HCMV Virion-Associated Glycoprotein UL14 is Critical for the Establishment of Infection in Epithelial Cells

Infection with Human Cytomegalovirus (HCMV) can result in a significant burden of disease in those that are immunocompromised or immunonaive. HCMV encodes a repertoire of glycoproteins that facilitate its extensive viral tropism, some of which remain to be characterized. Currently, there is no effective vaccine or cure for HCMV, therefore emphasizing the need to identify viral proteins of critical function. UL14 was selected as an ORF of interest due to its high scoring on an in silico prediction algorithm, as well as its conservation amongst human and primate CMVs. Our goal was to elucidate the function of this previously uncharacterized viral ORF.

We hypothesized that UL14 functions in the establishment of infection in epithelial cells, due to its predicted structural similarity to UL141. We first investigated the expression of the protein through the sequential generation of a 3X-Flag mutant, a Δ UL14 mutant, and a repair in the TB40/E background. UL14 is readily expressed and detected by means of Western blot in both fibroblast and epithelial cells. Furthermore, UL14 was demonstrated to be a glycosylated viral protein through Pngase and EndoH enzymatic digests. Additionally, we confirmed the presence of UL14 in fibroblast-derived virions via Western blotting. Growth curves revealed that in fibroblasts, UL14 is dispensable for infection; however, in epithelial cells, the deletion of UL14 results in attenuated viral growth. During the infection of epithelial cells, we observed that the deletion of UL14 significantly reduces the expression of IE1 when compared to wild-type infection, despite the same number of viral genomes being delivered to each cell. Additionally, when the Δ UL14 virus is propagated in UL14-expressing fibroblasts, the virus incorporates UL14 into the virion and rescues the epithelial IE1 phenotype. Taken together, our results suggest that UL14 functions in the early establishment of infection in an epithelial cell-specific manner.