Effects of HIV-1 MA domain on gag multimerization and viral assembly in mice.

HIV-1 MA 结构域对小鼠 gag 多聚化和病毒组装的影响。

基本信息

批准号：
7892980
负责人：
Mowgli Holmes
金额：
$ 4.14万
依托单位：
COLUMBIA UNIVERSITY HEALTH SCIENCES
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-07-07 至 2012-04-06
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7892980
关键词：
AIDS Vaccines Academic Medical Centers Achievement Acquired Immunodeficiency Syndrome Alanine Astrocytes Award Back Belief Binding Biological Assay Biological Models Biology Biotechnology Cell Line Cell fusion Cell membrane Cells Characteristics Child Cities Clinical Clinical Research Clinical Trials Confocal Microscopy Data Defect Development Doctor of Philosophy Drug resistance Educational process of instructing Embryo Enrollment Environment Epidemic Evolution Face Fellowship Flow Cytometry Fluorescence Resonance Energy Transfer Fluorescent Antibody Technique Fractionation Gagging Goals HIV HIV-1 Hospitals Human Immunology Individual Infection Institution Instruction Investigation Knowledge Laboratories Length Letters Life Life Cycle Stages Literature Location Membrane Membrane Microdomains Methods Microbiology Minority Modeling Molecular Molecular Biology Molecular Virology Monitor Mus Mutagenesis Mutation N-terminal Names Neuropathogenesis New York Phase Population Potassium Hydroxide Process Progress Reports Property Proteins Publications Registries Research Research Design Research Support Research Training Resistance Resource Sharing Role Safety Scanning Series Services Site Staining method Stains Students Techniques Testing Therapeutic Time Training Transfection Universities Vaccines Vertebrates Viral Viral Genome Virion Virus Woman Work base career design directed evolution embryonic stem cell experience gag Gene Products human embryonic stem cell human subject human subject protection improved in vivo interest macrophage medical schools mouse model mutant novel novel strategies novel therapeutics particle pre-doctoral professor programs research facility research study responsible research conduct skills stem therapeutic development therapeutic vaccine therapy development tool development vaccine development vaccine evaluation

AIDS Vaccines Academic Medical Centers Achievement Acquired Immunodeficiency Syndrome Alanine Astrocytes Award Back Belief Binding Biological Assay Biological Models Biology Biotechnology Cell Line Cell fusion Cell membrane Cells Characteristics Child Cities Clinical Clinical Research Clinical Trials Confocal Microscopy Data Defect Development Doctor of Philosophy Drug resistance Educational process of instructing Embryo Enrollment Environment Epidemic Evolution Face Fellowship Flow Cytometry Fluorescence Resonance Energy Transfer Fluorescent Antibody Technique Fractionation Gagging Goals HIV HIV-1 Hospitals Human Immunology Individual Infection Institution Instruction Investigation Knowledge Laboratories Length Letters Life Life Cycle Stages Literature Location Membrane Membrane Microdomains Methods Microbiology Minority Modeling Molecular Molecular Biology Molecular Virology Monitor Mus Mutagenesis Mutation N-terminal Names Neuropathogenesis New York Phase Population Potassium Hydroxide Process Progress Reports Property Proteins Publications Registries Research Research Design Research Support Research Training Resistance Resource Sharing Role Safety Scanning Series Services Site Staining method Stains Students Techniques Testing Therapeutic Time Training Transfection Universities Vaccines Vertebrates Viral Viral Genome Virion Virus Woman Work base career design directed evolution embryonic stem cell experience gag Gene Products human embryonic stem cell human subject human subject protection improved in vivo interest macrophage medical schools mouse model mutant novel novel strategies novel therapeutics particle pre-doctoral professor programs research facility research study responsible research conduct skills stem therapeutic development therapeutic vaccine therapy development tool development vaccine development vaccine evaluation

