Norovirus Infection of Dendritic Cells and Macrophages

描述（申请人提供）：人诺如病毒（诺瓦克型病毒）是世界范围内流行性非细菌性胃肠炎的主要病因。作为B类制剂，对其生物学进行分析是一项高度优先事项。然而，这些病毒一直难以研究，因为它们尚未离体培养。因此，关于这些病毒的生物学仍然存在许多基本问题。在2003年，我们报道了第一个鼠诺如病毒的发现（MNV-1，附录1，Karst等人，STAT 1依赖的先天免疫诺瓦克样病毒。2003.科学299：1575-8）。我们随后发现，这种有效的肠道病毒在体内对树突状细胞和巨噬细胞具有令人惊讶的向性，并利用这一信息开发了诺如病毒的第一个组织培养复制系统（附录2，Wobus等人，诺如病毒在细胞培养物中的复制揭示了对树突状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的空斑测定，空斑纯化的MNV-1，纯化毫克量的病毒，获得了感染性诺如病毒的第一个冷冻-EM图像，并证明MNV-1 RNA是感染性的，即使在不允许MNV-1感染的细胞中。后一结果表明，MNV-1的嗜性是在病毒RNA递送到细胞质中之前的感染步骤中确定的，例如结合、进入或脱壳。根据这些新数据，具体目标有两个目标。首先，我们建议定义一个病毒属，还没有被详细研究过的病毒进入的基本机制。其次，我们试图确定是否在这些细胞之间的基本机制的差异决定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。拟议中的研究将提供有关一个非常重要的病毒属（包括许多人类病原体）的结合、进入和脱壳的基本信息。

DESCRIPTION (provided by applicant): 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 includes many human pathogens.