HUMAN CYTOMEGALOVIRUS ENVELOPE GLYCOPROTEINS

病毒包膜糖蛋白在细胞膜上表达和/或被整合到包膜中，介导生命周期的关键步骤，如感染的进入、退出和细胞间传播。人类巨细胞病毒（HCMV）感染的一个标志性和决定性特征是它能够进入不同发育谱系的细胞类型并在细胞间传播。由于很少有细胞表面分子分布在所有这些细胞类型上，因此很可能有多种病毒糖蛋白参与HCMV的发病机制。序列分析预测了67种可能的糖蛋白，反映了利用多种病毒配体/细胞相互作用的潜力。HCMV包膜的生化分析显示存在至少10种糖蛋白，其中许多被组织成大的二硫连接复合物。然而，这些蛋白质中只有5个被映射到病毒基因组中。在描述病毒进入、离开和传播的机制之前，必须确定包膜的组成和编码包膜蛋白的基因。本文概述了一个实验计划，将确定两种主要HCMV包膜复合物gCII和gCIII的组成。这两种复合物都与病毒-细胞相互作用有关，并且都含有已识别的（分别为gM和gH/gL）和未识别的成分。这些复合物已知成分的单克隆抗体的可用性将允许纯化gCII和gCIII。单个复杂蛋白的进一步纯化和分离将先于微序列分析和未定位糖蛋白的遗传鉴定。这种方法的可行性是由重要的初步数据，关于识别以前未映射的组分gCIII。一旦新映射的蛋白质的试剂产生，复合物的组成将被验证。生物物理参数，如gCII和gCIII的分子质量和化学计量学将使用各种生化技术来确定。生物合成途径和感染细胞内的细胞定位将被表征。这些研究将促进我们对病毒粒子成熟和退出的理解，这是HCMV生命周期中存在巨大知识缺口的一个领域。在针对gCII和gCIII的未来功能分析的研究中，将通过单个组分的共表达来重组复合物。在研究过程中获得的信息对未来进一步了解HCMV的生命周期和发病机制至关重要，也可能作为未来抗病毒药物设计的基础。

Viral envelope glycoproteins expressed on cellular membranes and/or incorporated into the envelope, mediate critical steps in the lifecycle such as entry, egress and cell to cell spread of infection. A hallmark and defining feature of human cytomegalovirus (HCMV) infection, is its capacity to enter and spread cell to cell between cell types of distinct and divergent developmental lineages. Since few cell surface molecules are distributed on all of these cell types, it is likely that multiple viral glycoproteins participate in HCMV pathogenesis. Sequence analysis predicts 67 possible glycoproteins reflecting the potential to utilize multiple viral ligand/cellular interactions. Biochemical analysis of HCMV envelope revealed the presense of at least 10 glycoproteins, many of which are organized into large disulfide-linked complexes. However, only 5 of these proteins are mapped to the viral genome. Before the mechanism of viral entry, egress and spread can be delineated, the composition of the envelope must be defined and the genes encoding the envelope proteins identified. This outlines an experimental plan that will define the composition of two major HCMV envelop complexes, gCII and gCIII. Both of these complexes are implicated in virus-cell interactions and both contain identified (gM and gH/gL respectively) as well as unidentified components. The availability of monoclonal antibodies to the known constituents of these complexes will allow purification of gCII and gCIII. Further purification and separation of individual complex proteins will precede microsequence analysis and genetic identification of the unmapped glycoproteins. The feasibility of this approach is blostered by significant preliminary data regarding the identification of a previously unmapped component of gCIII. Once reagents to the newly mapped proteins are produced, the composition of the complexes will be verified. Biophysical parameters such as the molecular mass and stoichiometry of gCII and gCIII will be determined using a variety of biochemical techniques. The biosynthetic pathway and cellular localization within infected cells will be characterized. These studies will facilitate our understanding of virion maturation and egress, an area of the HCMV lifecycle with enormous gaps in knowledge. In studies aimed at future functional analysis of the gCII and gCIII, the complexes will be reconstituted by co-expression of the individual components. The information learned in the course of the studies is critical for future advances in the understanding of the HCMV lifecycle and pathogenesis and may also serve as the basis for future antiviral drug design.