GLYCOPROTEIN B AND CMV INFECTION

人巨细胞病毒（CMV）的糖蛋白B（gB）是病毒囊膜中含量最丰富的组分，是中和抗体的靶蛋白，也是一种具有潜在融合域和细胞粘附序列的关键基因产物。 我们研究的长期目标是确定CMV包膜蛋白与介导生命周期中关键步骤的细胞组分的分子相互作用，如进入，细胞间传播和病毒粒子成熟/外出。本提案的具体目标是定义CMV gB的结构/功能关系。在之前的资助期间，我们了解到gB是细胞感染所需的重要基因产物。 我们还发现gB是CMV的真正配体，与细胞表面上的一种或多种受体结合。gB与其受体的结合导致显著的细胞反应，如细胞内信号传导和基因表达的诱导，所有这些也可归因于病毒。在此资助期间，我们将扩展我们的新发现，并利用我们开发的试剂和功能测定的集合。在第一个目标中，将使用遗传学、细胞生物学和生物化学方法来鉴定gB受体。在第二个目标中，我们将在功能上表征在前一个资助期产生的一大组gB突变体。我们收集的接头插入突变体，均匀跨越gB ORF的特点是相对于更高的顺序结构，蛋白质折叠和细胞内运输。 将测试缺乏总体结构缺陷的突变体执行配体、信号传导和融合功能的能力。将通过假型化程序或通过分析纯化的蛋白质对突变蛋白进行分离评价，并通过将突变体重组到gB无效背景中，在病毒基因组背景下对其进行评估。该提案的最终目的是确定gB的结构。二硫键结构将通过生物化学方法确定。此外，通过与马萨诸塞州理工学院的Peter Kim博士合作，将在原子水平上解析gB结构。 这些实验将产生任何CMV包膜蛋白的第一个结构/功能图。由于gB是疫苗和抗病毒策略的确定靶点，因此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.