HUMAN CYTOMEGALOVIRUS DNA CLEAVAGE AND PACKAGING

描述（改编自申请人的摘要）： 病毒包括几种重要的人类病原体，包括单纯疱疹 病毒、水痘-带状疱疹病毒、爱泼斯坦-巴尔病毒、卡波西肉瘤相关 疱疹病毒和人巨细胞病毒（HCMV）。这些病毒中的DNA复制 产生大的多联体中间体，单位基因组从该中间体裂解 在特定的序列中包装成病毒颗粒。卤化 苯并咪唑核糖核苷是一类新的抗病毒药物， 这个过程不幸的是，我们对疱疹病毒裂解和 包装很少，这些药物的作用机制仍然未知。 利用鼠巨细胞病毒（MCMV），我们已经开始定义顺式元件 在切割位点被识别。用豚鼠巨细胞病毒 （GPCMV），我们已经证明，切割机制重复 病毒末端的序列。最近在GPCMV和HCMV中的发现表明， 苯并咪唑诱导过早切割以产生截短的基因组。在 本提案的目标1，我们将更准确地定义独联体 MCMV的切割/包装信号，并开始分析顺式元件 在HCMV中。在目标2中，通过实验阐明了切割和 包装将确定MCMV中顺式突变的表型， 细胞内复制DNA和细胞外病毒基因组的形成。 苯并咪唑诱导的截短基因组的积累将是 通过进一步分析所形成的新末端来研究， 衣壳组成和结构，并通过电子显微镜。提出的一种模式 重复复制的机制将通过分析 复制中间体，并通过构建基因突变， 切割/包装顺式元件。在目标3中，病毒切割/包装蛋白 将使用重组表达在体外和体内表征 并使用体外或透化细胞研究它们的功能 切割测定。由于切割和包装是高度保守的， 疱疹病毒，这一信息应直接适用于致病性 人类疱疹病毒，并可能揭示的作用机制， 苯并咪唑或促进靶向苯并咪唑的新化合物的发现。 切割和包装过程。

DESCRIPTION (Adapted from applicant's abstract): The Herpesviridae family of viruses includes several significant human pathogens, including herpes simplex virus, varicella-zoster virus, Epstein-Barr virus, Kaposi's sarcoma associated herpesvirus, and human cytomegalovirus (HCMV). DNA replication in these viruses produces large concatemeric intermediates, from which unit genomes are cleaved at specific sequences and packaged into viral particles. The halogenated benzimidazole ribonucleosides are a new class of antiviral drugs which block this process. Unfortunately, our understanding of herpesvirus cleavage and packing is scant and the mechanism of action of these drugs remains unknown. Using murine cytomegalovirus (MCMV), we have begun to define the cis elements that are recognized at the cleavage site. Using guinea pig cytomegalovirus (GPCMV), we have demonstrated that the mechanism of cleavage duplicates sequences at the viral termini. Recent findings in GPCMV and HCMV suggest that the benzimidazoles induce a premature cleavage to produce truncated genomes. In aim 1 of this proposal, we will more accurately define the cis cleavage/packaging signals of MCMV and initiate efforts to analyze cis elements in HCMV. In aim 2, experiments to elucidate the mechanisms of cleavage and packaging will define the phenotypes of cis mutations in MCMV with respect to formation of intracellular replicative DNAs and extracellular viral genomes. The benzimidazole-induced accumulation of truncated genomes will be investigated by further analysis of the novel ends formed, characterization of capsid composition and structure, and by electron microscopy. A model proposed for the mechanism of repeat duplication will be tested both by analysis of replicative intermediates and by construction of genetic mutations in cleavage/packaging cis elements. In aim 3, viral cleavage/packaging proteins will be characterized both in vitro and in vivo using recombinant expression and their functions studied using either in vitro or permeabilized cell cleavage assays. As cleavage and packaging are highly conserved among herpesviruses, this information should be directly applicable to pathogenic human herpesviruses and may reveal the mechanism of action of the benzimidazoles or facilitate the discovery of novel compounds that target the cleavage and packaging processes.