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MECHANISMS OF DENGUE VIRUS GENOME ENCAPSIDATION AND UNCOATING

登革热病毒基因组衣壳化和脱衣机制

基本信息

批准号：
9899735
负责人：
Andrea Gamarnik
金额：
$ 12.31万
依托单位：
FUNDACION INSTITUTO LELOIR
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-15 至 2022-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9899735
关键词：
3-Dimensional Address Affinity Affinity Chromatography Antiviral Agents Biochemical Biological Assay Biology Capsid Capsid Proteins Category A pathogen Cell Cycle Kinetics Cells Cellular biology Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Complex Culicidae Cytoplasm Data Dengue Dengue Infection Dengue Virus Development Disease Outbreaks Epidemic Event Family Flaviviridae Flavivirus Funding Genetic Genetic Translation Genome Human In Vitro Infection Integration Host Factors Intervention Life Cycle Stages Light Mass Spectrum Analysis Medical Methods Microscope Modeling Molecular Molecular Chaperones Nonstructural Protein Nucleocapsid Peptide Hydrolases Pharmacology Play Post-Translational Protein Processing Process Production Property Proteins Proteomics Public Health RNA RNA Binding RNA amplification Recombinants Reporter Research Personnel Resources Role Series Signal Transduction Small Interfering RNA Specificity Structure System Ubiquitination Vaccines Viral Viral Genome Viral Physiology Viral Proteins Virion Virus Assembly Virus Diseases Virus Replication arthropod-borne base design helicase innovation member novel nucleic acid binding protein particle protein complex protein function protein protein interaction public health relevance recombinant virus recruit small hairpin RNA tool viral RNA virology virus genetics

项目摘要

DESCRIPTION (provided by applicant): Dengue virus, a member of the Flaviviridae family, is a Category A pathogen that causes the most prevalent arthropod-borne viral illnesses in humans. The lack of in depth understanding of molecular mechanisms of viral replication and its interaction with the host cell, limits the development of antiviral strategies. The viral genome is an RNA molecule that plays multiple roles during viral replication. It serves as mRNA for translation, a template for RNA amplification, and substrate for encapsidation. A great deal has been learned in the last ten years about the mechanism of DENV genome replication. In contrast, the processes by which the genome is recruited by the capsid protein during viral particle formation and then released from the nucleocapsid into the cytoplasm during infection are two steps of the viral life cycle understudied for DENV and other flaviviruses. The viral capsid is a small highly basic protein that binds nucleic acids with low specificity. In addition, packaging signals have not been found in the viral RNA. Despite this, the viral genome is the only RNA encapsidated inside the particle. In this proposal, we will address mechanisms and define the machinery involved in dengue virus genome encapsidation and uncoating. To this end, we will combine our expertise in developing dengue virus genetic tools to dissociate overlapping functions in the viral genome together with proteomic approaches and biochemical studies. In Aim 1, we will investigate how the viral genome is freed into the cytoplasm by analyzing uncoating intermediates in infected cells and studying the fate of viral components during entry. In addition, a novel 3 dimensional single particle orbital tracking methods to trace the capsid protein during infection will be explored. In Aim 2, we will use genetic tools to dissec capsid protein requirements for particle formation and infectivity. It has been recently found that the dengue virus capsid protein suffers a variety of post-translational modification in infected cells. A comprehensive mass spectrometry analysis of capsid purified from infected cells and virions, together with the design of recombinant viruses will be used to define function of structural properties of capsid. In Aim 3, we will define the protein-protein interaction network fr dengue virus assembly and determine the function of the viral protein NS3 in this process. We will use a recently developed proteomic platform in the context of viral infections to define host components required for the assembly process. Dissecting the multiple functions and interactions of the capsid protein with host and viral components will shed light on fundamental aspects of dengue and other flavivirus replication. Importantly, the studies proposed will provide new information about viral processes still unexplored for antiviral intervention.

