Human cytomegalovirus alkaline nuclease (pUL98): potential antiviral target?

描述（由申请方提供）：人巨细胞病毒（HCMV）是疱疹病毒科病毒的一个成员，是一种重要的人类病原体，在移植和艾滋病患者中引起肺炎、失明和死亡，在先天性感染的新生儿中引起智力迟钝和听力损失。目前可用的抗病毒药物靶向病毒DNA聚合酶，但具有显著的毒性。HCMV编码的蛋白质pUL 98在体外具有内切酶和5 '-3'外切核酸酶活性。pUL 98在HCMV复制中的功能尚不清楚;然而，对其他疱疹病毒表达的同源核酸酶的研究表明，它们可能在DNA插入衣壳之前对复杂的多联体DNA复制中间体进行去分支。与噬菌体l reda（核酸外切酶）的相似性和促进由病毒单链DNA结合蛋白介导的链交换反应的能力进一步表明了在重组中的潜在作用，重组是疱疹病毒DNA复制的关键特征。最近的遗传学研究表明，UL 98基因是病毒复制所必需的;因此，pUL 98提出了一个有吸引力的抗病毒干预的目标。在本提案的目的1中，我们将使用诱变和重组蛋白表达鉴定对pUL 98的体外酶活性至关重要的活性位点氨基酸。在目标2中，我们将通过构建突变病毒基因组来确定这些活性对病毒复制的重要性，其中通过选择性活性位点突变使pUL 98酶活性失能。通过这些研究，我们将确定（1）pUL 98是否是HCMV复制所必需的，以及（2）pUL 98的溶核活性是否至关重要。证明pUL 98的酶活性对于HCMV复制是重要的，这将为进一步的研究提供强有力的理由，以通过确定酶活性位点结构和定制药物设计或通过使用体外酶测定法高通量筛选化合物文库来鉴定选择性抑制活性的化合物。这些研究可能导致新的pUL 98抑制剂，其可能在临床上单独或与现有的DNA聚合酶抑制剂组合用于治疗HCMV感染。人巨细胞病毒是美国出生缺陷的主要感染原因，也是艾滋病和移植患者的重要病原体。拟议的研究将探讨病毒碱性核酸酶（pUL 98）作为开发新型抗病毒药物的靶点的潜力。通过评估遗传灭活碱性核酸酶酶活性对病毒复制的影响，可以预测药理学抑制剂的潜在影响。证明这些活动是重要的病毒复制将提供强有力的理由，为未来的努力，以发现化合物的目标，这种蛋白质的活动。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV), a member of the herpesviridae family of viruses, is a significant human pathogen, causing pneumonitis, blindness, and death among transplant and AIDS patients and mental retardation and hearing loss among congenitally infected newborns. Currently available antivirals target the viral DNA polymerase but suffer from significant toxicities. HCMV encodes a protein, pUL98 that has both endonuclease and 5'-3' exonuclease activities in vitro. The function of pUL98 in HCMV replication is not known; however, studies of homologous nucleases expressed by other herpesviruses have suggested that they may function to debranch complex concatemeric DNA replicative intermediates prior to DNA insertion into capsids. Similarities to phage l reda (exonuclease) and the ability to promote strand-exchange reactions mediated by viral single-stranded DNA binding protein further suggest a potential role in recombination, a key feature of herpesvirus DNA replication. Recent genetic studies suggest that the UL98 gene is essential for viral replication; pUL98 therefore presents an attractive target for antiviral interventions. In aim 1 of this proposal we will identify active site amino acids critical for the in vitro enzymatic activities of pUL98 using mutagenesis and recombinant protein expression. In aim 2 we will determine the importance of these activities for viral replication by construction of mutant viral genomes in which pUL98 enzymatic activities are disabled by selective active site mutations. Through these studies we will determine if (1) pUL98 is essential for HCMV replication and (2) if it is pUL98's nucleolytic activities that are crucial. Demonstration that an enzymatic activity of pUL98 is important for HCMV replication will provide strong justification for further studies to identify compounds that selectively inhibit the activity, either through determination of the enzyme active site structure and custom drug design or through high throughput screening of compound libraries using in vitro enzymatic assays. Such studies could lead to novel pUL98 inhibitors that could be clinically useful in treating HCMV infections alone or in combination with existing DNA polymerase inhibitors. Human cytomegalovirus is the major infectious cause of birth defects in the United States and is an important pathogen in AIDS and transplant patients. The proposed studies will investigate the potential of the viral alkaline nuclease (pUL98) as a target for the development of novel antivirals. By evaluating the impact on viral replication of genetically inactivating the enzymatic activities of alkaline nuclease, the potential impact of pharmacological inhibitors can be predicted. Demonstration that these activities are important for viral replication would provide strong justification for future efforts towards discovery of compounds that target this protein's activities.