HCMV DNA REPLICATION GENES IDENTIFIED BY TRANSIENT ASSAY

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Human cytomegalovirus (HCMV) is a frequent opportunistic pathogen, causing extensive morbidity and mortality. It is the most common known congenital virus infection in the United States, sometimes with serious or fatal consequences; by one estimate 3000-4000 infants annually are born with symptomatic infection and suffer deafness, blindness, mental retardation, or death. In immunocompromised individuals HCMV infection or reactivation can be fatal. HCMV is a particular problem in AIDS patients, and may be a cofactor for HIV pathogenesis. Like other herpes viruses, CMV exhibits both lytic and latent phases. An understanding of the molecular mechanisms of HCMV DNA replication and their regulation is needed to aid in developing effective antiviral strategies. Lytic-phase DNA replication requires both trans-acting factors such as the virus- specified DNA polymerase, and a recently identified cis-acting sequence, the origin of DNA replication, oriLyt. The experiments proposed will identify the complete set of virus genes that carry out DNA replication, then characterize novel factors using biochemical and immunological methods. The specific aims are: (i) to define the minimal subset of HCMV genes necessary and sufficient for HCMV lyti-phase DNA replication; (ii) to express the defined genes and purify the gene products; and (iii) to use biochemical and immunological methods to characterize the purified proteins. The methods to be used include: (i) a transient transfection- complementation assay to map novel genes required for replication,a nd to confirm the role of candidate HCMV homologs of HSV type 1 replication genes; (ii) prokaryotic and eukaryotic systems for protein expression; and (iii) various biochemical techniques including electrophoresis, column and affinity chromatography, to purify and characterize the protein factors. The long term goal is to contribute to a comprehensive understanding of the molecular mechanisms and biological strategies of HCMV DNA replication, and to the development of an in vitro DNA replication system.