Cytomegalovirus Deubiquitinase pUL48: Identifying Substrate and Inhibitors

描述(申请人提供)：最近发现疱疹病毒群编码一种泛素特异性半胱氨酸蛋白酶，其生物学功能尚不清楚。它的催化结构域位于300到400个氨基酸序列中，这是每个疱疹病毒基因组编码的最大蛋白质的氨基末端。虽然它的活性对病毒复制并不是绝对必要的，但它似乎提高了这一过程的效率。我们发现这种脱泛素化酶(DUB)在全长蛋白质的背景下是活跃的，这导致了两个关键发现，这里提出的工作是基于这两个发现。首先，DUB是感染性病毒粒子的组成部分，其次，它是纯化的病毒粒子中存在的主要DUB活性，如果不是唯一的DUB活性的话。我们正在研究人类巨细胞病毒(HCMV，HHV5)中的疱疹病毒，因为这种性传播因子与艾滋病、器官移植、癌症化疗和由胎盘感染引起的出生缺陷的医学相关性越来越大。它也是典型的2-疱疹病毒，有必要识别和了解它与2-和3-疱疹病毒成员之间的差异。在人类巨细胞病毒中，具有DUB活性的蛋白由开放阅读框UL48编码，大小为253 kDa，被称为高分子量蛋白(HMWP)或pUL48。我们提出的具体目标直接来自于已发表的和初步研究的结果，这些研究旨在确定这种酶的生物学目的，并确定其作为抗病毒靶点的潜力。我们的直接目标是证实这一假设，即HCMV病毒粒子包含病毒Dub的底物(对了解功能和机制至关重要，但迄今尚未确定)，并开发一种能够适应高通量形式的方法来筛选HCMV Dub的抑制剂。我们将结合遗传、物理、生化、酶和生物学的方法，在一个多学科的环境中实现这些目标。与公共卫生相关：巨细胞病毒是一种疱疹病毒群病毒，它对免疫系统薄弱的人构成威胁，包括非常年轻和非常年长的人。这项研究针对的是最近发现的一种蛋白酶，它可以从蛋白质中去除泛素，但其作为抗病毒靶标的功能和潜力尚不清楚。我们建议在高通量分析中确定该酶的底物(S)并确定其活性的抑制剂。

DESCRIPTION (provided by applicant): Herpes-group viruses have recently been discovered to encode a ubiquitin-specific cysteine protease, whose biological function is unknown. Its catalytic domain lies within a 300 to 400 amino-acid sequence that is the amino terminal end of the largest protein encoded by each herpesvirus genome. Although its activity is not absolutely essential for virus replication, it appears to enhance the efficiency of the process. Our discovery that this deubiquitinylating enzyme (DUB) is active in the context of the full-length protein led to two key findings on which the work proposed here is based. First, the DUB is an integral constituent of infectious virions and second, it is the predominant if not sole DUB activity present in purified virions. We are studying the herpesvirus DUB in human cytomegalovirus (HCMV, HHV5) because this sexually transmissible agent is of increasing medical relevance in association with AIDS, organ transplantation, cancer chemotherapy, and birth defects resulting from transplacental infections. It is also the prototypic 2-herpesvirus and there is an underlying need to identify and understand differences between it and members of the 2- and 3- herpesviruses. In human cytomegalovirus the protein hosting the DUB activity is encoded by open reading frame UL48, is 253 kDa, and is called the high-molecular-weight protein (HMWP) or pUL48. The specific aims we propose follow directly from results of published and pilot studies done to define the biological purpose of this enzyme and determine its potential as an antiviral target. Our immediate aims are to substantiate the hypothesis that HCMV virions contain a substrate of the viral DUB (critical to understanding function and mechanism, but so far unidentified), and to develop an assay that can be adapted to high-throughput format to screen for inhibitors of the HCMV DUB. We will apply a combination of genetic, physical, biochemical, enzymological, and biological approaches to accomplish these aims in a multidisciplinary environment. PUBLIC HEALTH RELEVANCE: Cytomegalovirus is a herpes-group virus that is a threat to people with weakened immune systems, including the very young and the very old. This research is directed at a recently discovered protease that removes ubiquitin from proteins, but whose function and potential as an antiviral target are unknown. We propose to identify the substrate(s) of this enzyme and identify inhibitors of its activity in high-throughput assays.