FUNCTIONAL DOMAINS OF HSV-1 GLYCOPROTEINS GB AND GC

HSV-1 糖蛋白 GB 和 GC 的功能域

• 批准号: 2061729
• 负责人: THOMAS C HOLLAND
• 金额: $ 22.57万
• 依托单位: WAYNE STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-04-01 至 1996-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2061729
• 关键词: Alphaherpesvirinae; Herpes simplex disease; antibody neutralization test; cell fusion; cell membrane; gene expression; genetic mapping; genetic promoter element; genetic transcription; glycoproteins; laboratory mouse; laboratory rabbit; membrane proteins; molecular cloning; mutant; plasmids; protein biosynthesis; protein sequence; protein structure function; tissue /cell culture; virion; virus DNA; virus genetics; virus protein; virus replication

Herpes Simplex Virus Type 1 (HSV-1) is the cause of primary and recurrent infections of the mouth, gums, and lip, genital and ocular infections, generalized infections in neonates and immunodeficient individuals, and rare cases of encephalitis. The closely related virus HSV-2 is the cause of genital herpetic lesions and numerous neonatal infections. The process by which HSV-1 absorbs to and enters cells is not well understood. An understanding of this process may well lead to the development of effective and practical antiviral agents and/or immunization methods that block these early stages of infection. Several HSV-1 glycoproteins (gB, gC, gD, and gH) have been proven or are likely to be involved in these processes. Initial interaction of HSV-1 virions with the cell surface may be mediated by binding of viral glycoproteins to cell surface heparan sulfate. The identity of the viral adsorption protein that interacts with heparan sulfate is unclear, since virions lacking gB, gC, or gD all adsorb to cells. However, solubilized gB and gC bind to heparin. It has been suggested that initial binding to cells may be mediated by either glycoprotein. This hypothesis will be tested directly by the production of virions lacking both gB and gC and their use in adsorption experiments. Virions deficit in other combinations of glycoproteins will be tested as necessary. These virions will be produced by introduction of sites recognized by Factor Xa, a highly specific protease, or by recombination of existing glycoprotein deficient mutants. Once adsorbed, virions penetrate by fusion of the virion envelope with the plasma membrane. Three glycoproteins, gB, gC, and gH, have been shown to be essential for this process, although the exact roles of each are unknown. The penetration process is likely to be related to the induction of cell fusion by syncytial mutants of HSV-1, since many of the same glycoproteins have been implicated in each. Also involved in cell fusion is the UL53/syn1 gene. We have performed an extensive in vitro analysis on the protein specific by this gene. Significantly, we have found that the processed from of this protein has a hydrophobic N-terminal end, similar to the hydrophobic N- terminal fusion domains of other viral fusion proteins. It is likely that UL53 acts as a membrane fusogen in the cell fusion process and possibly in penetration as well. Extensive additional characterization of UL53 is proposed to augment our understanding of this molecule. These studies will include determination of the transmembrane structure of the UL53 protein, use of UL53 specific antisera to characterize the protein in vivo, sequencing of additional UL53 syncytial mutants, and production of new UL53 mutants. Finally, a transfection system for induction of syncytium formation by HSV-1 will be developed and used to identify the viral genes that are necessary and sufficient for cell fusion. This will eventually make it possible to probe the mechanism of cell fusion and determine its relationship to virion penetration.

1 型单纯疱疹病毒 (HSV-1) 是原发性和复发性疱疹的病因 口腔、牙龈、嘴唇、生殖器和眼部感染， 新生儿和免疫缺陷个体的全身感染，以及 脑炎的罕见病例。 密切相关的病毒 HSV-2 是病因 生殖器疱疹病变和许多新生儿感染。 过程 HSV-1 吸收并进入细胞的方式尚不清楚。 一个 对这个过程的理解很可能会导致有效的开发 以及阻断这些的实用抗病毒剂和/或免疫方法 感染的早期阶段。 几种 HSV-1 糖蛋白（gB、gC、gD 和 gH）已被证明或可能参与这些过程。 HSV-1 病毒粒子与细胞表面的初始相互作用可能是介导的 通过病毒糖蛋白与细胞表面硫酸乙酰肝素结合。 这 与乙酰肝素相互作用的病毒吸附蛋白的身份 硫酸盐尚不清楚，因为缺乏 gB、gC 或 gD 的病毒粒子都会吸附到 细胞。 然而，溶解的 gB 和 gC 与肝素结合。 它一直 表明与细胞的初始结合可能是由以下任一介导的 糖蛋白。 该假设将直接通过生产来检验 缺乏 gB 和 gC 的病毒粒子及其在吸附实验中的应用。 病毒体在其他糖蛋白组合中的缺陷将被测试为 必要的。 这些病毒体将通过引入位点来产生 由 Xa 因子（一种高度特异性的蛋白酶）或通过重组识别 现有的糖蛋白缺陷突变体。 一旦被吸附，病毒粒子就会渗透 通过病毒体包膜与质膜的融合。 三 糖蛋白 gB、gC 和 gH 已被证明对此至关重要 过程，尽管每个过程的确切作用尚不清楚。 渗透力 该过程可能与诱导细胞融合有关 HSV-1 的合胞体突变体，因为许多相同的糖蛋白已被 各有牵连。 UL53/syn1 基因也参与细胞融合。 我们对特定蛋白质进行了广泛的体外分析 这个基因。 值得注意的是，我们发现经过处理的 蛋白质有一个疏水性的N-末端，类似于疏水性的N-末端 其他病毒融合蛋白的末端融合结构域。 很可能是这样 UL53 在细胞融合过程中充当膜融合剂，并可能在 渗透力也一样。 UL53 的广泛附加特性是 旨在增强我们对这种分子的理解。 这些研究将 包括确定 UL53 蛋白的跨膜结构， 使用 UL53 特异性抗血清来表征体内蛋白质， 对其他 UL53 合胞体突变体进行测序，并生产新的 UL53 突变体。 最后，用于诱导合胞体的转染系统 HSV-1 的形成将被开发并用于鉴定病毒基因 这对于细胞融合是必要且充分的。 这最终将 使得探讨细胞融合机制并确定其融合成为可能 与病毒粒子穿透的关系。