FUNCTIONAL ANALYSIS OF GLYCOPROTEIN GD OF HERPES SIMPLEX VIRUS

单纯疱疹病毒糖蛋白GD的功能分析

• 批准号: 6104740
• 负责人: GARY H COHEN
• 金额: $ 12.07万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-12-01 至 2000-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6104740
• 关键词: affinity chromatography; conformation; enzyme linked immunosorbent assay; epitope mapping; gene mutation; genetic strain; glycoprotein structure; glycoproteins; glycosylation; herpes simplex virus 1; herpes simplex virus 2; immunoprecipitation; membrane fusion; monoclonal antibody; mutant; neurotropic virus; oligosaccharides; protein structure function; recombinant virus; site directed mutagenesis; transfection; virion; virus envelope; virus genetics; virus infection mechanism; virus protein; virus receptors; western blottings

Herpes simples viruses cause a variety of human diseases, including cold sores, eye and genital infections, neonatal infections and encephalitis. The virion envelope contains a number of glycoproteins which play important roles in HSV infection and pathogenesis. This grant is focused on glycoprotein D, which is essential for virus entry and is important for HSV neuroinvasiveness. Structural and immunological properties of gD make it an important candidate for a subunit vaccine against infections by HSV-1 (oral) and HSV-2 (genital). The objective of this study is to define the functions of gD and to relate them to its structure. The major approach in this renewal application is to construct mutations in the gD gene by site-directed mutagenesis and to test the ability of the altered gD protein to function biologically by use of a complementation assay. The structure of the mutated proteins expressed in transfected mammalian cells will be examined with a panel of monoclonal antibodies. Other properties of the expressed proteins, including aggregation, processing and transport to the cell surface will also be investigated. Changes which alter the ability of gD to function, but otherwise have little or no effect on these other properties, will identify important functional regions. Certain mutations in the gD gene will be recombined into the virus genome and the effect of these mutations on the phenotype of the recombinant viruses will be assayed in cell culture and in an animal model of neuroinvasion. There is good evidence that gD interacts with a specific cell receptor as an important step leading to virus-cell fusion, and two broad approaches will be used to examine this issue further. First, experiments will be carried out to determine if the initial interaction between HSV and the cell involves alterations in gD conformation, or in the conformation of other HSV glycoproteins. Experiments will examine the state of the glycoproteins immediately after infection with radiolabeled purified virus. In the second approach, experiments are designed to identify and characterize the cell surface molecule (receptor) with which gD interacts. The major experimental approach will be the use of a gD affinity column to isolate the cell receptor. The specific aims are: 1) to analyze the effect of specific mutations in the gD gene on the conformation, biological properties and function of the protein; 2) to determine if the conformation of gD or other viral glycoproteins changes as a result of virus entry; 3) to determine if gD mutations affecting cell entry also influence neuroinvasiveness; and 4) to identify cell surface molecules (receptors) which interact with HSV gD.

单纯疱疹病毒引起多种人类疾病，包括感冒 疮、眼睛和生殖器感染、新生儿感染和脑炎。 病毒体包膜含有大量的糖蛋白， 在HSV感染和发病机制中的重要作用。 这笔赠款的重点是 糖蛋白D，这是病毒进入所必需的， 单纯疱疹病毒神经侵袭性 的结构和免疫学特性 gD使其成为抗gD亚单位疫苗的重要候选者 HSV-1（口腔）和HSV-2（生殖器）感染。 的目的 研究的目的是定义gD的功能，并将它们与其 结构 在这种更新申请的主要方法是， 通过定点诱变在gD基因中构建突变， 测试改变的gD蛋白生物学功能的能力， 使用互补测定。 突变蛋白的结构 在转染的哺乳动物细胞中表达的蛋白将用一个板检查 单克隆抗体。 表达的蛋白质的其他性质， 包括聚集、加工和转运至细胞表面 也被调查。 改变gD功能的变化， 但对这些其它性质影响很小或没有影响， 确定重要的功能区。 gD基因的某些突变 会重组到病毒基因组中， 重组病毒表型上的突变将在 细胞培养和神经侵袭的动物模型中。 有好 证据表明gD与特定细胞受体相互作用， 导致病毒-细胞融合的步骤，将使用两种广泛的方法 进一步研究这个问题。 首先，将进行实验 以确定单纯疱疹病毒与细胞之间的初始相互作用是否涉及 gD构象或其他HSV构象的改变 糖蛋白 实验将检查糖蛋白的状态 感染后立即用放射性标记的纯化病毒。 在 第二种方法，设计实验来识别和表征 与gD相互作用的细胞表面分子（受体）。 主要 实验方法将是使用gD亲和柱分离 细胞受体。 具体目的是：1）分析 在gD基因的构象，生物学上的特定突变 蛋白质的性质和功能; 2）确定 gD或其他病毒糖蛋白的构象变化是由于 3）确定影响细胞进入的gD突变是否也 影响神经侵袭性;以及4）鉴定细胞表面分子 （受体）与HSV gD相互作用。