Herpesvirus Proteinase - Possible Target for Antivirals

描述(申请人提供)：疱疹病毒成熟蛋白是生产传染性病毒所必需的，是在衣壳组装过程中合成的具有多种功能的前体。自从12年前发现这种酶以来，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、尽管具有挑战性的酶的信息已经积累了大量。28 kDa的酶是由74 kDa的前体通过自身蛋白水解酶裂解而来的，首先在其羧端附近的(M)饱和位点，然后在(R)酶解位点。R-位切割使装配素从前体的大约44 kDa的羧基部分解脱出来。蛋白水解区在巨细胞病毒中称为集合素，已被克隆、纯化并在体外进行了研究。该酶被二聚体激活，但二聚体对有两个独立的活性部位，而且它的新折叠(7-链(桶)和催化三联体(Ser-His-His))明显不同于其他丝氨酸蛋白酶。目前还没有关于该前体的可比信息。 我们的目标是通过生化和遗传学研究，特别是对其前体的研究，了解更多关于这种病毒酶的知识。具体目的旨在通过以下方式帮助实现这一目标：(I)应用突变病毒来研究病毒感染期间该酶的机制，(Ii)开发一种可通过化学拯救在细胞内和体外启动其活性的蛋白水解酶突变体，(Iii)研究两个催化位点突变体形成对成熟裂解位点具有选择性专一性的互补复合体的能力，(Iv)利用最近的发现来研究前体的结构并将其与组装酶的酶性质进行比较，以及(V)研究两种形式的酶的二聚化的要求和结果。这项工作的结果有望为这一丝氨酸蛋白酶家族的新成员提供有用的新信息，并有助于开发将阻断其功能的抑制剂。还有可能在病毒蛋白水解酶机制和疱疹病毒复制方面产生有用的新信息。

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.