Norovirus Infection of Dendritic Cells and Macrophages

人类诺如病毒(诺沃克病毒)是流行非细菌性疾病的主要原因 世界上最严重的胃肠炎。作为B类特工，对他们的生物学分析是一个高度优先的问题。然而，这些 病毒一直很难研究，因为它们没有进行过体外培养。因此有很多 关于这些病毒的生物学，基本问题仍然存在。2003年，我们报告发现了 第一例小鼠诺沃克病毒(MNV-1，附录1，Karst等人，STAT1依赖的诺沃克样先天免疫 病毒。2003年。科学。299：1575-8)。我们随后发现，这种高效的肠道病毒具有 树突状细胞和巨噬细胞在体内的惊人取向，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等人，诺如病毒的细胞内复制 培养显示出对树突状细胞和巨噬细胞的趋向性。PLOS生物学。E432。EPub 2004年11月30日) 利用这个新的系统，我们建立了MNV-1的空斑检测方法，斑块纯化MNV-1，纯化的MNV-1 毫克量的病毒，获得了第一个具有传染性的诺沃克病毒的冷冻电子显微镜图像，并证明 MNV-1RNA具有传染性，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的嗜性是在感染过程中的各个步骤确定的，然后再将病毒RNA输送到 胞质：细胞质，如结合、进入或脱皮。 根据这些新数据，具体目标有两个。首先，我们建议定义 以前没有详细研究过的病毒属的病毒进入的基本机制。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 细胞嗜性。具体目标是： 目的1.定义MNV-1结合、进入和脱壳过程中涉及的病毒事件。 目的2.定义MNV-1结合、进入和去涂层过程中涉及的细胞事件。 目的3.确定MNV-1细胞趋向性的细胞和分子基础。 拟议的研究将提供关于绑定、进入和剥离涂层的基本信息 病毒的一个非常重要的属，包括许多人类病原体。

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.