Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the transcriptional regulation of MICA and PVR/CD155 expression.

Elimination of virus-infected cells by cytotoxic lymphocytes is triggered by activating receptors, among which NKG2D and DNAM-1/CD226 play an important role. Their ligands - MICA/B and ULBP1-6 (NKG2DL), Nectin-2/CD112 and PVR/CD155 (DNAM-1L) - are often induced on virus-infected cells, though some viruses, including Human Cytomegalovirus (HCMV), can block their expression. Here, we report that infection of different cell types with laboratory or low-passage HCMV strains upregulated MICA, ULBP3 and PVR, with NKG2D and DNAM-1 playing a role in NK cell-mediated lysis of infected cells. Inhibition of viral DNA replication with phosphonoformic acid did not prevent ligand upregulation, thus indicating that early phases of HCMV infection are involved in ligand increase. Indeed, the major immediate early (IE) proteins IE1 and IE2 stimulated the expression of MICA and PVR, but not ULBP3. IE2 directly activated MICA promoter, via its binding to an IE2-responsive element we identified within the promoter, and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR up-regulation, via a mechanism independent of IE DNA-binding activity. Finally, inhibiting IE protein expression during HCMV infection confirmed their involvement in ligand increase. We also investigated the contribution of the DNA damage response (DDR), a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ATM, ATR and DNA-PK kinases did not influence ligand expression. Overall, these data reveal that MICA and PVR are directly regulated by HCMV IE proteins, and this may be crucial for the onset of an early host anti-viral response.