Cell entry and transcription activation of non-enveloped dsRNA viruses

细胞进入和基因组复制是病毒生命周期的两个基本过程。原子的详细信息 与流感、艾滋病等有包膜的病毒不同，大型无包膜病毒的这些过程在很大程度上是未知的 和疱疹病毒。在细胞进入时，未被包膜的dsRNA病毒感知内部环境的变化 转录激活。我们已经研究了单层(细胞质)无包膜dsRNA病毒 多角体病毒(CPV)、双层(水生病毒)和三层(蓝舌病毒-或BTV-a 呼肠孤病毒科的成员)衣壳。因为它的分子生物学研究得很好，而且存在一种 反向遗传学系统，特别是BTV，是研究细胞进入和 这类病毒的转录。因此，这个项目的目标是使用最先进的低温电子显微镜 和断层扫描，以确定dsRNA病毒进入细胞和基因组转录的结构基础。 我们在BTV上的结果表明，VP5含有类似于膜融合蛋白的特征，并经历了 在低pH条件下发生显著构象变化，形成丝状三聚体结构。我们假设这是 丝状结构相互作用，随后在细胞进入时破坏内体膜。使用 呼肠孤病毒科的最简单成员(CPV)，我们最近确定了dsRNA基因组的组织和 转录酶复合体，并表明RNA转录激活是由SAM-1介导的。 依赖其封端蛋白的ATPase活性。这一结果，连同早先在CPV和 呼肠孤病毒科的其他成员认为，RNA转录活性与ATP水解(以及更多 最近与病毒ATPase活性)，导致了我们的第二个假设，BTV封顶蛋白(VP4)也 含有一种ATPase，它由外壳的去除触发，介导BTV RNA的激活 抄写。在目标1中，我们将用BTV病毒粒子和重组VP5(野生型和突变体)与 中性和低pH条件下的脂质体，观察病毒颗粒与脂类之间可能的分子相互作用 冷冻膜电子断层扫描(CryoET)。这种直接的结构数据--预计为纳米级 使用相位板和能量过滤技术以及亚层析平均技术的分辨率将测试我们的第一个 假设并阐明VP5是否以及如何穿透脂质体。AIMS 2-3将测试我们的假设 RNA转录的机制。首先，为了阐明VP5分离是如何触发构象变化的，我们将 确定BTV病毒粒子(触发前，所有转录底物)和核心(触发后)的原子结构 触发：仅用ATP或在转录条件下)通过二十面体重建(目标2)。第二，到 了解这些构象变化如何导致转录激活，我们将确定原子 核心转录酶复合体的结构和dsRNA基因组组织(转录，ATP) 和病毒粒子(所有转录底物)通过不对称重建(目标3)。这些研究将是 辅以基于结构的突变和我们建立的获得重组颗粒的方法。

Cell entry and genome replication are two essential processes of any viral life cycle. The atomic details of these processes are largely unknown for large non-enveloped viruses, unlike enveloped viruses like flu, AIDS and herpes viruses. Upon cell entry, non-enveloped dsRNA viruses sense environmental changes for internal transcription activation. 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 Reoviridae family) capsid. Because of its well studied molecular biology and the existence of a reverse genetics system, BTV in particular serves as a good model system for studying cell entry and transcription by such viruses. Thus, the goal of this project is to use state-of-the-art cryo electron microscopy and tomography to determine the structural basis of dsRNA virus cell entry and genome transcription. Our results on BTV show that VP5 contains features similar to membrane fusion proteins and undergoes significant conformational changes at low pH to form a filamentous trimer structure. We hypothesize that this filamentous structure interacts and subsequently breaks endosomal membrane during cell entry. Using the simplest member (CPV) of the Reoviridae, we recently determined organization of dsRNA genome and transcriptional enzyme complex, and showed that RNA transcription activation is mediated by the SAM- dependent ATPase activity of its capping protein. This result, together with earlier observations in CPV and other members of the Reoviridae that RNA transcription activities were coupled with ATP hydrolysis (and more recently with viral ATPase activity), lead to our second hypothesis that the BTV capping protein (VP4) also contains an ATPase, which, triggered by the removal of outer shell, mediates activation of BTV RNA transcription. In Aim 1, we will incubate BTV virions and recombinant VP5 (wild-type and mutants) with liposomes at neutral and low pH and observe possible molecular interactions between viral particles and lipid membrane with cryo electron tomography (cryoET). Such direct structural data – expected at nanometer resolution with phase plate and energy-filtering technologies and subtomogram averaging – will test our first hypothesis and clarify whether and how VP5 penetrates liposomes. Aims 2-3 will test our hypothesis on the mechanism of RNA transcription. First, to clarify how VP5 detachment triggers conformational changes, we will determine the atomic structures of the BTV virion (pre-triggering, all transcription substrates) and cores (post- triggering: with only ATP or under transcribing condition) by icosahedral reconstruction (Aim 2). Second, to learn how these conformational changes lead to transcription activation, we will determine the atomic structures of transcriptional enzyme complex and dsRNA genome organization of cores (transcribing, ATP) and virions (all transcription substrates) by asymmetric reconstruction (Aim 3). These studies will be complemented by structure-based mutagenesis with our established method to obtain recombinant particles.