描述（由申请人提供）：登革热病毒是黄病毒科的一员，是一种 A 类病原体，可引起人类最流行的节肢动物传播的病毒性疾病。缺乏对病毒复制的分子机制及其与宿主细胞相互作用的深入了解，限制了抗病毒策略的发展。病毒基因组是一种在病毒复制过程中发挥多种作用的RNA分子。它充当翻译的 mRNA、RNA 扩增的模板和衣壳化的底物。在过去十年中，人们对 DENV 基因组复制机制有了很多了解。相比之下，基因组在病毒颗粒形成过程中被衣壳蛋白招募，然后在感染过程中从核衣壳释放到细胞质中的过程是登革热病毒和其他黄病毒正在研究的病毒生命周期的两个步骤。病毒衣壳是一种小的高碱性蛋白质，以低特异性结合核酸。此外，病毒RNA中尚未发现包装信号。尽管如此，病毒基因组是颗粒内唯一包裹的 RNA。在本提案中，我们将解决登革热病毒基因组衣壳化和脱壳所涉及的机制并定义机制。为此，我们将把我们在开发登革热病毒遗传工具方面的专业知识与蛋白质组学方法和生化研究结合起来，以分离病毒基因组中的重叠功能。在目标 1 中，我们将通过分析受感染细胞中的脱壳中间体并研究进入过程中病毒成分的命运，研究病毒基因组如何释放到细胞质中。此外，还将探索一种新颖的 3 维单粒子轨道跟踪方法来追踪感染过程中的衣壳蛋白。在目标 2 中，我们将使用遗传工具来剖析颗粒形成和感染性的衣壳蛋白需求。最近发现登革热病毒衣壳蛋白在受感染的细胞中遭受多种翻译后修饰。对从感染细胞和病毒颗粒中纯化的衣壳进行全面的质谱分析，以及重组病毒的设计将用于定义衣壳结构特性的功能。在目标 3 中，我们将定义登革热病毒组装的蛋白质-蛋白质相互作用网络，并确定病毒蛋白质 NS3 在此过程中的功能。我们将在病毒感染的背景下使用最近开发的蛋白质组平台来定义组装过程所需的宿主组件。剖析衣壳蛋白的多种功能以及与宿主和病毒成分的相互作用将揭示登革热和其他黄病毒复制的基本方面。重要的是，拟议的研究将提供有关尚未探索的抗病毒干预病毒过程的新信息。

项目成果

期刊论文数量（29）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Dengue Virus Capsid-Protein Dynamics in Live Infected Cells Studied by Pair Correlation Analysis.

DOI：
10.1007/978-1-0716-1879-0_8
发表时间：
2021-10
期刊：
Methods in molecular biology
影响因子：
0
作者：
M. Gabriel;Ignacio Sallaberry;Guadalupe S. Costa Navarro;E. Gratton;A. Gamarnik;L. Estrada
通讯作者：
M. Gabriel;Ignacio Sallaberry;Guadalupe S. Costa Navarro;E. Gratton;A. Gamarnik;L. Estrada

Novel ATP-independent RNA annealing activity of the dengue virus NS3 helicase.

DOI：
10.1371/journal.pone.0036244
发表时间：
2012
期刊：
PloS one
影响因子：
3.7
作者：
Gebhard LG;Kaufman SB;Gamarnik AV
通讯作者：
Gamarnik AV

In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.

DOI：
10.1038/s41598-021-03854-z
发表时间：
2021-12-24
期刊：
Scientific reports
影响因子：
4.6
作者：
Sallaberry I;Luszczak A;Philipp N;Navarro GSC;Gabriel MV;Gratton E;Gamarnik AV;Estrada LC
通讯作者：
Estrada LC

Structural and functional analysis of dengue virus RNA.

DOI：
10.1002/0470058005.ch9
发表时间：
2006-01-01
期刊：
Novartis Foundation symposium
影响因子：
0
作者：
Alvarez, Diego E;Lodeiro, Maria F;Gamarnik, Andrea V
通讯作者：
Gamarnik, Andrea V

Insights into the product release mechanism of dengue virus NS3 helicase.

DOI：
10.1093/nar/gkac473
发表时间：
2022-07-08
期刊：
NUCLEIC ACIDS RESEARCH
影响因子：
14.9
作者：
Adler, Natalia S.;Cababie, Leila A.;Sarto, Carolina;Cavasotto, Claudio N.;Gebhard, Leopoldo G.;Estrin, Dario A.;Gamarnik, Andrea, V;Arrar, Mehrnoosh;Kaufman, Sergio B.
通讯作者：
Kaufman, Sergio B.