Human Cytomegalovirus (HCMV) causes morbidity and mortality in neonates and in immunocompromised individuals. HCMV encodes at least four genes (UL33, UL78, US27, and US28) with homology to G-protein coupled receptors (GPCRs). Studies of M78, the murine homologue of UL78, have shown that M78 is an integral protein in the envelope of virions. Mutations in this gene result in a viral growth defect and reduced expression of an immediate early gene, m123. Interestingly, the de novo expression of M78 is not required to mediate this effect suggesting that the M78 protein present in the virion envelope is sufficient for the efficient expression of m123. This research proposal will: 1.) Determine the expression pattern of UL78 mRNA and protein, 2.) Determine the growth properties of a UL78 deleted mutant virus and 3.) Determine whether the presence of UL78 protein by itself, or in the presence of a CC chemokine (RANTES, MCP-1, MIP-1alpha and MIP-1beta), influences the expression of HCMV and/or cellular genes. Protein expression and localization will be determined by Western blot analysis and/or epitope tagging of the C-terminal domain of UL78. Mutant virus will be generated in an infectious HCMV bacterial artificial chromosome (BAC) and, if necessary, utilizing a complementing cell-line. Growth properties will be determined at a high and low multiplicity of infection in several primary or life-extended cell types. Effects on HCMV or cellular gene expression patterns can be monitored using a viral gene array or cellular affymetrix array respectively, and confirmed by Northern blot analysis.
