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

PLoS Pathogens, Mar 2015

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.

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

March Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase Junjie Zhang 0 1 2 Shanping He 0 1 2 Yi Wang 0 1 2 Kevin Brulois 0 1 2 Ke Lan 0 1 2 Jae U. Jung 0 1 2 Pinghui Feng 0 1 2 0 1 Department of Molecular Microbiology and Immunology, Norris Comprehensive Cancer Center, Keck School of Medicine, University of Southern California, Los Angeles, California, United States of America, 2 Institut Pasteur of Shanghai, Chinese Academy of Sciences , Shanghai , P.R. China 1 Funding: This work is supported by grants from American Cancer Society (RGS-11-162-01-MPC), NIH (CA134241 and DE021445) to PF and (CA180779) to JUJ. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript 2 Editor: Bala Chandran, Rosalind Franklin University of Medicine and Science , UNITED STATES 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 NFATdependent 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. - Competing Interests: The authors have declared that no competing interests exist. G protein-coupled receptors (GPCRs) constitute the largest family of proteins that transmit signal across plasma membrane. Herpesviral GPCRs (vGPCRs) activate diverse signaling cascades and are implicated in viral pathogenesis (e.g., tumor development). In contrast to cellular GPCRs that are chiefly regulated via cognate ligand-association, vGPCRs are constitutively active independent of ligand-binding. vGPCRs provide useful tools to dissect signal transduction from plasma membrane receptors to nuclear transcription factors. To probe the activation of nuclear factor of T cells (NFAT), we demonstrate that vGPCRs target the ER calcium ATPase to increase cytosolic calcium concentration and activate NFAT. Inhibition of NFAT activation impairs tumor formation induced by vGPCRs, implying the antitumor therapeutic potential via disabling NFAT activation. Herpesviruses are ubiquitous pathogens and their infections contribute to a number of malignancies in humans [1]. The lymphotropic gamma herpesviruses, including Kaposis sarcomaassociated herpesvirus (KSHV, also known as HHV-8) and Epstein-Barr virus (EBV or HHV4), are large DNA tumorigenic viruses [2]. Remarkably, these viruses have pirated a number of cellular genes to assist the completion of crucial steps of infection cycle consisting of lytic replication and latent infection. Under immuno-compromised conditions, uncontrolled replication of these viral pathogens results in aberrant cell proliferation that is associated with and underpinned by inflammation [3,4]. Discovered by Yuan Chang, Patrick Moore and their coworkers in 1994, KSHV is the etiological agent of Kaposis sarcoma (KS), primary effusion lymphoma (PEL) and multicentric Castlemans disease (MCD) [5,6]. It is believed that KS is of endothelial origin, whereas PEL and MCD are malignancies of lymphoid cells. Among genes pirated by human herpesviruses, G protein-coupled receptor (GPCR) is a common target and implicated in viral pathogenesis [7]. All gamma herpesviruses express one GPCR homologue, while genomes of beta-herpesviruses contain up to four copies of GPCR [8,9]. Herp (...truncated)


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Junjie Zhang, Shanping He, Yi Wang, Kevin Brulois, Ke Lan, Jae U. Jung, Pinghui Feng. Herpesviral G Protein-Coupled Receptors Activate NFAT to Induce Tumor Formation via Inhibiting the SERCA Calcium ATPase, PLoS Pathogens, 2015, Volume 11, Issue 3, DOI: 10.1371/journal.ppat.1004768