PHYSICAL ELUCIDATION OF VIRUS ASSEMBLY

描述(改编自申请者摘要)：对此知之甚少 病毒蛋白质和核糖核酸在血浆上组装的过程 宿主细胞膜。调查人员一直在研究这一过程 并发现这些络合物可以被重组以提供 在形态上类似于未成熟病毒粒子集合的颗粒和 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 提议，他们将测试一种模型的病毒组装，包括最初的 涉及GAG的几个结构域的膜相互作用，被取代 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，肉豆蔻化在将病毒Gag定向到 膜表面。此外，他们还提出了一个高度保守的区域 衣壳结构域传递发生的构象变化 膜结合并发现该区域的突变严重损害 集合。GAG释放MA导致蛋白质接触改变的处理 通过蛋白质的特定部分进行调节。这就削弱了 MA与膜表面的相互作用，从而允许它被输送到 宿主细胞核在新一轮感染期间。要确定 可能作为设计目标的核蛋白复合体的性质 在抗病毒药物中，斯卡拉塔博士建议使用GAG，一种病毒蛋白酶， 以及在病毒5‘端含有囊化信号的RNA 基因组定义功能重组的大分子相互作用 核衣壳。申请者将首先通过描述In的特征来建模 野生型Gag体外组装的构象、取向和 利用荧光能量转移的肉豆蔻化作用；核蛋白 组装将通过凝胶移位、离心和保护 核酸酶降解；功能能力将通过PR法进行评估； 有限的蛋白质分解和核酸酶的水解会将改变的局部 分别在GAG和RNA中的区域。其次，申请者会比较 在这个高度保守的区域有突变的GAG蛋白的组装。 第三，申请者将研究MA区域在弱化中的作用 膜相互作用。拟议的研究使我们有可能确定 装配和设计装配所需的关键交互作用 抑制剂。

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.