拯救各种SeV突变体的能力，我们建议揭示呼吸道上皮细胞中vRNP运输和病毒组装过程中涉及的细胞和病毒因子。我们将确定负责vRNP运输的细胞机制和蛋白质，以及介导该过程的病毒蛋白质。在这个项目中，我们将揭示SeV和相关的人呼吸道副粘病毒的组装过程，特别是细胞内囊泡运输途径在vRNP运输和病毒体在上皮细胞顶面出芽位点组装中的作用。这项研究将揭示呼吸道病毒组装的未知关键过程，有望为开发安全的减毒疫苗或抗病毒药物提供有价值的数据。 公共卫生关系：虽然副流感病毒和呼吸道合胞病毒是婴幼儿呼吸道病毒性疾病的主要病因，但目前尚无疫苗或治疗药物。该资助计划的研究将揭示病毒组装和形成所需的病毒-宿主相互作用，这将为新型疫苗和抗病毒试剂的设计提供重要意义。