GLYCOPROTEIN B AND CMV INFECTION

人巨细胞病毒（HCMV）糖蛋白B （gB）是病毒包膜中最丰富的成分，是中和抗体的靶标，是具有潜在融合结构域和细胞粘附序列的关键基因产物。包膜糖蛋白是组织趋向性的主要决定因素，也是病毒进入、细胞-细胞扩散和病毒粒子出口/成熟的介质。我们研究的长期目标是确定与HCMV包膜蛋白和细胞成分的分子相互作用，这些分子相互作用介导了生命周期中的关键步骤，如进入，细胞间传播和病毒粒子成熟/退出。我们了解到gB是细胞感染所必需的基因产物。我们发现，gB是HCMV的一个真正的配体，与细胞表面的一种或多种受体结合。gB与其受体的相互作用具有生物学相关性，因为当gB结合位点被占据时，病毒进入被阻断。gB与其受体的结合会导致剧烈的细胞反应，如细胞内信号传导和基因表达的诱导，所有这些都可归因于病毒。我们将通过定义响应gB和病毒而激活的信号效应物的组成来进一步表征gB信号通路的性质。这些实验将揭示受体候选物，这些受体候选物将使用新发现的HCMV进入缺陷细胞系进行功能测试。表达克隆策略也被设计用于分离编码HCMV进入受体的cDNA克隆。为了准备结构/功能分析，我们在整个gB中制作了一个大的广义突变面板。所有突变体都具有高阶结构、蛋白质折叠和细胞内运输的特征。我们还开发了一种新颖和创新的表达策略来对突变体进行功能测试。一种缺乏其单包膜基因并编码绿色荧光蛋白的修饰水泡性口炎病毒可以用gB和gB突变体进行假型。该表达系统的浓缩、膜锚定展示具有许多实验优势，将用于从配体、信号和融合活性方面对gB突变体进行功能表征。这些实验将产生首个HCMV包膜蛋白的结构/功能图谱。第三项研究将进一步确定已知的HCMV和gB结合伙伴膜联蛋白II的作用。膜联蛋白II在病毒进入中缺乏明确的作用，我们将使用膜联蛋白II功能缺陷的跨显性细胞系来验证gB：膜联蛋白II相互作用对病毒进入和病毒粒子成熟很重要的假设。由于gB是疫苗和抗病毒策略的确定靶点，我们的研究结果将对HCMV疾病的治疗和预防具有重要意义。

Glycoprotein B (gB) of human cytomegalovirus (HCMV) 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. Envelope glycoproteins are primary determinants of tissue tropism and the mediators of virus entry, cell-cell spread and egress/maturation of virions. The long term goal of our research is to define the molecular interactions with HCMV envelope proteins and cellular components that mediate critical steps in the lifecycle such as entry, cell to cell spread and virion maturation/egress. We learned that gB is an essential gene product required for infection of cells. We discovered that gB is a authentic ligand for HCMV, binding to one or more classes of receptors on the surface of cells. The interaction of gB with its receptor(s) is biologically relevant since virus entry was blocked when gB binding sites were occupied. 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. We will further characterize the nature of the gB signaling pathway by defining the composition of signaling effectors activated in response to gB and the virus. These experiments will disclose receptor candidates that will be functionally tested using a newly identified HCMV entry-defective cell line. Expression cloning strategies are also designed to isolate a cDNA clone encoding an HCMV entry receptor. In preparation for structure/function analysis, we produced a large panel of generalized mutations throughout gB. All the mutants were characterized with respect to higher order structure, protein folding and intracellular trafficking. We have also developed a novel and innovative expression strategy to functionally test the mutants. A modified vesciular stomatitis virus lacking its single envelope gene and encoding a green fluorescent protein can be pseudotyped with gB and gB mutants. The concentrated, membrane-anchored display of this expression system has many experimental advantages and will be used to functionally characterize gB mutants in terms of ligand, signaling and fusogenic activities. These experiments will yield the first structure/functional map of any HCMV envelope protein. A third line of investigation will further characterize the role of a known HCMV and gB binding partner, annexin II. Annexin II lacks a demonstrated role in virus entry and we will test the hypothesis that the gB: annexin II interaction is important for egress and virion maturation using trans-dominant cell lines that are annexin II functionally deficient. 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 HCMV disease.