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
基本信息
- 批准号:10054968
- 负责人:
- 金额:$ 39.16万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:ATP HydrolysisATP phosphohydrolaseAnimalsAntiviral AgentsAquareovirusesBacteriaBindingBiochemicalBiological ModelsBluetongue virusCapsidCellsCessation of lifeChimeric ProteinsClosure by clampComplementCoupledCryo-electron tomographyCryoelectron MicroscopyCytoplasmCytoplasmic Polyhedrosis VirusesDataDevelopmentDouble Stranded RNA VirusDouble-Stranded RNAEconomicsElectronsEndocytosisEnzymesEventExcisionExhibitsFamilyFamily memberFlu virusGenetic TranscriptionGenomeGlucosyltransferaseGoalsGuanosine TriphosphateHIVHerpesviridaeIncubatedLeadLearningLife Cycle StagesLinkLiposomesLiteratureLivestockMeasuresMediatingMembraneMembrane FusionMembrane LipidsMessenger RNAMethodsModelingMolecularMolecular BiologyMolecular ConformationMultienzyme ComplexesMutagenesisPenetrationPhasePilot ProjectsProcessProteinsRNARNA CapsRNA chemical synthesisRNA-Directed RNA PolymeraseRecombinantsReoviridaeResolutionRotavirusS-AdenosylmethionineSiteStructureSystemTechnologyTestingTranscriptional ActivationTranscriptional RegulationVaccinesViralVirionVirusVirus Replicationbaseelectron tomographyenvironmental changeinsightmembermutantnanometer resolutionnucleoside triphosphatasenull mutationparticleplant fungipreferencereceptor bindingreconstructionreverse geneticssocialthree dimensional structureviral RNAvirus corevirus envelope
项目摘要
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。
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Z Hong ZHOU其他文献
Z Hong ZHOU的其他文献
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{{ truncateString('Z Hong ZHOU', 18)}}的其他基金
A Mid-Level 200kV Instrument for Single-Particle cryoEM
用于单粒子冷冻电镜的中级 200kV 仪器
- 批准号:
10436739 - 财政年份:2022
- 资助金额:
$ 39.16万 - 项目类别:
In situ structures of three components essential to human cytomegalovirus pathogenesis: genome-packaging machinery, capsid-associated tegument and prefusion glycoprotein complexes
人类巨细胞病毒发病机制所必需的三个成分的原位结构:基因组包装机制、衣壳相关的外皮和融合前糖蛋白复合物
- 批准号:
10395617 - 财政年份:2019
- 资助金额:
$ 39.16万 - 项目类别:
In situ structures of three components essential to human cytomegalovirus pathogenesis: genome-packaging machinery, capsid-associated tegument and prefusion glycoprotein complexes
人类巨细胞病毒发病机制所必需的三个成分的原位结构:基因组包装机制、衣壳相关的外皮和融合前糖蛋白复合物
- 批准号:
10595938 - 财政年份:2019
- 资助金额:
$ 39.16万 - 项目类别:
In situ structures of three components essential to human cytomegalovirus pathogenesis: genome-packaging machinery, capsid-associated tegument and prefusion glycoprotein complexes
人类巨细胞病毒发病机制所必需的三个成分的原位结构:基因组包装机制、衣壳相关的外皮和融合前糖蛋白复合物
- 批准号:
10597018 - 财政年份:2019
- 资助金额:
$ 39.16万 - 项目类别:
Direct Detection Device for atomic resolution cryoEM of macromolecular complexes
大分子复合物原子分辨率冷冻电镜直接检测装置
- 批准号:
8640787 - 财政年份:2014
- 资助金额:
$ 39.16万 - 项目类别:
Genome structure, transcription and packaging of dsRNA viruses
双链RNA病毒的基因组结构、转录和包装
- 批准号:
10554343 - 财政年份:2012
- 资助金额:
$ 39.16万 - 项目类别:
Cellular attachment, penetration and transport of non-enveloped dsRNA viruses
无包膜 dsRNA 病毒的细胞附着、渗透和运输
- 批准号:
8531141 - 财政年份:2012
- 资助金额:
$ 39.16万 - 项目类别:
Cellular attachment, penetration and transport of non-enveloped dsRNA viruses
无包膜 dsRNA 病毒的细胞附着、渗透和运输
- 批准号:
8304894 - 财政年份:2012
- 资助金额:
$ 39.16万 - 项目类别:
Genome structure, transcription and packaging of dsRNA viruses
双链RNA病毒的基因组结构、转录和包装
- 批准号:
10449147 - 财政年份:2012
- 资助金额:
$ 39.16万 - 项目类别:
Genome Structure, Transcription and Packaging of dsRNA Viruses
dsRNA 病毒的基因组结构、转录和包装
- 批准号:
10820018 - 财政年份:2012
- 资助金额:
$ 39.16万 - 项目类别: