HUMAN CYTOMEGALOVIRUS ENVELOPE GLYCOPROTEINS

病毒包膜糖蛋白在细胞膜上表达和/或结合到包膜上，在病毒生命周期的关键步骤中起中介作用，如进入、离开和感染的细胞间传播。人类巨细胞病毒(HCMV)感染的一个标志和定义特征是它能够在不同和不同发育谱系的细胞类型之间进入细胞并在细胞之间传播。由于在所有这些细胞类型上分布的细胞表面分子很少，很可能是多种病毒糖蛋白参与了HCMV的致病过程。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。对HCMV包膜的生化分析表明，至少存在10种糖蛋白，其中许多糖蛋白被组织成大的二硫键连接的复合体。然而，这些蛋白质中只有5个被映射到病毒基因组上。在描述病毒进入、输出和传播的机制之前，必须确定包膜的组成和编码包膜蛋白的基因。这份报告概述了一项实验计划，该计划将确定两种主要的HCMV包膜复合体gCII和gCIII的组成。这两个复合体都与病毒-细胞相互作用有关，都含有已识别的成分(分别为Gm和Gh/gl)以及未识别的成分。抗这些复合体已知成分的单抗的出现将使gCII和gCIII的纯化成为可能。在对未定位的糖蛋白进行微序列分析和遗传鉴定之前，将对单个复杂蛋白进行进一步的纯化和分离。关于鉴定以前未映射的gCIII组分的重要初步数据证明了这种方法的可行性。一旦新绘制的蛋白质的试剂被生产出来，复合体的组成将得到验证。生物物理参数，如gCII和gCIII的分子质量和化学计量，将使用各种生化技术来确定。将描述感染细胞内的生物合成途径和细胞定位。这些研究将有助于我们理解病毒粒子的成熟和出口，这是人类巨细胞病毒生命周期中的一个领域，在知识上存在巨大差距。在未来对gCII和gCIII进行功能分析的研究中，将通过单独成分的共同表达来重组这些复合体。在研究过程中获得的信息对于未来在理解巨细胞病毒生命周期和发病机制方面的进展至关重要，也可能成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.