GLYCOPROTEIN B AND CMV INFECTION

人巨细胞病毒(CMV)糖蛋白B(GB)是病毒包膜中含量最丰富的成分，是中和抗体的靶标，是具有潜在融合结构域和细胞黏附序列的关键基因产物。我们研究的长期目标是确定CMV包膜蛋白与细胞成分的分子相互作用，这些成分介导着生命周期中的关键步骤，如进入、细胞到细胞的扩散和病毒粒子的成熟/离开。这项建议的具体目标是定义CMV GB的结构/功能关系。在之前的资助期间，我们了解到GB是细胞感染所必需的基因产物。我们还发现，Gb是CMV的可靠配体，与细胞表面的一种或多种受体结合。GB与其受体(S)的结合导致了戏剧性的细胞反应，如细胞内信号传递和基因表达的诱导，所有这些也可归因于病毒。在这一资助期间，我们将扩大我们的新发现，并利用我们开发的试剂和功能分析的集合。在第一个目标中，将使用遗传学、细胞生物学和生化方法来鉴定GB受体。在第二个目标中，我们将从功能上描述在前一个资助期产生的大量GB突变株。我们收集的连接子插入突变体统一跨越GB ORF，在高阶结构、蛋白质折叠和细胞内转运方面进行了特征分析。缺乏严重结构缺陷的突变体将被测试其执行配基、信号和融合功能的能力。突变蛋白将通过分离、伪分型程序或对纯化蛋白的分析进行评估，并将通过将突变基因重新组合成GB零背景来在病毒基因组的背景下进行评估。该提案的最终目标是定义GB的结构。二硫键结构将通过生物化学方法确定。此外，GB结构的解析将通过与麻省理工学院的Peter Kim博士合作在原子水平上解决。这些实验将产生任何CMV包膜蛋白的第一个结构/功能图谱。由于GB是疫苗和抗病毒策略的确定目标，我们的研究结果将对GMV疾病的治疗和预防具有重要意义。

Glycoprotein B (gB) of human cytomegalovirus (CMV) is the most abundant component of the virus envelope, a target of neutralizing antibodies and a key gene product that possesses potential fusogenic domains and cellular adhesion sequences. The long term goal of our research is to define the molecular interactions of CMV envelope proteins with cellular components that mediate critical steps in the lifecycle such as entry, cell to cell spread and virion maturation/egress. The specific goal of this proposal is to define the structure/function relationship of CMV gB. In the previous funding period, we learned that gB is an essential gene product required for infection of cells. We also discovered that gB is a authentic ligand for CMV, binding to one or more classes of receptors on the surface of cells. Engagement of gB with its receptor(s) results in dramatic cellular responses such as intracellular signaling and induction of gene expression, all of which are also attributable to the virus. In this funding period, we will extend our novel findings and exploit the collection of reagents and functional assays we have developed. In the first aim, genetic, cell biological and biochemical approaches will be used to identify the gB receptor. In the second aim, we will functionally characterize a large panel of gB mutants produced in the previous funding period. Our collection of linker-insertion mutants which uniformly spans the gB ORF has been characterized with respect to higher order structure, protein folding and intracellular trafficking. Mutants lacking gross structural defects will be tested for their ability to perform ligand, signaling and fusion functions. The mutant proteins will be appraised in isolation, via pseudotyping procedures or by analysis of purified proteins, and they will be assessed in the context of the viral genome by recombining the mutants into a gB null background. The final aim of the proposal seeks to define gB's structure. The disulfide bond structure will be determined by biochemical means. In addition, resolution of the gB structure will be resolved at the atomic level via a collaboration with Dr. Peter Kim at Massachusetts Institute of Technology. These experiments will yield the first structure/functional map of any CMV envelope protein. Since gB is an identified target of vaccine and antiviral strategies, our research findings will be of great significance in efforts to treat and prevent GMV disease.