HERPESVIRUS PROTEINASE--POSSIBLE TARGET FOR ANTIVIRAL

巨细胞病毒编码成熟蛋白酶的基因已被发现， 鉴定、克隆和测序。 该基因长度为1，770 bp， 产生具有约85 kDa的M的蛋白质，如估计的 通过聚丙烯酰胺凝胶电泳。 酶的活性位点似乎是 位于蛋白质氨基的一半--可能在两个区域内 在其他同源蛋白中高度保守的蛋白质， 疱疹病毒 这种蛋白酶的底物是一种蛋白质， 组装蛋白前体。 有证据表明， 成熟装配蛋白的前体是疱疹病毒所必需的 成熟 因此，干扰这种裂解事件将是 预期具有有效的抗病毒作用。 具体地点， 病毒蛋白酶切割其底物已被确定， 利用蛋白酶的克隆基因的转染测定及其 已经开发了底物来监测裂解事件。 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酶，并评估其潜力， 抗病毒药物开发的新靶点。 列出了五个具体目标 其试图（i）验证酶从大得多的 通过在推定的共有酶“释放”位点切割前体;（ii） 确定酶中功能重要的氨基酸 “释放”和底物“成熟”裂解位点，并确定 特征区分两者;（iii）描绘活性位点域的 蛋白酶，并确定其中功能重要的氨基酸， 区域;（iv）开发无细胞系统，以允许更快，更通用， 和蛋白酶的定量测定;和（v）产生大量的 用于生物化学和晶体学研究的活性病毒蛋白酶。 这项工作的结果预计将提供宝贵的线索， 开发广谱抗疱疹病毒药物， 成熟蛋白酶 也有可能有用的新信息 将在病毒蛋白酶机制的一般领域获得， 疱疹病毒复制的特定区域。

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.