Norovirus Infection of Dendritic Cells and Macrophages

人诺如病毒（诺瓦克型病毒）是引起非细菌性流行病的主要原因 世界上最严重的肠胃炎作为B类制剂，对其生物学进行分析是一项高度优先事项。但这些 病毒难以研究，因为它们没有被离体培养。因此许多 关于这些病毒的生物学仍然存在一些基本问题。2003年我们报道了 第一种鼠诺如病毒（MNV-1，附录1，Karst等，STAT 1依赖性天然免疫对诺瓦克样 病毒2003.科学299：1575-8）。我们随后发现，这种有效的肠道病毒具有 树突状细胞和巨噬细胞在体内的惊人的向性，并利用这一信息开发了第一个 诺如病毒的组织培养复制系统（附录2，Wobus等，诺如病毒在细胞中的复制 培养物揭示了树突细胞和巨噬细胞的向性。PLOS Biology. E432 Epub 2004年11月30日）。 使用这种新的系统，我们已经开发了MNV-1的噬斑测定，噬斑纯化的MNV-1，纯化的 毫克数量的病毒，获得了第一个传染性诺如病毒的冷冻电镜图像，并证明， MNV-1 RNA具有感染性，即使在不允许MNV-1感染的细胞中也是如此。后一种结果 表明MNV-1嗜性是在病毒RNA递送到细胞中之前的感染步骤中确定的。 细胞质，如结合，进入，或脱壳。 根据这些新数据，具体目标有两个目标。首先，我们建议定义 病毒进入病毒属的基本机制，以前没有详细研究过。第二、 我们试图确定细胞之间这些基本机制的差异是否决定了MNV-1 细胞向性具体目标是： 目标1。定义涉及MNV-1结合、进入和脱壳的病毒事件。 目标二。定义参与MNV-1结合、进入和脱膜的细胞事件。 目标3。确定MNV-1细胞嗜性的细胞和分子基础。 拟议的研究将提供有关结合，进入和涂层的基本信息， 包括许多人类病原体的非常重要的病毒属。

Human noroviruses (type virus Norwalk) are the major cause of epidemic non-bacterial gastroenteritis in the world. As Category B agents, analysis of their biology is a high priority. However, these viruses have been difficult to study because they have not been cultured ex vivo. Therefore many fundamental questions remain about the biology of these viruses. In 2003 we reported the discovery of the first murine norovirus (MNV-1, Appendix 1, Karst et al., STAT1-dependent innate immunity to a Norwalk-like virus. 2003. Science. 299:1575-8). We have subsequently found that this efficient enteric virus has a surprising tropism for dendritic cells and macrophages in vivo, and used this information to develop the first tissue culture replication system for a norovirus (Appendix 2, Wobus et al., Replication of a Norovirus in cell culture reveals a tropism for dendritic cells and macrophages. PLOS Biology. E432. Epub Nov 30 2004). Using this novel system we have developed a plaque assay for MNV-1, plaque purified MNV-1, purified milligram amounts of virus, obtained the first cryo-EM image of an infectious norovirus, and proved that MNV-1 RNA is infectious, even in cells that are not permissive for MNV-1 infection. This latter result indicates that MNV-1 tropism is determined at steps in infection prior to delivery of viral RNA into the cytoplasm such as binding, entry, or uncoating. Based on these new data, there are two goals of the Specific Aims. First, we propose to define fundamental mechanisms of viral entry for a viral genus that has not been studied in detail before. Second, we seek to define whether differences in these fundamental mechanisms between cells determine MNV-1 cell tropism. The Specific Aims are: Aim 1. Define the viral events involved in MNV-1 binding, entry, and uncoating. Aim 2. Define the cellular events involved in MNV-1 binding, entry, and uncoating. Aim 3. Determine the cellular and molecular basis of MNV-1 cell tropism. The proposed studies will provide fundamental information about the binding, entry, and uncoating of a very important genus of viruses that incudes many human pathogens.