A mechanism of human cytomegalovirus for establishing latency through inhibition of HCMV UL16 expression by hcmv-miR-US33-5p

Human cytomegalovirus (HCMV) is the only beta herpesvirus that can encode microRNA (miRNA). As one of the 26 HCMV miRNAs, hcmv-miR-US33-5p has been reported to inhibit viral DNA synthesis and DNA replication via downregulation of the host gene syntaxin 3. Here, we tested the luciferase activity of 8 other putative target mRNAs of hcmv-miR-US33-5p, which were identified via hybrid PCR, 7 of which decreased following the over expression of hcmv-miRNA-US33-5p. A viral gene, HCMV UL16, was confirmed to be a direct target of hcmv-miR-US33-5p since both luciferase activity and protein levels were decreased by hcmv-miR-US33-5p overexpression. Moreover, by using a reconstituted virus with UL16 deletion, we found that UL16 is not an essential gene for the ability of hcmv-miR-US33-5p to downregulate DNA replication. UL16 plays a critical role in NK cell evasion by sequestering NKG2D ligands in the endoplasmic reticulum (ER) and reducing their cell surface expression. We demonstrated that over expression of hcmv-miR-US33-5p induced some NKG2D ligands independent of other HCMV genes and decreased the interaction between MICB and UL16. These stimulating NKG2D ligands can be recognized by NKG2D and subsequently activate NK cells. Our results provide insight into the mechanisms by which HCMV promotes latency through the inhibition of UL16 and the stimulation of NKG2D ligand expression by hcmv-miR-US33-5p.