Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly
病毒组装中逆转录病毒 Gag-RNA 相互作用的分子机制
基本信息
- 批准号:10656231
- 负责人:
- 金额:$ 42.81万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 财政年份:2020
- 资助国家:美国
- 起止时间:2020-08-20 至 2025-06-30
- 项目状态:未结题
- 来源:
- 关键词:AddressAdoptedAnimalsAutomobile DrivingBindingBiochemicalBiologicalBiological ModelsBiophysical ProcessBiophysicsCRISPR/Cas technologyCancer EtiologyCell NucleusCell membraneCellsCellular biologyChromatinComplexCytoplasmCytoplasmic GranulesDNADataDiseaseDomestic FowlsGeneticGenetic TranscriptionGenomeGoalsHIVHost DefenseHumanImmunologic Deficiency SyndromesInfectionIntegration Host FactorsIntracellular TransportKineticsKnowledgeLaboratoriesLiquid substanceMeasuresMediatingMembraneModelingMolecularMorphogenesisNuclearNuclear EnvelopeNuclear ExportOrganellesPathway interactionsPharmaceutical PreparationsPhasePhysical condensationProcessProductionPropertyProteomicsPublishingRNARNA BindingRNA SplicingRNA VirusesResearch Project GrantsRetroviridaeRetrovirus ProteinsRibonucleoproteinsRoleRous sarcoma virusSiteSourceStructural ProteinStructureTestingThinkingTranscriptTravelViralVirionVirus AssemblyWorkbiophysical analysisbiophysical propertiesbiophysical techniquesdeep sequencingexperimental studyfluorescence imaginggag Gene Productsgenomic RNAimaging approachimaging modalityimaging studyinnovationinsightintegration sitelive cell imagingmicroscopic imagingparticlerecruitsingle moleculetraffickingtranscription factorultra high resolutionviral RNAviral genomicsvirus host interaction
项目摘要
Abstract
Retroviruses are positive-sense, single-stranded RNA viruses that cause cancers and severe
immunodeficiency diseases in animals and humans, including human immunodeficiency virus. For over a
century, Rous sarcoma virus (RSV), which causes cancer in domestic fowl, has served as a powerful model
system to dissect the molecular basis of retroviral replication, including retrovirus assembly. Gag, the major
structural protein of retroviruses, orchestrates the assembly of virus particles that bud from the plasma
membrane of infected cells. To initiate particle assembly, Gag selectively binds unspliced viral RNA as the
source of genomic RNA in virions. This proposal focuses on the mechanism by which Gag selects genomic
RNA, addressing fundamental, unanswered questions in the field: (i) where in the cell does the initial contact
between Gag and viral RNA occur; (ii) how does Gag selectively recruit unspliced viral RNA for packaging
when it comprises only ~1% of the total RNA in an infected cell; and (iii) what are the properties of Gag-viral
RNA complexes that promote transport through the cell to the plasma membrane for particle release? Because
virus particles bud from the plasma membrane, it was originally thought that initial Gag-genomic RNA
interactions occurred in the cytoplasm. Our laboratory discovered that RSV Gag undergoes nuclear trafficking,
which is required for efficient genomic viral RNA packaging. This finding raised the possibility that Gag binds
genomic RNA in the nucleus, which challenges the dogma for how retroviruses package their genomes.
Our imaging and biophysical studies have revealed that the RSV Gag protein forms discrete nuclear,
cytoplasmic, and plasma membrane foci that have properties of biological condensates, which have recently
been shown to be important in regulating cell biology processes and virus-host interactions. We have observed
that the Gag nuclear foci colocalize with unspliced viral RNA, suggesting that RSV Gag initially binds genomic
RNA in the nucleus. In Aim 1, we will determine whether RSV Gag binds genomic RNAs at transcription sites
using super-resolution live cell imaging, deep sequencing, single molecule fluorescence imaging, and
CRISPR-based approaches. In Aim 2, we will use biophysical approaches to examine whether Gag-genomic
RNA complexes form biological condensates that adopt properties of membrane-less organelles and undergo
liquid-liquid phase separation. We will test the hypothesis that Gag-genomic RNA condensates remain tightly
packed as they cross the nuclear envelope and traffic through the cytoplasm to the plasma membrane, where
virus particles are assembled. Together, these experiments will move the field forward with new insights into
how retroviruses select their RNA genomes and transport them to the plasma membrane for budding.
