STUDIES ON THE PENTAMERIZATION OF SV40 VP1 IN VITRO

SV40 VP1五聚化的体外研究

• 批准号: 6107382
• 负责人: EDITTE GHARAKHANIAN
• 金额: --
• 依托单位: CALIFORNIA STATE UNIVERSITY LONG BEACH
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-08-01 至 1999-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6107382
• 关键词: SDS polyacrylamide gel electrophoresis; capsid; cell nucleus; genetic transcription; genetic translation; intermolecular interaction; intracellular transport; protein transport; sedimentation; simian virus 40; site directed mutagenesis; virus protein

Simian Virus 40 (SV40) belongs to the papovavirus family of oncogenic DNA animal viruses. The nuclear site of assembly of the virus as well as the nuclear activities of its large-Tumor-Antigen, have rendered it an ideal model system for the study of nuclear localization and nuclear localization signals. Regulated nuclear protein import of different cellular proteins, including the rel oncoprotein, the glucocorticoid receptor, and the dorsal early developmental protein, is essential to normal cell growth and development. Understanding the nuclear transport machinery is a first step towards studying disease states caused by mislocalization of nuclear proteins. The SV40 capsid is comprised of three structural proteins --Vp1, Vp2, and Vp3. Vp1 is the major capsid protein of SV40 accounting for at least 70% of the total virion protein by weight. VP1 forms pentamers in the cytoplasm of the infected cells and is then transported to the nucleus. The chemical nature of the bonds that stabilize the VP1 pentamers has not been established. We have been interested in identifying the nature of the interactions that stabilize the VP1 pentamers, as well as studying the role of pentamerization in the nuclear transport of VP1. In the past year, results of VP1 reduction/alkylation experiments obtained in our laboratory strongly implicate the involvement of disulfide bridges in the formation of Vp1 pentameters in vitro. SV40 Vp1 contains seven cysteine residues, any or several of which could be involved in disulfide linkage(s). We have initiated studies to identify Vp1 cysteine residues involved in those interactions. Based on a model of how two internal domains of single Vp1 peptide chain interact to form an intramolecularly folded Vp1 protein and may be available for intermolecular interactions with other Vp1's. Oligo-directed mutagenesis of Cys10 and Cys50 is presently in progress. In vitro protein-protein interaction assays will enable a rapid screening of generated mutants. Eventually, all seven cysteines of Vp1 will be mutagenized and assayed. Mutants defective in multimerization in vitro will be further studies for the extent and efficiency of their nuclear transport in vivo. The project provides an excellent opportunity for student involvement in both basic and advanced molecular biological technique, as well as for development of skills and interests in biomedical research. Currently, 3 MBRS students are involved in these studies in our laboratory. They will be presenting their results shortly at National meetings including the MBRS annual symposium and the Annual Meeting of the Society for Cell Biology.

猴病毒40（SV 40）属于乳多空病毒科的致癌DNA 动物病毒 病毒的核组装位点以及 其大肿瘤抗原的核活性，使其成为理想的 研究核定位和核动力学的模型系统 定位信号 调节不同的核蛋白输入 细胞蛋白，包括rel癌蛋白，糖皮质激素 受体和背侧早期发育蛋白，是必不可少的， 正常的细胞生长和发育。 了解核运输 机械是研究由疾病引起的疾病状态的第一步 核蛋白的错误定位。 SV 40衣壳由三种结构蛋白--Vp 1、Vp 2和Vp 3--组成。 Vp 3。 VP 1是SV 40的主要衣壳蛋白，约占SV 40的70%以上 占总病毒体蛋白的重量。 VP 1在细胞中形成五聚体。 感染细胞的细胞质，然后被运送到细胞核。 稳定VP 1五聚体的键的化学性质还没有改变。 确立了习 我们一直在研究 稳定VP 1五聚体的相互作用，以及研究 五聚体化在VP 1核转运中的作用。 过去 2009年，我们在我们的实验室中获得了VP 1还原/烷基化实验的结果， 实验室强烈暗示二硫桥参与了 在体外形成Vp 1五聚体。 SV 40 Vp 1含有7个半胱氨酸 残基，其中任何一个或几个可能涉及二硫化物 链接。 我们已经开始研究，以确定Vp 1半胱氨酸残基 参与这些互动。 基于两个内部模型 单个Vp 1肽链的结构域相互作用形成分子内 折叠的Vp 1蛋白，并可用于分子间相互作用 与其他VP 1的。 Cys 10和Cys 50的寡核苷酸定向诱变是 正在进行中体外蛋白质-蛋白质相互作用测定将 能够快速筛选产生的突变体。 最终，所有七个 将对Vp 1的半胱氨酸进行诱变并进行测定。 突变体缺陷， 体外多聚化的程度和程度有待进一步研究 其在体内的核运输效率。 该项目为学生参与提供了一个极好的机会， 基础和先进的分子生物学技术，以及 发展生物医学研究的技能和兴趣。 目前， 3名MBRS学生参与了我们实验室的这些研究。 他们 不久将在国家会议上介绍他们的成果， MBRS年度研讨会和细胞学会年会 生物学