The Human Cytomegalovirus US27 Gene Product Constitutively Activates Antioxidant Response Element-Mediated Transcription through G_βγ, Phosphoinositide 3-Kinase, and Nuclear Respiratory Factor 1

Abstract

Human cytomegalovirus (HCMV) is a widespread pathogen that modulates host chemokine signaling during persistent infection in the host. HCMV encodes four proteins with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs): US27, US28, UL33, and UL78. Each of the four receptors modulates host CXCR4 signaling. US28, UL33, and UL78 impair CXCR4 signaling outcomes, while US27 enhances signaling, as evidenced by increased calcium mobilization and cell migration to CXCL12. To investigate the effects of US27 on CXCR4 during virus infection, fibroblasts were infected with bacterial artificial chromosome-derived clinical strain HCMV TB40/E- (wild type [WT]), mutants lacking US27 (TB40/E--US27Δ [US27Δ]) or all four GPCRs (TB40 E--allΔ), or mutants expressing only US27 but not US28, UL33, or UL78 (TB40/E--US27 [US27]). CXCR4 gene expression was significantly higher in WT- and US27-infected fibroblasts. This effect was evident at 3 h postinfection, suggesting that US27 derived from the parental virion enhanced CXCR4 expression. Reporter gene assays demonstrated that US27 increased transcriptional activity regulated by the antioxidant response element (ARE), and small interfering RNA treatment indicated that this effect was mediated by NRF-1, the primary transcription factor for CXCR4. Increased translocation of NRF-1 into the nucleus of WT-infected cells compared to mock- or US27Δ-infected cells was confirmed by immunofluorescence microscopy. Chemical inhibitors targeting Gγ and phosphoinositide 3-kinase (PI3K) ablated the increase in ARE-driven transcription, implicating these proteins as mediators of US27-stimulated gene transcription. This work identifies the first signaling pathway activated by HCMV US27 and may reveal a novel regulatory function for this orphan viral receptor in stimulating stress response genes during infection. Human cytomegalovirus (HCMV) is the most common congenital infection worldwide, causing deafness, blindness, and other serious birth defects. CXCR4 is a human chemokine receptor that is crucial for both fetal development and immune responses. We found that the HCMV protein US27 stimulates increased expression of CXCR4 through activation of the transcription factor nuclear respiratory factor 1 (NRF-1). NRF-1 regulates stress response genes that contain the antioxidant response element (ARE), and HCMV infection is associated with increased expression of many stress response genes when US27 is present. Our results show that the US27 protein activates the NRF-1/ARE pathway, stimulating higher expression of CXCR4 and other stress response genes, which is likely to be beneficial for virus replication and/or immune evasion.

Deciphering the Potential Coding of Human Cytomegalovirus: New Predicted Transmembrane Proteome

“…On the contrary, a group of proteins encoded by the genes UL33, UL78 and the genes from the unique short (US) region US12-US21, US27 and US28 proteins were predicted to have more than five TM regions by all three methods. In fact, TM regions of these genes, such as the members of US12 family and the proteins with homology to the chemokine receptor family of G protein-coupled receptors (GPCRs): US27 and US28, have been previously described supporting our results [ 108 , 109 , 121 ].…”

“…The GPCR-7 TM family includes four genes coding for proteins with seven predicted TM domains: US27, US28, UL33 and UL78. This family includes G protein-coupled surface receptors with an important role in immunomodulation that transmit an intracellular signal when binding to an extracellular ligand [ 109 , 124 ]. Within this family only UL78 (protein from the unknown core proposed in this work) remains uncharacterized.…”

Viral G protein-coupled receptors as modulators of cancer hallmarks

“…1) [43,44]. UL33, US27 and US28 exhibit constitutive G protein-dependent signaling [45][46][47]. Where UL33 and US28 promiscuously couple to G proteins (Gα s , Gα i and Gα q , and Gα i , Gα q and Gα 12/13 , respectively) [45,][47-51], US27 signaling is mediated via Gβγ heterodimers but the corresponding Gα class remains unknown [46].…”

Viral G Protein–Coupled Receptors: Attractive Targets for Herpesvirus-Associated Diseases

“…Finally, all four HCMV-encoded GPCRs seem to influence CXCR4 signaling and expression. Coexpression of US27 and CXCR4 leads to enhanced CXCR4 expression and signaling (Arnolds et al, 2013;Boeck et al, 2018). UL33, UL78, and US28 expression, on the other hand, downregulate CXCR4 expression and signaling (Tadagaki et al, 2012;Frank et al, 2016).…”