Global genetic analysis of the human cytomegalovirus

描述（申请人提供）：人类巨细胞病毒（HCMV）编码超过200个假定的基因。大量病毒基因的功能仍然未知。我的长期目标是了解病毒基因如何在感染过程中发挥作用，以及它们如何调节宿主-病毒相互作用以促进感染。从这些研究中获得的知识将有助于更好地理解HCMV发病机制的分子机制。构建了携带HCMV菌株AD169全基因组的感染性细菌人工染色体（BAC） pAD/Cre。本课题将对pAD/Cre进行转座子随机突变，系统地将插入物引入病毒基因组。转座子插入突变体将被表征，病毒基因将被分类为(i)必需的，（ii）可有可无的，（iii）非必需的，但对人类成纤维细胞的最佳生长是必需的。将构建一组互补细胞系，以繁殖具有基本基因缺陷的突变病毒。此外，将建立一项协议，以确认生长缺陷是由假定的基因丢失直接导致的。最后，生长缺陷突变病毒的一个子集将被充分表征，以描述以前未被研究的潜在基因的功能。

DESCRIPTION (provided by applicant): Human cytomegalovirus (HCMV) encodes more than 200 putative genes. The function of a substantial number of the viral genes remains unknown. My long-term goal is to understand how the viral genes function during infection and how they regulate the host-virus interactions to facilitate infection. The knowledge obtained from these studies will lead to a better understanding of the molecular mechanism of HCMV pathogenesis. An infectious bacterial artificial chromosome (BAC), pAD/Cre, has been constructed that carries the complete genome of the HCMV strain AD169. In this proposal, transposon random mutagenesis will be carried out on pAD/Cre to systematically introduce insertions into the viral genome. The transposon insertion mutants will be characterized and the viral genes will be classified as being (i) essential, (ii) dispensable, and (iii) non-essential but required for optimal growth in human fibroblasts. A set of complementing cell lines will be constructed to propagate the mutant viruses defective in essential genes. In addition, a protocol will be established to confirm that the growth defect is a direct result of loss of the presumed gene. Finally, a subset of growth-defective mutant viruses will be fully characterized to delineate the function of the underlying genes that have not been previously studied. The work will be initiated in Dr. Thomas Shenk's lab at Princeton University. The lab is fully equipped for carrying out research in molecular biology and virology. The Shenk lab, the department and the university provide a vibrant intellectually stimulating and interdisciplinary research environment for the candidate's professional development. The department also provides all core facilities required for the success of this proposal. The graduate work as well as the former postdoctoral training of the candidate has well prepared him to perform the proposed studies. At completion of the mentored phase of the award, the candidate will establish his own lab at a research-oriented academic institution to pursue research on HCMV replication and pathogenesis.