PHYSICAL ELUCIDATION OF VIRUS ASSEMBLY

描述（改编自申请人的摘要）：关于 病毒蛋白质和RNA在血浆上组装的过程 宿主细胞的膜。 调查人员一直在研究这一过程 并发现这些复合物可以重构， 在形态上类似于未成熟病毒体组装的颗粒， 功能上，从而提供了一种方法来研究，在分子水平上， 组装途径导致感染性颗粒形成。 在这 他们将测试一种病毒组装的模型， 涉及Gag的几个结构域的膜相互作用， 蛋白质-蛋白质和蛋白质-RNA的相互作用。 他们提出，肉豆蔻酰化在将病毒Gag定位在 膜表面 此外，他们还提出，一个高度保守的区域， 衣壳结构域转导在细胞分裂时发生的构象变化， 膜联合，并发现该区域的突变严重损害了 组装件. 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，从而使其被转运到 在新一轮的感染过程中宿主细胞的核。 以识别 核蛋白复合物的性质，可能是设计的目标 Scarlata博士建议使用病毒蛋白酶Gag， 和在病毒的5'端含有腺苷酸化信号的RNA， 基因组来定义功能重建的大分子相互作用 核衣壳 申请人将首先通过表征 野生型Gag的体外组装在构象、取向和 利用荧光能量转移的肉豆蔻酰化作用;核蛋白 将通过凝胶转移、离心和保护来监测组装， 核酸酶降解;通过PR试验评估功能活性; 并且有限的蛋白质水解和核酸酶水解将定位改变的 分别位于Gag和RNA中。 二、申请人会比较 在这个高度保守的区域中具有突变的Gag蛋白的组装。 第三，申请人将审查“千年评估”的一个区域在削弱 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 抑制剂的

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.