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. ? ?
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