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

人巨细胞病毒碱性核酸酶（pUL98）：潜在的抗病毒靶点？

• 批准号: 7313743
• 负责人: Michael A McVoy
• 金额: $ 20.56万
• 依托单位: VIRGINIA COMMONWEALTH UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7313743
• 关键词: Acquired Immunodeficiency Syndrome; Active Sites; Amino Acids; Antiviral Agents; Bacteriophages; Biochemical; Biological Assay; Blindness; Capsid; Cessation of life; Complex; Congenital Abnormality; Custom; Cytomegalovirus; Cytomegalovirus Infections; DNA; DNA Polymerase Inhibitor; DNA biosynthesis; DNA-Directed DNA Polymerase; Development; Disabled Persons; Drug Design; Enzymes; Escherichia coli; Essential Genes; Evaluation; Exonuclease; Facility Construction Funding Category; Family; Future; Gastritis; Genes; Genetic; Genetic Recombination; Genome; HIV; Herpesviridae; Human; In Vitro; Intervention; Lead; Libraries; Mediating; Mental Retardation; Mutagenesis; Mutation; Newborn Infant; Patients; Pneumonia; Proteins; Range; Reaction; Recombinant Proteins; Retinitis; Role; SS DNA BP; Simplexvirus; Structure; Testing; Toxic effect; Transplant Recipients; Transplantation; United States; Viral; Viral Genes; Viral Genome; Viral Physiology; Virus; base; congenital infection; deafness; endonuclease; genetic analysis; hearing impairment; high throughput screening; inhibitor/antagonist; member; mutant; novel; nuclease; pathogen; protein expression; recombinant virus; spleen exonuclease; viral alkaline nuclease

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.

描述(申请人提供)：人类巨细胞病毒(HCMV)是疱疹病毒科病毒家族的成员，是一种重要的人类病原体，可导致移植患者和艾滋病患者的肺炎、失明和死亡，以及先天性感染的新生儿的智力低下和听力损失。目前可用的抗病毒药物针对病毒DNA聚合酶，但存在显著的毒性。人巨细胞病毒编码一种蛋白质pUL98，在体外具有内切酶和5‘-3’外切酶活性。PUL98在HCMV复制中的作用尚不清楚，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串联DNA复制中间产物进行去分支。与噬菌体L Reda(核酸外切酶)的相似性以及促进病毒单链DNA结合蛋白介导的链交换反应的能力进一步表明，重组可能在重组中发挥作用，这是疱疹病毒DNA复制的关键特征。最近的遗传学研究表明，UL98基因对病毒复制是必不可少的；因此，pUL98是抗病毒干预的一个有吸引力的靶点。在本提案的目标1中，我们将通过诱变和重组蛋白表达来鉴定对pUL98体外酶活性至关重要的活性部位氨基酸。在目标2中，我们将通过构建突变的病毒基因组来确定这些活性对病毒复制的重要性，在突变的病毒基因组中，pUL98的酶活性因选择性活性位点突变而失效。通过这些研究，我们将确定(1)pUL98是否是巨细胞病毒复制所必需的，(2)是否是pUL98‘S的核溶解活性是至关重要的。证明pUL98的酶活性对HCMV复制是重要的，这将为进一步的研究提供强有力的证据，以确定选择性抑制该活性的化合物，无论是通过确定酶活性部位结构和定制药物设计，还是通过使用体外酶分析高通量筛选化合物文库。这样的研究可能导致新的pUL98抑制剂，可能在临床上用于单独治疗HCMV感染或与现有的DNA聚合酶抑制剂联合使用。人类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。这项拟议的研究将探讨病毒碱性核酸酶(PUL98)作为新型抗病毒药物开发目标的潜力。通过评估碱性核酸酶基因失活对病毒复制的影响，可以预测药物抑制剂的潜在影响。证明这些活性对病毒复制是重要的，这将为未来发现针对该蛋白质活性的化合物提供强有力的理由。