摘要
逆转录病毒是正义单链RNA病毒,可导致癌症和严重的癌症。
在动物和人类中的免疫缺陷疾病,包括人类免疫缺陷病毒。了一个多
世纪以来,引起家禽癌症的劳斯肉瘤病毒(Rous sarcoma virus,RSV
系统剖析逆转录病毒复制的分子基础,包括逆转录病毒装配。加格少校
逆转录病毒的结构蛋白,协调从血浆中出芽的病毒颗粒的装配
感染细胞的膜。为了启动颗粒组装,Gag选择性地结合未剪接的病毒RNA,因为Gag是一种非剪接的RNA。
病毒体中基因组RNA的来源。该建议侧重于Gag选择基因组的机制,
RNA,解决该领域未解答的基本问题:(i)细胞中的何处进行初始接触
Gag和病毒RNA之间发生;(ii)Gag如何选择性地招募未剪接的病毒RNA进行包装
当它仅占感染细胞总RNA的~1%时;以及(iii)GAG病毒的特性是什么
RNA复合物,促进运输通过细胞到质膜的颗粒释放?因为
病毒颗粒从质膜出芽,最初认为最初的GAG基因组RNA
相互作用发生在细胞质中。我们的实验室发现RSV Gag经历了核贩运,
这是有效的基因组病毒RNA包装所必需的。这一发现增加了Gag结合
基因组RNA在细胞核中,这挑战了逆转录病毒如何包装其基因组的教条。
我们的成像和生物物理学研究揭示了RSV Gag蛋白形成离散的核,
细胞质和质膜病灶,具有生物凝聚物的性质,最近
已经证明在调节细胞生物学过程和病毒-宿主相互作用中是重要的。我们观察到
Gag核灶与未剪接的病毒RNA共定位,表明RSV Gag最初结合基因组
核中的RNA。在目标1中,我们将确定RSV Gag是否在转录位点结合基因组RNA
使用超分辨率活细胞成像,深度测序,单分子荧光成像,
基于CRISPR的方法在目标2中,我们将使用生物物理方法来检查是否存在GAG-基因组
RNA复合物形成生物浓缩物,其采用无膜细胞器的特性,
液-液相分离我们将检验这样的假设,即GAG-基因组RNA浓缩物保持紧密
当它们穿过核膜并穿过细胞质到达质膜时,
病毒颗粒被组装。这些实验将共同推动该领域的发展,
逆转录病毒如何选择它们的RNA基因组并将其转运到质膜上进行出芽。
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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Leslie J Parent的其他文献
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{{ truncateString('Leslie J Parent', 18)}}的其他基金
Role of biomolecular condensates in regulating HIV-1 viral ribonucleoprotein complex formation in the setting of substance use disorder
物质使用障碍中生物分子缩合物在调节 HIV-1 病毒核糖核蛋白复合物形成中的作用
- 批准号:
10398171 - 财政年份:2021
- 资助金额:
$ 42.81万 - 项目类别:
Role of biomolecular condensates in regulating HIV-1 viral ribonucleoprotein complex formation in the setting of substance use disorder
物质使用障碍中生物分子缩合物在调节 HIV-1 病毒核糖核蛋白复合物形成中的作用
- 批准号:
10831594 - 财政年份:2021
- 资助金额:
$ 42.81万 - 项目类别:
Role of biomolecular condensates in regulating HIV-1 viral ribonucleoprotein complex formation in the setting of substance use disorder
物质使用障碍中生物分子缩合物在调节 HIV-1 病毒核糖核蛋白复合物形成中的作用
- 批准号:
10228358 - 财政年份:2021
- 资助金额:
$ 42.81万 - 项目类别:
Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly
病毒组装中逆转录病毒 Gag-RNA 相互作用的分子机制
- 批准号:
10797635 - 财政年份:2020
- 资助金额:
$ 42.81万 - 项目类别:
Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly
病毒组装中逆转录病毒 Gag-RNA 相互作用的分子机制
- 批准号:
10241537 - 财政年份:2020
- 资助金额:
$ 42.81万 - 项目类别:
Molecular Mechanisms of Retroviral Gag-RNA interactions in Virus Assembly
病毒组装中逆转录病毒 Gag-RNA 相互作用的分子机制
- 批准号:
10441591 - 财政年份:2020
- 资助金额:
$ 42.81万 - 项目类别:
Nuclear Trafficking of the Retroviral Gag Protein
逆转录病毒 Gag 蛋白的核运输
- 批准号:
7901230 - 财政年份:2009
- 资助金额:
$ 42.81万 - 项目类别:
Nuclear Trafficking of the Retroviral Gag Protein
逆转录病毒 Gag 蛋白的核运输
- 批准号:
6611470 - 财政年份:1998
- 资助金额:
$ 42.81万 - 项目类别:
Nuclear Trafficking of the Retroviral Gag Protein
逆转录病毒 Gag 蛋白的核运输
- 批准号:
8072188 - 财政年份:1998
- 资助金额:
$ 42.81万 - 项目类别:
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