Analysis of cytomegalovirus DNA cleavage/packaging genes

描述（由应用程序提供）。人类巨细胞病毒（HCMV）是一种具有重要临床意义的疱疹病毒，迫切需要安全有效的抗病毒药物。尽管切割和包装为新型抗病毒药物提供了一个有吸引力的靶点，并且已经发现了几个切割/包装抑制剂家族，但我们对疱疹病毒切割和包装以及这些化合物如何起作用的基本了解仍然很少。亲本赠款代表了鉴定在裂解和包装过程中被识别的顺式元件、进行裂解和包装的蛋白质以及这一协调过程发生的机制的承诺。在母体提案中，描述了在体外表达HCMV切割/包装蛋白并表征其生化功能的实验。由于构建能够支持切割/包装基因中HCMV突变体复制的互补细胞系迄今尚未成功，因此涉及构建病毒突变体的遗传方法未包括在亲本资助中。中克隆HCMV基因组的出现促进了重组病毒构建的快速方法，为使用互补细胞系进行传统遗传分析提供了一种替代方法。可以在大肠杆菌中创建具有致命缺失的基本病毒基因的bacmedium克隆基因组，并使用高度快速和可靠的遗传工具（如Tn7转位）将互补基因顺式恢复到突变基因组中，我们最近使用Tn7转位来补充HCMV ie2突变体（Hahn等人，J. Virol）。冰毒。（待印）。因此，现在有了一个高通量系统来评估切割/包装基因内的小靶向突变对突变病毒生长/不生长的影响。这本身将为定义这些蛋白质的功能域提供关键信息。此外，如果构建了足够数量的小突变，一些突变无疑会表现出部分生长缺陷。与这些生长缺陷相关的表型特征可以在没有补充细胞的情况下进行，并且应该为这些蛋白质的功能域提供有价值的线索。最后，鉴定恢复高效生长的第二位点突变可能为切割/包装蛋白之间的功能相互作用提供遗传证据。

DESCRIPTION (provided by applica.nt): Human cytomegalovirus (HCMV) is a herpesvirus of considerable clinical importance and for which there exists a strong need for safe and effective antivirals. Although cleavage and packaging presents an attractive target for novel antivirals and several families of cleavage/packaging inhibitors have been discovered, our basic understanding of herpesvirus cleavage and packaging, and how these compounds act, remains scant. The parent grant represents an undertaking to identify the cis elements that are recognized during the cleavage and packaging process, the proteins that carry out cleavage and packaging, and the mechanism by which this concerted process occurs. In the parent proposal, experiments are described to express HCMV cleavage/packaging proteins and to characterize their biochemical functions in vitro. Because construction of complementing cell lines able to support replication of HCMV mutants in cleavage/packaging genes has thusfar been unsucessful, genetic approaches involving construction of viral mutants were not included in the parent grant. The advent of bacmid-cloned HCMV genomes facilitates rapid methods for recombinant virus construction that provide an alternative to conventional genetic analysis using complementing cell lines. Bacmid-cloned genomes with lethal deletions of essential viral genes can be created in E. coli and complementing genes can be restored to the mutant genomes in cis using highly rapid and reliable genetic tools such as Tn7 transposition, which we recently used to complement an HCMV ie2 mutant (Hahn et al., J. Virol. Meth., in press). Thus, a high throughput system is now available to evaluate the effects of small targeted mutations within cleavage/packaging genes in terms of growth/no-growth of the mutant viruses. This in itself will provide information critical to defining functional domains within these proteins. Moreover, if a sufficient number of small mutations are constructed, some will undoubtedly exhibit partial growth defects. Characterization of the phenotypes associated with these growth defects can proceed without complementing cells and should provide valuable clues to the functional domains within these proteins. Finally, identidication of second-site mutations that restore efficient growth may provide genetic evidence for functional interations between cleavage/packaging proteins.