The object of this proposal is to further characterize cytomegalovirus protease and in so doing to aid in the development of antiviral inhibitors. The central hypothesis is that because protease function is intimately associated with capsid structure and assembly, studying the catalytic properties of recombinant protease does not allow prediction of the antiviral effects of protease inhibitors to be made with any accuracy. This application proposes to construct strains of murine cytomegalovirus (MCMV) in which the protease/assembly gene has been mutated and to characterize those mutants with respect to a variety of properties, including replication, morphology, and protein-protein interactions in the virion capsid. A second part of the proposal is to purify native MCMV protease and compare it with recombinant protease to try to obtain an enzyme that can be used to more accurately study the mechanisms of action of PR antivirals. A chimeric MCMV containing a human CMV (HCMV) protease will be constructed and tested for the ability to grow in mice. If successful, this will provide a way to test HCMV antivirals in vivo and to correlate the results with those from analyses of recombinant and native enzymes.
