Paramyxovirus Assembly

描述(申请人提供)：由被包裹的RNA病毒引起的呼吸道病毒感染，如副流感和呼吸道合胞病毒，是一个主要的公共卫生问题。这些病毒在上呼吸道和/或下呼吸道感染和复制，并通过呼吸道传播。这些呼吸道病毒从极化的上皮细胞的顶端表面发芽。显然，病毒在呼吸道上皮细胞中的有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，特别是病毒结构成分是如何运输到质膜组装/萌发部位的，目前尚不清楚，部分原因是缺乏一种方法来监测病毒成分在活体感染细胞中的运输。在这项提案中，我们将使用仙台病毒(SeV)，一种特征最好的副粘病毒，来揭示病毒核衣壳(VRNP)是如何运输到组装部位的。利用反向遗传系统，我们成功地挽救了L蛋白带有增强型绿色荧光蛋白的重组SeV(rSeV-LeGFP)。这种病毒使我们能够监测vRNP在活体感染细胞中的实时运输。利用该病毒，我们成功地记录了vRNP在感染细胞中通过微管的移动。更多的数据有力地表明，Rab11a调控的细胞内囊泡转运途径参与了vRNP的转运。充分利用rSeV-LeGFP和我们拯救各种SeV突变体的能力，我们打算揭示vRNP在呼吸道上皮细胞运输和病毒组装过程中涉及的细胞和病毒因素。我们将确定负责vRNP运输的细胞机制和蛋白质，以及介导这一过程的病毒蛋白质。在这个项目中，我们将揭示SeV和相关的人类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运输和病毒粒子在上皮细胞顶端表面萌发位置组装中的作用。这项研究将阐明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。

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.