Analysis of cytomegalovirus DNA cleavage/packaging genes

描述：人类巨细胞病毒(HCMV)是一种具有相当重要临床意义的疱疹病毒，目前迫切需要安全有效的抗病毒药物。尽管裂解和包装是新型抗病毒药物的一个有吸引力的靶点，并且已经发现了几个裂解/包装抑制剂家族，但我们对疱疹病毒裂解和包装以及这些化合物如何作用的基本了解仍然很少。母体赠款代表对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发生这一协调过程的机制的承诺。在父母的建议中，描述了表达HCMV裂解/包装蛋白的实验，并在体外表征其生化功能。由于到目前为止，构建能够支持在切割/包装基因中复制巨细胞病毒突变体的补充细胞系是不成功的，因此涉及构建病毒突变体的遗传方法不包括在父母赠款中。杆状克隆人巨细胞病毒基因组的出现促进了重组病毒的快速构建，为利用互补细胞系进行常规遗传分析提供了一种替代方法。巴克米德克隆的基因组具有致命的必要病毒基因的删除可以在大肠杆菌中创建，补充基因可以使用高度快速和可靠的遗传工具，例如TN7转座，在顺式病毒中恢复到突变基因组，我们最近用它来补充HCMV IE2突变体(Hahn等人，J.Virol)。冰毒，印刷中)。因此，现在有了一个高通量的系统来评估切割/包装基因内的小靶向突变对突变病毒生长/不生长的影响。这本身将提供对确定这些蛋白质中的功能结构域至关重要的信息。此外，如果构建了足够数量的小突变，其中一些无疑会出现部分生长缺陷。与这些生长缺陷相关的表型鉴定可以在不补充细胞的情况下进行，并应该为这些蛋白质中的功能域提供有价值的线索。最后，鉴定恢复有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学证据。

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.