Mechanisms of Norovirus Entry

描述(由申请人提供)：肠道病毒感染在全球范围内导致严重的发病率和死亡率。其中，人类诺如病毒(HuNoV)研究严重不足，因为它们不在小动物或现成的细胞培养模型中生长，也没有针对性的疾病预防和控制策略。关于这些病毒如何进入细胞的基本问题的答案仍然未知。对于诺如病毒来说，病毒生命周期中受体结合和进入的初始事件是决定哪些细胞类型和组织可以被感染的关键，并最终决定感染的结果。然而，诺沃克病毒进入机制的知识在设计抗击疾病或传播的治疗或战略时至关重要。小鼠诺如病毒(MNV)与人新城疫病毒关系密切，具有许多生物学和分子特征。与HuNov不同的是，MNV可以进行反向工程，在组织培养中复制，并提供一个小型动物模型系统。因此，MNV模型首次提供了一个易于处理的系统来阐明诺如病毒进入的病毒和细胞决定因素。目前的提议将检验中心假设，即MNV-1衣壳P区的特定残基与宿主细胞表面的部分成分相互作用，这种相互作用导致病毒感染细胞。为了验证这一假设，我们设计了以下特定目标：1)确定唾液酸在MNV-1附着过程中的作用；2)确定MNV-1进入的病毒决定因素；3)确定MNV-1进入小鼠巨噬细胞和树突状细胞的细胞机制。用于解决这些目的的方法包括对病毒衣壳蛋白进行定向突变，然后进行体外和体内感染研究、荧光显微镜以及分子和细胞生物学方法。这些目标的成功完成可能会揭示抗病毒药物设计的新靶点。它将促进我们对诺沃克病毒的生命周期以及病毒如何劫持细胞器感染巨噬细胞和树突状细胞，特别是胃肠道的细胞的理解。因此，这些研究将推动病毒学、细胞生物学和免疫学领域的发展。公共卫生相关性：诺沃克病毒在人类中的感染导致“胃型流感”的频繁爆发，但目前还没有疫苗或治疗方法可供使用。对于诺如病毒来说，生命周期的第一步对于确定哪些细胞被感染至关重要。因此，我们的目标是提高我们对宿主和病毒在诺沃克病毒感染早期事件中的作用的理解，希望找到抗病毒药物开发的新靶点。

DESCRIPTION (provided by applicant): Enteric virus infections cause significant morbidity and mortality worldwide. Of these, human noroviruses (HuNoV) are severely understudied as they do not grow in small animals or readily available cell culture models and no directed disease prevention and control strategies exist. Answers to fundamental questions as to how these viruses enter cells remain unknown. For noroviruses, the initial events in the viral life cycle of receptor binding and entry are key in determining which cell types and tissues can be infected and ultimately the outcome of an infection. However, the knowledge of norovirus entry mechanisms is pivotal in designing therapies or strategies to combat disease or spread. Murine norovirus (MNV) is closely related to HuNoV and shares many biological and molecular characteristics. In contrast to HuNoV, MNV can be reverse engineered, replicates in tissue culture and provides a small animal model system. Therefore, the MNV model provides a tractable system to elucidate the viral and cellular determinants of norovirus entry for the first time. The current proposal will test the central hypothesis that specific residues in the P domain of the MNV-1 capsid interact with moieties on the host cell surface and this interaction leads to virus infection of cells. The following specific aims are designed to test this hypothesis: 1) Define the role of sialic acid during MNV-1 attachment; 2) Identify viral determinants of MNV-1 entry; 3) Define cellular mechanisms of MNV-1 entry into murine macrophages and dendritic cells. The methods used to address these aims include targeted mutagenesis of the viral capsid protein followed by infection studies in vitro and in vivo, fluorescence microscopy as well as molecular and cell biological approaches. Successful completion of these aims may reveal novel targets for antiviral drug design. It will advance our understanding of the norovirus life cycle and how viruses hijack the cellular machinery to infect macrophages and dendritic cells, particularly those of the GI tract. As such these studies will advance the fields of virology, cell biology and immunology. PUBLIC HEALTH RELEVANCE: Norovirus infections in humans cause frequent outbreaks of "stomach-flu", but no vaccines or treatments are available for use. For noroviruses, the first steps in the life cycle are critical in determining which cells are infected. Therefore, our goal is to improve our understanding of the role of both the host and the virus in these early events during norovirus infection in the hopes of identifying new targets for antiviral drug development.