HERPESVIRUS PROTEINASE--POSSIBLE TARGET FOR ANTIVIRAL

疱疹病毒蛋白酶——抗病毒的可能靶点

• 批准号: 3148046
• 负责人: D Wade Gibson
• 金额: $ 18.44万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1997-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3148046
• 关键词: Herpesviridae; active sites; antiserum; antiviral agents; cell free system; cytomegalovirus; endopeptidases; enzyme biosynthesis; enzyme mechanism; enzyme structure; gene deletion mutation; human tissue; laboratory rabbit; molecular site; protein sequence; proteolysis; site directed mutagenesis; transfection /expression vector; ultracentrifugation; virus protein; virus replication; zymogens

A cytomegalovirus gene encoding a maturational proteinase has been identified, cloned, and sequenced. The gene is 1,770 bp in length and gives rise to a protein that has an M of approximately 85 kDa, as estimated by polyacylamide gel electrophoresis. The enzyme active site appears to be located in the amino one-half of the protein -- probably within two domains that are highly conserved in the homologous proteins of other herpesviruses. The substrate of this proteinase is a protein referred to as the assembly protein precursor. There is evidence that cleavage of the precursor to the mature assembly protein is essential for herpesvirus maturation. Therefore, interference with this cleavage event would be expected to have a potent antiviral effect. The specific site at which the viral proteinase cleaves its substrate has been determined, and a transfection assay utilizing the cloned genes for the proteinase and its substrate has been developed to monitor the cleavage event. Studies proposed in this application are intended to further characterize this herpesvirus maturational proteinase, and to evaluate its potential as a new target for antiviral drug development. Five specific aims are listed which seek to (i) verify that the enzyme is freed from a much larger precursor by cleavage at a putative consensus enzyme "release" site; (ii) identify the functionally important amino acids in both the enzyme "release" and substrate "maturational" cleavage sites, and determine what features distinguish the two; (iii) delineate the active site domain of the proteinase and identify functionally important amino acids within that region; (iv) develop a cell-free system to permit quicker, more versatile, and quantitative assays of the proteinase; and (v) produce large amounts of the active viral proteinase for biochemical and crystallographic studies. Results of this work are anticipated to provide valuable leads for developing perhaps broad spectrum antiherpesvirus drugs targeted to the maturational proteinase. It is also likely that useful new information will be gained in the general area of viral proteinase mechanisms, and in the specific area of herpesvirus replication.

一种编码成熟蛋白酶的巨细胞病毒基因已经被 鉴定、克隆和测序。该基因全长1,770个核苷酸， 产生一种蛋白质，据估计，M约为85 kDa 经聚丙烯酰胺凝胶电泳法测定。酶的活性部位似乎是 位于蛋白质一半的氨基上--可能在两个区域内 它们在其他生物的同源蛋白中高度保守 疱疹病毒。这种蛋白水解酶的底物是指 作为组装蛋白的前体。有证据表明乳沟的分裂 成熟组装蛋白的前体对疱疹病毒是必不可少的 成熟。因此，对这种切割事件的干扰将是 预计会有很强的抗病毒作用。的特定地点。 病毒蛋白水解酶裂解其底物已经确定，并且一种 利用所克隆的基因进行蛋白水解酶及其蛋白的转染法 已经开发了底物来监测切割事件。 本申请中提出的研究旨在进一步表征 这种疱疹病毒成熟蛋白水解酶，并评价其作为 抗病毒药物开发的新靶点。列出了五个具体目标 它们寻求(I)验证酶是否从更大的 前体在假定的共识酶“释放”部位被切割；(2) 鉴定这两种酶中具有重要功能的氨基酸 “释放”和底物“成熟”的裂解位点，并决定什么 特征区分了两者；(Iii)描述了 并鉴定其中具有重要功能的氨基酸 区域；(4)发展无牢房系统，以便更快、更多用途、 和定量检测；和(V)产生大量的 用于生化和结晶学研究的活性病毒蛋白酶。 这项工作的结果有望为以下工作提供有价值的线索 可能正在开发广谱的抗疱疹病毒药物 成熟蛋白水解酶。也有可能是有用的新信息 将在病毒蛋白水解酶机制的一般领域获得，并在 疱疹病毒复制的特定区域。