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PHYSICAL ELUCIDATION OF VIRUS ASSEMBLY

病毒组装的物理解析

基本信息

批准号：
6384328
负责人：
Suzanne F Scarlata
金额：
$ 19.36万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
1998
资助国家：
美国
起止时间：
1998-05-01 至 2003-04-30
项目状态：
已结题

项目摘要

DESCRIPTION (Adapted from applicant's abstract): Little is known about the process through which viral proteins and RNA assemble on the plasma membranes of host cells. The investigators have been studying this process in vitro and find that these complexes can be reconstituted to give particles that resemble immature virion assemblies morphologically and functionally, and thus provide a way to study, at the molecular level, the assembly pathway leading to infectious particle formation. In this proposal, they will test a model for viral assembly that involves initial membrane interactions involving several domains of Gag, that are replaced with protein-protein and protein-RNA interactions as assembly proceeds. They propose that myristylation plays a role in orienting viral Gag on the membrane surface. Further, they propose that a highly conserved region in the capsid domain transduces the conformational changes that occur upon membrane association and find that mutations in this region severely impair assembly. Processing of Gag releases MA causing altered protein contacts mediated through a particular portion of the protein. This serves to weaken MA interaction with the membrane surface, thus allow it to be transported to the host cell nucleus during a new round of infection. To identify properties of the nucleoprotein complexes that might be targeted for design of anti-viral agents, Dr. Scarlata proposes using Gag, the viral protease, and RNA containing the encapsidation signal in the 5' end of the viral genome to define the macromolecular interactions of functional reconstituted nucleocapsids. The applicant will first model by characterizing the in vitro assembly of wild type Gag in terms of conformation, orientation, and role of myristylation using fluorescence energy transfer; nucleoprotein assembly will be monitored by gel-shift, centrifugation, and protection from nuclease degradation; functional competence will be assessed by PR assay; and limited proteolysis and hydrolysis with nucleases will localize altered regions in Gag and RNA, respectively. Second, the applicant will compare the assembly of Gag proteins with mutations in this highly conserved region. Third, the applicant will examine the role of a region of MA in weakening membrane interactions. The proposed study makes it possible to identify critical interactions required for assembly and to design assembly inhibitors.

描述（改编自申请人的摘要）：对此知之甚少病毒蛋白和 RNA 在血浆上组装的过程宿主细胞的膜。研究人员一直在研究这个过程体外实验并发现这些复合物可以重构以给出形态上类似于未成熟病毒体组装体的颗粒功能上，从而提供了一种在分子水平上研究导致感染性颗粒形成的组装途径。在这个提案中，他们将测试一个病毒组装模型，其中涉及初始涉及 Gag 多个域的膜相互作用，这些域被替换随着组装的进行，蛋白质-蛋白质和蛋白质-RNA 相互作用。他们提出，肉豆蔻酰化在病毒 Gag 的定位中发挥着作用。膜表面。此外，他们提出，一个高度保守的区域衣壳结构域转导发生的构象变化膜关联并发现该区域的突变严重损害集会。 Gag 的加工释放 MA 导致蛋白质接触改变通过蛋白质的特定部分介导。这有助于削弱 MA与膜表面相互作用，从而使其被运输至新一轮感染期间宿主细胞核。识别可能作为设计目标的核蛋白复合物的特性抗病毒药物中，斯卡拉塔博士建议使用病毒蛋白酶 Gag，和病毒 5' 端含有衣壳信号的 RNA 基因组来定义功能重组的大分子相互作用核衣壳。申请人将首先通过表征野生型 Gag 的构象、方向和结构的体外组装利用荧光能量转移的肉豆蔻酰化作用；核蛋白组装将通过凝胶转移、离心和保护来监测核酸酶降解；功能能力将通过 PR 测定进行评估；核酸酶的有限蛋白水解和水解作用将局部改变分别位于 Gag 和 RNA 中。其次，申请人会比较在这个高度保守的区域组装带有突变的 Gag 蛋白。第三，申请人将研究 MA 区域在弱化中的作用膜相互作用。拟议的研究可以确定装配和设计装配所需的关键交互抑制剂。