Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase

Abstract

G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal to regulate an array of fundamental biological processes. Viruses deploy diverse tactics to hijack and harness intracellular signaling events induced by GPCR. Herpesviruses encode multiple GPCR homologues that are implicated in viral pathogenesis. Cellular GPCRs are primarily regulated by their cognate ligands, while herpesviral GPCRs constitutively activate downstream signaling cascades, including the nuclear factor of activated T cells (NFAT) pathway. However, the roles of NFAT activation and mechanism thereof in viral GPCR tumorigenesis remain unknown. Here we report that GPCRs of human Kaposi’s sarcoma-associated herpesvirus (kGPCR) and cytomegalovirus (US28) shortcut NFAT activation by inhibiting the sarcoplasmic reticulum calcium ATPase (SERCA), which is necessary for viral GPCR tumorigenesis. Biochemical approaches, entailing pharmacological inhibitors and protein purification, demonstrate that viral GPCRs target SERCA2 to increase cytosolic calcium concentration. As such, NFAT activation induced by vGPCRs was exceedingly sensitive to cyclosporine A that targets calcineurin, but resistant to inhibition upstream of ER calcium release. Gene expression profiling identified a signature of NFAT activation in endothelial cells expressing viral GPCRs. The expression of NFAT-dependent genes was up-regulated in tumors derived from tva-kGPCR mouse and human KS. Employing recombinant kGPCR-deficient KSHV, we showed that kGPCR was critical for NFAT-dependent gene expression in KSHV lytic replication. Finally, cyclosporine A treatment diminished NFAT-dependent gene expression and tumor formation induced by viral GPCRs. These findings reveal essential roles of NFAT activation in viral GPCR tumorigenesis and a mechanism of “constitutive” NFAT activation by viral GPCRs.

Species-Specific Deamidation of cGAS by Herpes Simplex Virus UL37 Protein Facilitates Viral Replication

“…Immunoprecipitation was carried out as previously described (Zhang et al, 2015a). Briefly, THP-1 cells were infected with HSV-1 FLAG-UL37 recombinant virus (MOI = 0.5) for 16 h. Cells were harvested and lysed with NP40 buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1% NP-40, 1 mM EDTA, 5% glycerol) supplemented with a protease inhibitor cocktail (Roche).…”

Hijacking GPCRs by viral pathogens and tumor

“…Although being a homologue of human CXCR2, vGPCR induces a set of downstream signaling distinct from those of cellular chemokine receptors. While human chemokine receptors predominantly signal via Gα i proteins, vGPCR promiscuously couples to multiple Gα proteins and activates diverse downstream signaling cascades, such as phospholipase C (PLC) [22], NFAT [50, 51], MAPK [52, 53], PI3K-AKT [54] and Hippo-YAP [55]. Activation of phosophoinositide 3-kinase (PI3K) occurs largely through Gα i [54], while activation of YAP is dependent on Gα q/11 and Gα 12/13 [55].…”

“…However, it was shown that vGPCR can mobilize calcium release from the ER compartment via inhibiting the Sarco/Endoplasmic reticulum calcium ATPase (SERCA) [51]. The possible cooperation between Gα q and SERCA signaling warrants further investigation.…”

“…The possible cooperation between Gα q and SERCA signaling warrants further investigation. Nonetheless, it is known that the potent activation of NFAT by vGPCR promotes the secretion of IL-8, Angiopoietin-2 (ANGPT2) and the expression of other NFAT-dependent proteins such as RCAN1 and COX-2, all of which are directly or indirectly implicated in angiogenesis [51, 75]. Similar to NF-κB activation induced by vGPCR, NFAT activation and factors secreted by vGPCR-expressing cells constitute a feed-forward circuit that fuels NFAT activation and chemokine production.…”

“…Conceivably, negative regulators may curtail the hyper-activation of NFAT, given that hyper-activation of NFAT in endothelial cells induces precocious cell death [76]. Given the inflammatory nature of KS tumors, it is not surprising to find that inhibiting NFAT activation impairs vGPCR-induced tumorigenesis in a xenograph mouse model [51]. …”