Loss of the Human Cytomegalovirus US16 Protein Abrogates Virus Entry into Endothelial and Epithelial Cells by Reducing the Virion Content of the Pentamer.

The human cytomegalovirus (HCMV) US12 gene family encodes a group of predicted seven-transmembrane proteins whose functions have yet to be established. While inactivation of individual US12 members in laboratory strains of HCMV does not affect viral replication in fibroblasts, disruption of the US16 gene in the low-passage-number TR strain prevents viral growth in endothelial and epithelial cells. In these cells, the US16-null viruses fail to express immediate early (IE), early (E), and late (L) viral proteins due to a defect which occurs prior to IE gene expression. Here, we show that this defective phenotype is a direct consequence of deficiencies in the entry of US16-null viruses in these cell types due to an impact on the gH/gL/UL128/UL130/UL131A (pentamer) complex. Indeed, viral particles released from fibroblasts infected with US16-null viruses were defective for the pentamer, thus preventing entry during infections of endothelial and epithelial cells. A link between pUS16 and the pentamer was further supported by the colocalization of pUS16 and pentamer proteins within the cytoplasmic viral assembly compartment (cVAC) of infected fibroblasts. Deletion of the C-terminal tail of pUS16 reproduced the defective growth phenotype and alteration of virion composition as US16-null viruses. However, the pentamer assembly and trafficking to the cVAC were not affected by the lack of the C terminus of pUS16. Coimmunoprecipitation results then indicated that US16 interacts with pUL130 but not with the mature pentamer or gH/gL/gO. Together, these results suggest that pUS16 contributes to the tropism of HCMV by influencing the content of the pentamer into virions.IMPORTANCE Human cytomegalovirus (HCMV) is major pathogen in newborns and immunocompromised individuals. A hallmark of HCMV pathogenesis is its ability to productively replicate in an exceptionally broad range of target cells. The virus infects a variety of cell types by exploiting different forms of the envelope glycoprotein gH/gL hetero-oligomers, which allow entry into many cell types through different pathways. For example, incorporation of the pentameric gH/gL/UL128/UL130/UL131A complex into virions is a prerequisite for infection of endothelial and epithelial cells. Here, we show that the absence of US16, a thus far uncharacterized HCMV multitransmembrane protein, abrogates virus entry into endothelial and epithelial cells and that this defect is due to the lack of adequate amounts of the pentameric complex in extracellular viral particles. Our study suggests pUS16 as a novel viral regulatory protein important for shaping virion composition in a manner that influences HCMV cell tropism.