Global genetic analysis of the human cytomegalovirus

描述(申请人提供)：人巨细胞病毒(HCMV)编码200多个假定基因。大量病毒基因的功能仍不清楚。我的长期目标是了解病毒基因在感染过程中如何发挥作用，以及它们如何调节宿主与病毒的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致病的分子机制。构建了携带巨细胞病毒AD169株全基因组的感染性细菌人工染色体pAD/Cre。在这项建议中，转座子随机突变将在PAD/Cre上进行，以系统地将插入到病毒基因组中。将确定转座子插入突变体的特征，并将病毒基因分类为(I)必需的，(Ii)可有可无的，和(Iii)非必需的，但对人成纤维细胞的最佳生长是必需的。将构建一组互补的细胞系，以繁殖存在必要基因缺陷的突变病毒。此外，还将建立一项协议，以确认生长缺陷是假定基因丢失的直接结果。最后，将对生长缺陷突变病毒的子集进行充分的表征，以描绘以前没有研究过的潜在基因的功能。 这项工作将在普林斯顿大学托马斯·申克博士的实验室启动。该实验室装备齐全，可以开展分子生物学和病毒学研究。申克实验室、系和大学为候选人的专业发展提供了一个充满活力、激发智力和跨学科的研究环境。该部还提供了这项提议成功所需的所有核心设施。应聘者的研究生工作以及以前的博士后培训为他进行拟议的研究做好了充分的准备。在完成该奖项的指导阶段后，候选人将在一家以研究为导向的学术机构建立自己的实验室，从事HCMV复制和发病机制的研究。

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.