Herpesvirus Proteinase - Possible Target for Antivirals

描述（由申请人提供）：疱疹病毒成熟蛋白酶对感染性病毒的生产至关重要，在衣壳组装过程中作为具有多种功能的前体合成。自12年前发现以来，它作为抗病毒药物的可能靶标受到了相当大的关注，在过去的7年里，关于这种有趣的酶的大量信息已经积累起来，尽管具有挑战性。28-kDa酶是由74-kDa前体通过自身蛋白水解裂解产生的，首先在其羧基端附近的(M)饱和位点，然后在(R)释放位点。r位点的裂解使组装蛋白脱离前体约44 kda的羧基部分。巨细胞病毒的蛋白水解结构域称为组装素，已被克隆、纯化并在体外进行了研究。该酶是由二聚体激活的，但二聚体对有两个独立的活性位点，而且它与其他丝氨酸蛋白酶有明显的不同，它具有新的7链（桶状）和催化三联体（Ser-His-His）。没有关于该前体的可比信息。

DESCRIPTION (provided by applicant): The herpesvirus maturational protease is essential for the production of infectious virus and is synthesized as a precursor that has multiple functions during capsid assembly. Since its discovery 12 years ago, it has received considerable attention as a possible target for antivirals, and in the past 7 years a wealth of information about this interesting, albeit challenging, enzyme has accumulated. The 28-kDa enzyme is derived from a 74-kDa precursor by autoproteolytic cleavage, first at the (M)aturational site near its carboxyl end, and then at the (R)elease site. R-site cleavage frees assemblin from the approximately 44-kDa carboxyl portion of the precursor. The proteolytic domain, called assemblin in cytomegaolvirus, has been cloned, purified, and studied in vitro. The enzyme is activated by dimerization but the dimer pair has two separate active sites, moreover, it differs remarkably from other serine proteases by its new fold (7-stranded ( barrel) and catalytic triad (Ser-His-His). No comparable information is available about the precursor. Our objective is to learn more about this viral enzyme through biochemical and genetic studies, with particular attention to its precursor. The specific aims are intended to help accomplish that objective by (i) applying mutant viruses to study the mechanism of this protease during virus infection, (ii) developing a protease mutant whose activity can be switched on in cells and in vitro by chemical rescue, (iii) investigating the ability of two catalytic-site mutants to form a complementation complex with selective specificity for the maturational-cleavage site, (iv) taking advantage of recent findings to investigate the structure of the precursor and compare its enzymatic properties with those of assemblin, and (v) investigating requirements and consequences of dimerization by both forms of the enzyme. Results of this work are anticipated to provide useful new information about this novel member of the serine proteinase family, and contribute to development of inhibitors that will block its function. It is also likely that useful new information will be generated in the areas of viral proteinase mechanisms and herpesvirus replication.