Cellular attachment, penetration and transport of non-enveloped dsRNA viruses

描述（由申请人提供）：许多无包膜病毒引起毁灭性的人类疾病。这些病毒进入宿主细胞的机制知之甚少，尽管可能与研究充分的包膜病毒如流感，HIV和疱疹病毒相似。我们已经研究了具有单层（细胞质多角体病毒- CPV）、双层（水生呼肠孤病毒）和三层（蓝舌病毒-或BTV -呼肠孤病毒科的环状病毒属的成员）衣壳的无包膜dsRNA病毒。由于BTV的细胞生物学已被充分研究，而且它具有独立的附着蛋白和穿透蛋白，因此BTV尤其可作为研究此类病毒进入细胞和转运的良好模型系统。因此，该项目的长期目标是通过冷冻电子显微镜（cryoEM）揭示无包膜病毒感染的结构基础，特别是病毒附着和进入的过程，以及子代病毒颗粒的细胞转运。 我们的7A分辨率cryoEM结构的预穿透BTV病毒粒子表明存在一个中央三螺旋束和18个两亲性螺旋的表面上的BTV穿透蛋白（VP 5），类似于在某些方面的I类融合蛋白的包膜病毒。氨基酸的二级结构的这种分配构成了一组需要在原子分辨率下进行测试的假设。此外，通过使用低pH值，我们已经将病毒转化为其穿透状态，并可视化长“倒钩”的开花，我们假设它们是展开的两亲性螺旋。此外，我们假设VP 5内的二硫键参与展开机制。我们的cryoEM结构还表明，细胞附着蛋白（VP 2）有两个结合位点，一个用于唾液酸，一个用于未知的靶分子，可能是整合素。此外，我们的初步数据表明，非结构蛋白NS 1具有病毒释放的作用，并形成高度有序的螺旋小管在锌离子依赖的方式，从而提供了一个机会，结构研究，以解释其作用。拟议的研究将 我们将通过以下四个具体目标来检验这些假设：（1）通过确定天然BTV病毒粒子的原子结构，我们将检验我们的假设，即无包膜BTV病毒的VP 5穿透蛋白在其核心处具有三螺旋束，在其表面上具有18个两亲性螺旋，并且在关键位置处具有二硫键。(2)从天然BTV病毒粒子的原子结构 在唾液酸存在下，我们将检验我们的假设，即VP 2附着蛋白具有唾液酸结合位点。(3)通过确定BTV颗粒的（开花）渗透状态（低pH值）的结构，我们将测试我们假设的展开机制。此外，我们将进行基于结构的诱变研究，以补充结构研究，以建立触发展开的机制。(4)从BTV非结构蛋白NS 1在螺旋小管中的原子结构，随后通过功能和基于结构的诱变实验，我们将了解NS 1如何调节病毒释放。这些机制和结构将与其他包膜和无包膜病毒（包括上述病毒）的机制和结构相关。

DESCRIPTION (provided by applicant): Many non-enveloped viruses cause devastating human diseases. The mechanisms of entry of these viruses into host cells are poorly understood, although there are likely analogies with well-studied enveloped viruses like flu, HIV and herpesvirus. We have studied non-enveloped dsRNA viruses with a single-layered (cytoplasmic polyhedrosis virus - CPV), a double-layered (aquareovirus), and a triple-layered (bluetongue virus - or BTV -- a member of the Orbivirus genus of the Reoviridae family) capsid. Because its cell biology is well studied, and because it has separate attachment and penetration proteins, BTV in particular serves as a good model system for studying cell entry and transport by such viruses. Thus, the long term goal of this project is to uncover, by cryo electron microscopy (cryoEM), the structural basis of non-enveloped virus infection, particularly the processes of viral attachment and entry, as well as cellular transport of progeny viral particles. Our 7A-resolution cryoEM structure of the pre-penetration BTV virion suggests the presence of a central three-helix bundle and 18 amphipathic helices on the surface of the BTV penetration protein (VP5), similar in some respects to Class I fusion proteins of enveloped viruses. This assignment of amino acids to secondary structures constitutes a set of hypotheses begging to be tested at atomic resolution. Moreover, by use of low pH, we have transformed the virus to its penetration state and visualized the blossoming of long 'barbs' that we hypothesize to be unfurled amphipathic helices. Further, we hypothesize involvement of a disulfide bond within VP5 in the unfurling mechanism. Our cryoEM structure also suggests that the cell attachment protein (VP2) has two binding sites, one for sialic acid and one for an unknown target molecule, perhaps an integrin. In addition, our preliminary data shows that non-structural protein NS1 has a role on virus release and forms highly ordered helical tubules in a Zn2+-dependent manner, thus providing an opportunity for structural studies to explain its role. The proposed studies will test these hypotheses by carrying out four specific aims: (1) By determining the atomic structure of the native BTV virion, we will test our hypothesis that the VP5 penetration protein of the non-enveloped BTV virus has a three-helix bundle at its core, 18 amphipathic helices on its surface, and a disulfide bond in a critical position. (2) From the atomic structure of the native BTV virion in the presence of sialic acid, we will test our hypothesis that the VP2 attachment protein has a sialic-acid binding site. (3) By determining the structure of the (blossomed) penetration state (low pH) of the BTV particle, we will test our hypothesized unfurling mechanism. Moreover, we will carry out structure-based mutagenesis studies to complement structural studies to establish mechanisms for triggering the unfurling. (4) From the atomic structure of BTV non-structural protein NS1 in helical tubules, followed by functional and structure-based mutagenesis experiments, we will learn how NS1 regulates virus release. These mechanisms and structures will be correlated with those of other enveloped and non-enveloped viruses, including the ones cited above.