A MOLECULAR BASIS FOR THE RHESUS CYTOMEGALOVIRUS MODEL

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Human cytomegalovirus remains a significant health problem. Great strides have been made in understanding the basic molecular biology of HCMV lytic infection of cells in culture and, although much is yet to be learned, many tools to study HCMV lytic infection are available. However, comprehensive studies of the latent phase and pathogenesis are difficult or impossible in tissue culture, and these aspects of HCMV biology are less well understood. Small animal models, most notably murine CMV, have been developed to study pathogenesis and latency, and have proven invaluable. These models are nevertheless significantly divergent from the human system in several respects. Many genes have diverged beyond ready recognition. Regulatory elements are not always conserved. Aspects of pathogenesis differ. For these reasons, they are not adequate to address some questions. In contrast, the rhesus CMV (RhCMV) is very similar to HCMV. The limited sequence data available show that the overall genome is roughly colinear with the human virus, with a few notable differences. Even genes encoding regulatory proteins that are quite divergent in small animal models are relatively well conserved in RhCMV. Most importantly, infection in rhesus macaques appears to recapitulate in many important details infection by HCMV of the human host. Therefore, RhCMV is hypothesized to be an ideal model for the study of those aspects of CMV pathogenesis that cannot be addressed in the small animal models. Unfortunately, the basic molecular information required to confirm this hypothesis and efficiently exploit this system is not yet available. The specific aims are to: 1) establish an ordered set or sets of cosmid clones containing the entire RhCMV genome, 2) determine and analyze the complete nucleotide sequence of the RhCMV genome, and 3) develop systems for generating recombinants that will enable efficient construction of mutant viruses whose biological properties can then be assessed in vivo. These aims will be addressed using standard molecular cloning and sequencing methods, and methods already applied to other herpesviruses to construct mutants. The long-term goal of the work proposed here is to build the knowledge base that will enable efficient application of the RhCMV system to otherwise intractable problems in studies of CMV pathogenesis and latency.