Cell entry and transcription activation of non-enveloped dsRNA viruses

无包膜 dsRNA 病毒的细胞进入和转录激活

• 批准号: 10054968
• 负责人: Z Hong ZHOU
• 金额: $ 39.16万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-16 至 2022-01-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10054968
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Animals; Antiviral Agents; Aquareoviruses; Bacteria; Binding; Biochemical; Biological Models; Bluetongue virus; Capsid; Cells; Cessation of life; Chimeric Proteins; Closure by clamp; Complement; Coupled; Cryo-electron tomography; Cryoelectron Microscopy; Cytoplasm; Cytoplasmic Polyhedrosis Viruses; Data; Development; Double Stranded RNA Virus; Double-Stranded RNA; Economics; Electrons; Endocytosis; Enzymes; Event; Excision; Exhibits; Family; Family member; Flu virus; Genetic Transcription; Genome; Glucosyltransferase; Goals; Guanosine Triphosphate; HIV; Herpesviridae; Incubated; Lead; Learning; Life Cycle Stages; Link; Liposomes; Literature; Livestock; Measures; Mediating; Membrane; Membrane Fusion; Membrane Lipids; Messenger RNA; Methods; Modeling; Molecular; Molecular Biology; Molecular Conformation; Multienzyme Complexes; Mutagenesis; Penetration; Phase; Pilot Projects; Process; Proteins; RNA; RNA Caps; RNA chemical synthesis; RNA-Directed RNA Polymerase; Recombinants; Reoviridae; Resolution; Rotavirus; S-Adenosylmethionine; Site; Structure; System; Technology; Testing; Transcriptional Activation; Transcriptional Regulation; Vaccines; Viral; Virion; Virus; Virus Replication; base; electron tomography; environmental change; insight; member; mutant; nanometer resolution; nucleoside triphosphatase; null mutation; particle; plant fungi; preference; receptor binding; reconstruction; reverse genetics; social; three dimensional structure; viral RNA; virus core; virus envelope

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.

进入细胞和基因组复制是任何病毒生命周期的两个基本过程。原子的细节 与流感、艾滋病等有包膜病毒不同， 和疱疹病毒。在进入细胞后，无包膜dsRNA病毒感知环境变化， 转录激活我们已经研究了具有单层（细胞质）的无包膜dsRNA病毒， 多角体病毒- CPV），双层（水生呼肠孤病毒）和三层（蓝舌病毒-或BTV - a 呼肠孤病毒科成员）衣壳。由于它的分子生物学研究得很好， 反向遗传学系统，BTV特别用作研究细胞进入的良好模型系统， 这些病毒的转录。因此，本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。 我们对BTV的研究结果表明，VP 5含有类似于膜融合蛋白的特征， 在低pH值下发生显著的构象变化以形成丝状三聚体结构。我们假设这 丝状结构相互作用并随后在细胞进入期间破坏内体膜。使用 呼肠孤病毒科最简单的成员（CPV），我们最近确定了dsRNA基因组的组织， 转录酶复合物，并表明RNA转录激活是由SAM介导的- 依赖于其加帽蛋白的ATP酶活性。这一结果，加上先前在CPV和 呼肠孤病毒科的其他成员认为RNA转录活性与ATP水解偶联（以及更多 最近与病毒ATP酶活性），导致我们的第二个假设，即BTV加帽蛋白（VP 4）也 含有ATP酶，其由外壳的去除触发，介导BTV RNA的活化 转录。在目标1中，我们将BTV病毒粒子和重组VP 5（野生型和突变体）与 脂质体在中性和低pH值，观察病毒颗粒和脂质之间可能的分子相互作用 冷冻电子断层扫描（cryoET）。这种直接的结构数据-预计在纳米 分辨率与相位板和能量过滤技术和subtomography平均-将测试我们的第一个 假设并阐明VP 5是否以及如何穿透脂质体。目标2-3将检验我们的假设， RNA转录机制。首先，为了阐明VP 5分离如何引发构象变化，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， 触发：仅用ATP或在转录条件下）通过二十面体重建（Aim 2）。二是 了解这些构象变化如何导致转录激活，我们将确定原子 转录酶复合物的结构和核心的dsRNA基因组组织（转录，ATP） 和病毒体（所有转录底物）通过不对称重建（目的3）。这些研究报告将 用我们建立的方法通过基于结构的诱变进行补充以获得重组颗粒。