KINETICS AND MECHANISMS OF VIRUS-CELL MEMBRANE FUSION

病毒-细胞膜融合的动力学和机制

• 批准号: 3138945
• 负责人: NEJAT A DUZGUNES
• 金额: $ 21.07万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-02-01 至 1993-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3138945
• 关键词: AIDS; T lymphocyte; Vesiculovirus; acidity /alkalinity; calorimetry; cell membrane; cholesterol; coulometry; electron microscopy; erythrocyte membrane; fluorescence microscopy; fluorescent dye /probe; glycolipids; glycoproteins; human immunodeficiency virus 1; liposomes; membrane activity; membrane fusion; membrane proteins; parainfluenza virus type 1; phospholipids; surface antigens; synthetic peptide; tissue /cell culture; virus cytopathogenic effect; virus envelope; virus infection mechanism

Lipid enveloped viruses infect their host cell by fusing with either the plasma membrane (Sendai virus), or the endosome membrane after endocytosis (influenza and vesicular stomatitis viruses). Human immunodeficiency virus (HIV) infects CD4+ cells by binding to the CD4 antigen and induces extensive cell-cell fusion. Whether HIV fuses with the plasma membrane or the endosome is not known. We will investigate the kinetics of fusion of all these viruses with their host cells, by means of fluorescence assays. We will determine the role of the host (target) membrane components in fusion with the viruses, by selective removal of particular molecules or by using liposomes as target membranes. Using a theoretical analysis, we will establish the rate constants of adhesion and fusion of the viruses with the target membranes, under various conditions. The fusion activity of the viruses is mediated by envelope glycoproteins. We will reconstitute these proteins in phospholipid vesicles, to produce "virosomes" which have the same requirements for fusion (such as pH) as the intact virus. We will then study the effect of the membrane composition of virosomes on their fusion with a number of target membranes. We will also use the virosomes to deliver encapsulated macromolecules into the cytoplasm of cultured cells in a pH- dependent manner. We will couple the bromelain digestion fragment of the influenza hemagglutinin (or pH-sensitive synthetic peptides) to liposomes, and determine whether the resulting proteoliposomes fuse with target membranes as a function of pH. Certain hydrophobic sections of the envelope glycoproteins are thought to be involved in the fusion of the virus with the target membrane. We will examine whether synthetic peptides corresponding to these sections penetrate target membranes and induce fusion. We will determine the kinetics of fusion of HIV with T-cell lines and clones. In particular, we will ascertain the involvement in fusion of the CD4 molecule and the envelope protein gp160 of HIV, by means of monoclonal antibodies and by reconstituting these proteins in phospholipid vesicles. These studies will elucidate the molecular mehanisms of viral infection, and indicate means by which infection may be prevented.

脂质包膜病毒通过与其中之一融合来感染宿主细胞 质膜（仙台病毒）或内体膜 内吞作用后（流感病毒和水泡性口炎病毒）。 人类免疫缺陷病毒 (HIV) 通过以下方式感染 CD4+ 细胞： 与 CD4 抗原结合并诱导广泛的细胞-细胞融合。 HIV是否与质膜或内体融合 不知道。 我们将研究所有这些病毒与 他们的宿主细胞，通过荧光测定。 我们将 确定宿主（目标）膜成分的作用 通过选择性去除特定的病毒与病毒融合 分子或使用脂质体作为靶膜。 使用 理论分析，我们将建立速率常数 病毒与目标膜的粘附和融合， 在各种条件下。 病毒的融合活性是由包膜介导的 糖蛋白。 我们将在磷脂中重建这些蛋白质 囊泡，产生具有相同特征的“病毒体” 融合为完整病毒的要求（例如 pH）。 我们将 然后研究病毒体膜成分的影响 关于它们与许多目标膜的融合。 我们还将使用病毒体来递送封装的 大分子进入pH-培养细胞的细胞质 依赖方式。 我们将耦合菠萝蛋白酶消化 流感血凝素片段（或pH敏感的 合成肽）至脂质体，并确定是否 由此产生的蛋白脂质体与靶膜融合作为 pH 值的函数。 包膜糖蛋白的某些疏水部分是 被认为参与病毒与靶标的融合 膜。 我们将检查是否合成肽 对应于这些部分穿透目标膜并且 诱导融合。 我们将确定 HIV 与 T 细胞系融合的动力学 和克隆人。 特别是，我们将确定参与 CD4分子与包膜蛋白gp160的融合 HIV，通过单克隆抗体和重组 这些蛋白质存在于磷脂囊泡中。 这些研究将阐明病毒的分子机制 感染，并指出可能感染的方式 阻止了。