Human cytomegalovirus (HCMV) is a herpesvirus pathogen that causes life threatening infections in people with compromised immune systems, and congenital defects in newborn children. HCMV depends on its viral envelope glycoproteins to infect human cells. A pair of viral glycoproteins, gH and gL, form a complex, gH/gL, that plays crucial roles in entry of HCMV. As is also the case for Epstein Barr virus (EBV) and human herpesvirus 6 (HHV-6), HCMV virions contain two alternative forms of gH/gL. In HCMV, one of these is a complex with gO, gH/gL/gO, and the other is a complex with UL128, UL130 and UL131, gH/gL/UL128-131. The gH/gL/gO complex suffices for infection of fibroblasts, while gH/gL/UL128-131 is necessary for infection of epithelial and endothelial cells. Viral factors that regulate the relative abundance of alternative gH/gL complexes have not been previously described for any herpesvirus. Intriguingly, we have discovered that HCMV encodes an ER-resident glycoprotein, UL148, which may represent the first example of such a factor. UL148 strongly influences the relative amounts of the two gH/gL complexes on virions, and has large effects on cell tropism.
The Specific Aims of this project are to define the influence of UL148 on cell tropism and on maturation of gH/gL complexes, and to identify and characterize protein-protein interactions involving UL148. To carry out these aims, UL148 null mutants of multiple HCMV strains will be characterized, and whether variation in the UL148 gene contributes to the well-documented differences in tropism between HCMV strains will be determined. Studies will also be conducted to determine the effects on tropism and gH/gL when the UL148 is restored to laboratory-adapted HCMV strain AD169, which otherwise lacks the gene. Proteins that co-purify with UL148 during immunoprecipitation from infected cells will be identified by Western blotting and mass spectrometry. Protein interactions involving UL148 and its effects on maturation of gH/gL complexes will be characterized in uninfected cells ectopically expressing the proteins. Chimeric UL148 proteins, in which specific domains of UL148 are swapped with topologically similar proteins, and mutant UL148 proteins, lacking either its ectodomain or cytoplasmic tail, will be used to define the domains necessary for its functions and interactions. Recombinant viruses will be constructed using infectious bacterial artificial chromosome clones of HCMV and sequence manipulation techniques in E. coli. Western blotting, pulse-chase labeling, and enzymatic approaches will be used to visualize the influence of UL148 on gH/gL complexes. Cell tropism will be measured by comparing infectivity on epithelial cells, endothelial cells and fibroblasts. Completion of the project will result in new information that addresses gaps in the current understanding of how HCMV cell tropism is regulated. The resulting findings may have implications for HCMV vaccines, and for understanding the cell tropism of other medically important herpesviruses, such as HHV-6 and EBV, which likewise deploy alternative gH/gL complexes.
Human cytomegalovirus (HCMV) is a leading cause of birth defects and of dangerous opportunistic infections in immunocompromised individuals, and hence, poses a serious threat to public health. The ability of the virus to infect human cells is controlled in large part by two alternate forms of a virion protein complex called gH/gL. This project will define the biological function of an HCMV protein, UL148, that regulates the relative abundance of the two gH/gL complexes, and strongly influences the ability of the virus to infect human cells.
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