Analysis of cytomegalovirus DNA cleavage/packaging genes

描述（由applica.nt提供）：人巨细胞病毒（HCMV）是一种具有相当临床重要性的疱疹病毒，对安全有效的抗病毒药物存在强烈需求。 虽然裂解和包装提出了一个有吸引力的目标，新的抗病毒药物和几个家庭的裂解/包装抑制剂已被发现，我们的基本理解疱疹病毒裂解和包装，以及如何这些化合物的作用，仍然很少。 母基金代表了一项承诺，以确定在切割和包装过程中识别的顺式元件，进行切割和包装的蛋白质，以及这种协调过程发生的机制。 在母提案中，描述了表达HCMV切割/包装蛋白并在体外表征其生化功能的实验。 由于能够支持切割/包装基因中HCMV突变体复制的补充细胞系的构建迄今尚未成功，因此涉及构建病毒突变体的遗传方法不包括在母基金中。 杆粒克隆的HCMV基因组的出现促进了重组病毒构建的快速方法，其提供了使用互补细胞系的常规遗传分析的替代方案。 可以在大肠杆菌中创建具有致命病毒必需基因缺失的Bacmid-cloned基因组。大肠杆菌中，并且互补基因可以使用高度快速和可靠的遗传工具如Tn 7转座以顺式恢复到突变体基因组中，我们最近使用Tn 7转座来补充HCMV ie 2突变体（Hahn等人，J. Virol.冰毒，出版中）。 因此，高通量系统现在可用于评估切割/包装基因内的小靶向突变对突变病毒生长/不生长的影响。 这本身将提供定义这些蛋白质内的功能结构域的关键信息。 此外，如果构建了足够数量的小突变，一些无疑会表现出部分生长缺陷。 与这些生长缺陷相关的表型的表征可以在不补充细胞的情况下进行，并且应该为这些蛋白质内的功能结构域提供有价值的线索。 最后，鉴定第二个位点的突变，恢复有效的增长可能提供切割/包装蛋白之间的功能相互作用的遗传证据。

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.