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项目摘要

The achievement of a robust pathogenic infection of mice with HIV-1 resembling natural HIV-1 infection in people would greatly facilitate understanding of HIV-1 biology in vivo, development of AIDS vaccines, and testing of new therapeutics. Early studies conducted in cell lines indicated that there are major blocks to HIV-1 replication in mouse cells at several points in the viral life cycle, but recent research suggests that HIV-1 infection of primary mouse cells and mice in vivo is in principle possible. This research indicates that the major impediment limiting HIV-1 replication in mouse cells is a defect in the assembly of the viral Gag polyprotein that reduces Gag binding to the plasma membrane and viral particle export. Research has implicated the membrane-binding function of the MA protein within Gag in this defect; however, early multimerization steps are required before Gag is able to bind to membranes at all. The overall goal of this fellowship application is to continue the investigation of a novel defect in cytosolic Gag-Gag interaction in mouse cells that may influence Gag membrane binding, assembly and virion export. Preliminary results using FRET and fractionation techniques indicate that the presence of MA in Gag impedes early Gag multimerization steps in mouse cells. These results support the hypothesis that the murine restriction of HIV- 1 assembly is due to inhibition of Gag-Gag multimerization prior to membrane binding, as a result of interactions between MA and factors present in mouse cells. Identification and mutagenesis of the regions in MA involved in this restriction may improve Gag multimerization in mouse cells, facilitate virion export, and improve HIV-1 replication in mice. This hypothesis will be tested in three Specific Aims: 1) To study Gag-Gag interactions in mouse cells using FRET and functional assays to pinpoint the roles of MA and NC in Gag multimerization; 2) To identify the sites of HIV-1 assembly in mouse cells, using staining and cell fusion techniques; and 3) To identify MA mutants with enhanced multimerization by construction and functional testing of MA deletion and alanine scanning mutants, and to test whether such mutants cloned into full-length virus are able to support the full replication cycle of HIV-1. The proposed studies will employ virological and molecular biology methods; flow cytometry, confocal microscopy, FRET and immunofluorescence techniques; and primary macrophage and astrocyte cultures. This research is aimed at understanding and circumventing the block to HIV-1 replication in mouse cells, and may help in the development of a powerful model of HIV-1 infection in mice. Such a model would be an enormous step forward in the search for vaccines and therapeutics for HIV-1 infection.

HIV-1类似于天然HIV-1感染的小鼠的强大致病感染实现在人中，人们将极大地了解体内HIV-1生物学，艾滋病疫苗的开发以及测试新疗法。在细胞系中进行的早期研究表明，有主要障碍病毒生命周期中几个点的小鼠细胞中的HIV-1复制，但最近的研究表明原理是原代小鼠细胞和小鼠的HIV-1感染原则上是可能的。这项研究表明小鼠细胞中HIV-1复制的主要障碍是病毒堵嘴组装中的缺陷多蛋白会降低与质膜和病毒颗粒导出的插孔结合。研究有在此缺陷中，含义了MA蛋白在GAG中的膜结合功能。但是，早在GAG能够完全与膜结合之前，需要进行多聚步骤。总体目标奖学金的应用是继续研究胞质堵嘴相互作用中新型缺陷可能影响插科打膜结合，组装和病毒体导出的小鼠细胞。初步结果使用FRET和分级技术表明，GAG中MA的存在阻碍了早期堵嘴小鼠细胞中的多聚化步骤。这些结果支持以下假设。 1组装是由于抑制膜结合之前的GAG-GAG多聚化的抑制作用，因此 MA与小鼠细胞中存在的因素之间的相互作用。区域的识别和诱变在参与该限制的MA中，可以改善小鼠细胞中的插孔多聚化，促进病毒体导出，并改善小鼠的HIV-1复制。该假设将以三个特定目的进行检验：1）使用FRET和功能测定法以查明MA和NC的作用在堵嘴中多聚化； 2）使用染色和细胞识别小鼠细胞中HIV-1组装的位点融合技术； 3）确定通过构造和 MA缺失和丙氨酸扫描突变体的功能测试，并测试该突变体是否克隆全长病毒能够支持HIV-1的完整复制周期。拟议的研究将采用病毒学和分子生物学方法；流式细胞仪，共聚焦显微镜，fret和免疫荧光技术；以及原发性巨噬细胞和星形胶质细胞培养物。这项研究旨在理解和规避小鼠细胞中HIV-1复制的块，并可能有助于开发小鼠HIV-1感染模型。这样的模型将是寻找HIV-1感染的疫苗和治疗剂迈出了巨大的一步。