Distinct Roles for Human Cytomegalovirus Immediate Early Proteins IE1 and IE2 in the Transcriptional Regulation of MICA and PVR/CD155 Expression

Abstract

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, that is, MHC class I–related chain (MIC) A/B and UL16-binding proteins (ULBP)1–6 (NKG2D ligand), Nectin-2/CD112, and poliovirus receptor (PVR)/CD155 (DNAM-1 ligand), are often induced on virus-infected cells, although some viruses, including human CMV (HCMV), can block their expression. In this study, 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 that we identified within the promoter and that is conserved among different alleles of MICA. Both IE proteins were instead required for PVR upregulation 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, a pathway activated by HCMV and implicated in ligand regulation. However, silencing of ataxia telangiectasia mutated, ataxia telangiectasia and Rad3–related protein, and DNA-dependent protein kinase 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 antiviral response.

Human Cytomegalovirus Tegument Protein pp65 (pUL83) Dampens Type I Interferon Production by Inactivating the DNA Sensor cGAS without Affecting STING

“…Cells and viruses. Primary HFFs (ATCC strain SCRC-1041) and human embryonic kidney 293 (HEK 293) cells (Microbix Biosystems Inc.) were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) supplemented with 10% fetal calf serum (FCS) (Sigma-Aldrich) as previously described (39,64). The HCMVs used in this study were all bacterial artificial chromosome (BAC) clones.…”

Regulation of NKG2D-Dependent NK Cell Functions: The Yin and the Yang of Receptor Endocytosis

“…Moreover, DDR-mediated pathway may be activated upon infection by several viruses, including herpes viruses, adenoviruses and retroviruses, and is responsible for NKG2DL up-regulation. However, while a direct link between these two phenomena has been established for HIV-1 [ 25 , 26 ], up-regulation of MICA upon human cytomegalovirus (HCMV) infection appears to be independent from DDR induction and is directly promoted by viral proteins [ 27 ]. In cancer cells, genotoxic stress increases the sensitivity of tumor cells to NK cell-mediated lysis by the induction of NKG2DL expression mainly through DDR activation [ 19 , 20 , 21 ].…”

P2Y2 purinergic receptor modulates virus yield, calcium homeostasis, and cell motility in human cytomegalovirus-infected cells

“…Additionally, we have previously shown that IE1 and IE2 expressed in this model cooperate to activate expression of different target genes (46). IE1 and IE2 proteins are known to have distinct functions (52), but at this point the mechanistic basis for the inhibitory effect of IE2 on P2Y2 expression outside the context of infection is unclear. IE1 protein expression alone induces P2Y2 RNA levels by a factor of about 16 ( Fig.…”