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

描述（由申请人提供）：许多非包膜病毒导致毁灭性的人类疾病。这些病毒进入宿主细胞的机制尚不清楚，尽管它们可能与被充分研究的包膜病毒（如流感病毒、艾滋病毒和疱疹病毒）有相似之处。我们研究了具有单层（细胞质多角体病毒- CPV），双层（水囊病毒）和三层（蓝舌病毒-或BTV -呼肠孤病毒科的一种眶病毒属）衣壳的非包膜dsRNA病毒。由于其细胞生物学已被充分研究，并且由于其具有独立的附着和渗透蛋白，因此BTV尤其可作为研究此类病毒进入细胞和运输的良好模型系统。因此，该项目的长期目标是通过低温电子显微镜（cryoEM）揭示非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运输。

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.