FUSION DOMAINS OF HSV-1 PROTEINS

HSV-1 蛋白质的融合域

• 批准号: 3455132
• 负责人: Konstantin G Kousoulas
• 金额: $ 9.56万
• 依托单位: LOUISIANA STATE UNIV A&M COL BATON ROUGE
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-12-01 至 1994-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3455132
• 关键词: DNA replication; Herpes simplex disease; Herpesviridae; cell fusion; fusion gene; gene complementation; gene mutation; genetic mapping; genetic markers; genetic transcription; glycoprotein biosynthesis; glycoproteins; mutagens; nucleic acid sequence; protein sequence; protein transport; simian virus 40; suppressor mutations; transposon /insertion element; virus DNA; virus protein

Herpes viruses are important pathogens of humans and animals. They enter eukaryotic cells by fusion of their viral envelope with the cellular membrane, and spread partly by inducing fusion of adjacent cellular membranes. Our long term goal is to understand the mechanisms of viral-entry into cells and virus- induced cell fusion. HSV-1 glycoproteins B(gB), D(gD) and H(gH) and a hydrophobic protein with membrane spanning potential termed the Fusion Factor (FF) are involved in these fusion events. Viruses that carry single mutations in gB (gB-Syn3) and FF (FF- Syn1) cause extensive fusion of infected cells and enter faster than wild-type viruses. We propose to elucidate interactions among these proteins that are important for virus-entry and virus-induced cell fusion. Our approach is two-fold: first, a genetic component focuses on the identification of domains of viral proteins that interact with gB and FF via the isolation of intergenic and intragenic suppressor mutations of the gB-Syn1, and FF-Syn3 mutations and other mutations that enhance fusion at low pH conditions. Intragenic and intergenic suppressor mutations of the Syn1 and Syn3 loci will be isolated from viruses grown in the presence of mutagens. In addition, we will isolate mutations in HFEM(tsB5; gB-Syn3) and mP(MP; FF-Syn1) viruses that enable them to cause extensive fusion at pH 6.5. These mutations will be mapped on the viral genome in marker-rescue and marker- transfer experiments and mutations will be identified by DNA sequencing. Second, a physical component aims to assess the effect of co-expression of gB, gD, gH and FF on their synthesis, subcellular localization and transport to the cell surface and further aims to develop a virus-dependent model system for cell fusion to investigate physical interactions among these proteins. Glycoproteins gB, gB(Syn3), gD, gH, fusion factor(FF), and FF(Syn1) will be cloned into SV40 transient expression vectors and co-expressed in COS cells and Vero cells to define different combinations of these proteins that cause cell fusion. A permanent cell line will be constructed expressing a combination of proteins that cause cell fusion under inducible conditions. The synthesis, maturation and transport of each protein to the cell surface will be examined. Amino-terminal and carboxy- terminal truncations of each of the proteins will be used in complementation experiments to define interacting domains. A cell line transformed with a DNA fragment that codes for the FF protein will be used to isolate a FF-null virus and the intracellular distribution of the FF will be investigated.

疱疹病毒是人类和动物的重要病原体。 他们通过融合病毒包膜进入真核细胞 使用细胞膜，部分通过诱导融合而扩散 相邻的细胞膜。 我们的长期目标是 了解病毒进入细胞和病毒的机制 诱导细胞融合。 HSV-1糖蛋白B（GB），D（GD）和H（GH） 和疏水蛋白具有跨膜的潜在蛋白 融合因子（FF）参与了这些融合事件。 在GB（GB-Syn3）和FF（FF-）中携带单个突变的病毒 SYN1）引起被感染的细胞的广泛融合并更快地进入 比野生型病毒。 我们建议阐明相互作用 在这些蛋白质中，对于病毒入门很重要 病毒诱导的细胞融合。 我们的方法是两个方面：首先 遗传成分侧重于识别域的识别 通过分离与GB和FF相互作用的病毒蛋白 GB-SYN1的基因间和基因内抑制突变 以及FF-Syn3突变和其他增强融合的突变 低pH条件。 基因内和基因间抑制剂 Syn1和Syn3基因座的突变将从病毒中分离出来 在诱变剂存在下生长。 此外，我们将隔离 HFEM（TSB5； GB-SYN3）和MP（MP; FF-SYN1）病毒中的突变 使它们能够在pH 6.5处引起广泛的融合。 这些突变 将在标记和标记中的病毒基因组上映射 传输实验和突变将通过DNA鉴定 测序。 第二，物理成分旨在评估 GB，GD，GH和FF共表达对它们的合成的影响， 亚细胞定位并转运到细胞表面， 进一步的旨在开发依赖病毒的模型系统 融合研究这些蛋白质之间的物理相互作用。 糖蛋白GB，GB（SYN3），GD，GH，融合因子（FF）和 FF（SYN1）将被克隆到SV40瞬态表达向量中 并在COS细胞和Vero细胞中共表达以定义不同的 这些引起细胞融合的蛋白质的组合。 一个 将构建永久性细胞系以表达组合 在诱导型条件下引起细胞融合的蛋白质。 每种蛋白质的合成，成熟和转运 将检查细胞表面。 氨基末端和羧基 - 每种蛋白质的末端截断将用于 互补实验以定义相互作用的域。 一个 细胞系用代码为FF的DNA片段转换 蛋白质将用于隔离FF无效病毒，并 将研究FF的细胞内分布。