The herpesviral antagonist m152 reveals differential activation of STING ‐dependent IRF and NF ‐κB signaling and STING 's dual role during MCMV infection
2019; Springer Nature; Volume: 38; Issue: 5 Linguagem: Inglês
10.15252/embj.2018100983
ISSN1460-2075
AutoresMarkus Stempel, Baca Chan, Vanda Juranić Lisnić, Astrid Krmpotić, Josephine Hartung, Søren R. Paludan, Nadia Füllbrunn, Niels A. W. Lemmermann, Melanie M. Brinkmann,
Tópico(s)Mosquito-borne diseases and control
ResumoArticle29 January 2019Open Access Source DataTransparent process The herpesviral antagonist m152 reveals differential activation of STING-dependent IRF and NF-κB signaling and STING's dual role during MCMV infection Markus Stempel Markus Stempel orcid.org/0000-0002-9240-3987 Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Baca Chan Baca Chan Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Vanda Juranić Lisnić Vanda Juranić Lisnić Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Search for more papers by this author Astrid Krmpotić Astrid Krmpotić Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Search for more papers by this author Josephine Hartung Josephine Hartung Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Søren R Paludan Søren R Paludan orcid.org/0000-0001-9180-4060 Department of Biomedicine, Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Nadia Füllbrunn Nadia Füllbrunn Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Niels AW Lemmermann Niels AW Lemmermann orcid.org/0000-0001-9190-6497 Institute for Virology and Research Center for Immunotherapy, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Melanie M Brinkmann Corresponding Author Melanie M Brinkmann [email protected] orcid.org/0000-0001-5431-6527 Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany Search for more papers by this author Markus Stempel Markus Stempel orcid.org/0000-0002-9240-3987 Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Baca Chan Baca Chan Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Vanda Juranić Lisnić Vanda Juranić Lisnić Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Search for more papers by this author Astrid Krmpotić Astrid Krmpotić Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia Search for more papers by this author Josephine Hartung Josephine Hartung Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Søren R Paludan Søren R Paludan orcid.org/0000-0001-9180-4060 Department of Biomedicine, Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Nadia Füllbrunn Nadia Füllbrunn Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Search for more papers by this author Niels AW Lemmermann Niels AW Lemmermann orcid.org/0000-0001-9190-6497 Institute for Virology and Research Center for Immunotherapy, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany Search for more papers by this author Melanie M Brinkmann Corresponding Author Melanie M Brinkmann [email protected] orcid.org/0000-0001-5431-6527 Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany Search for more papers by this author Author Information Markus Stempel1, Baca Chan1, Vanda Juranić Lisnić2, Astrid Krmpotić2, Josephine Hartung1, Søren R Paludan3, Nadia Füllbrunn1, Niels AW Lemmermann4 and Melanie M Brinkmann *,1,5 1Viral Immune Modulation Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany 2Center for Proteomics, Faculty of Medicine, University of Rijeka, Rijeka, Croatia 3Department of Biomedicine, Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark 4Institute for Virology and Research Center for Immunotherapy, University Medical Center of the Johannes Gutenberg-University Mainz, Mainz, Germany 5Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany *Corresponding author. Tel: +49 531 6181 3069; E-mail: [email protected] The EMBO Journal (2019)38:e100983https://doi.org/10.15252/embj.2018100983 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cytomegaloviruses (CMVs) are master manipulators of the host immune response. Here, we reveal that the murine CMV (MCMV) protein m152 specifically targets the type I interferon (IFN) response by binding to stimulator of interferon genes (STING), thereby delaying its trafficking to the Golgi compartment from where STING initiates type I IFN signaling. Infection with an MCMV lacking m152 induced elevated type I IFN responses and this leads to reduced viral transcript levels both in vitro and in vivo. This effect is ameliorated in the absence of STING. Interestingly, while m152 inhibits STING-mediated IRF signaling, it did not affect STING-mediated NF-κB signaling. Analysis of how m152 targets STING translocation reveals that STING activates NF-κB signaling already from the ER prior to its trafficking to the Golgi. Strikingly, this response is important to promote early MCMV replication. Our results show that MCMV has evolved a mechanism to specifically antagonize the STING-mediated antiviral IFN response, while preserving its pro-viral NF-κB response, providing an advantage in the establishment of an infection. Synopsis The murine cytomegalovirus (MCMV) protein m152 specifically antagonizes the cGAS-STING mediated antiviral type I IFN response but not STING-mediated NF-κB signaling to promote CMV infection. MCMV m152 selectively dampens STING-mediated IRF3, but not STING-mediated NF-κB activation. m152 and STING interact via their respective ER-luminal domains and traffic together from the ER to the Golgi compartment. STING trafficking is slowed down in the presence of m152, resulting in a delayed type I IFN response to MCMV infection. The m152-mediated delay of the type I IFN response is advantageous for MCMV and promotes its replication in vitro and in vivo. STING-dependent NF-κB activation from the ER is beneficial for early MCMV transcription. Introduction Host defense against infection requires the early recognition of invading pathogens by pattern recognition receptors (PRR). DNA derived from pathogens, such as DNA viruses, is a potent pathogen-associated molecular pattern (PAMP), which can be detected by DNA sensors and thereby trigger the production of type I interferons (IFN) and proinflammatory cytokines. Although several DNA sensors have been described, the cyclic guanosine monophosphate-adenosine monophosphate (cGAMP) synthase (cGAS) is considered the major sensor of cytosolic DNA (Sun et al, 2013). DNA binding to cGAS leads to the production of the second messenger 2′-3′-cGAMP, which then directly binds to the endoplasmic reticulum (ER)-resident protein stimulator of interferon genes (STING) (Ishikawa & Barber, 2008). STING is composed of four transmembrane domains and a long cytoplasmic C terminus (Ouyang et al, 2012). Upon activation, STING undergoes dimerization via its C-terminal domain and then translocates from the ER to the Golgi compartment, where it binds to and is phosphorylated by the TANK-binding kinase 1 (TBK1) leading to phosphorylation and activation of the transcription factor interferon regulatory factor 3 (IRF3) and type I IFN transcription (Liu et al, 2015). Moreover, STING can also activate nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB)-dependent signaling; however, the exact mechanism and subcellular compartment from where this signaling pathway is activated remains poorly understood. Previous findings suggest that STING activates canonical and non-canonical NF-κB activation via the TNF receptor associated factor 6 (TRAF6)-TBK1 axis and TRAF3, respectively (Abe & Barber, 2014). STING is essential for the innate immune response to a variety of viral pathogens. Herpes simplex virus type 1 (HSV-1) was the first DNA virus reported to induce the cGAS-STING pathway (Li et al, 2013). Mice lacking cGAS or STING were shown to be susceptible to HSV-1 infection (Reinert et al, 2016). Similar observations were made for several other herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV) (Ma et al, 2015), human cytomegalovirus (HCMV) (Paijo et al, 2016), and murine cytomegalovirus (MCMV) (Lio et al, 2016; Chan et al, 2017). The initial burst of type I IFN production upon MCMV infection was shown to be dependent on STING (Lio et al, 2016). It follows then that herpesviruses would have evolved discreet mechanisms to overcome this pathway, which is an important source of the potent antiviral type I IFN response. Through millions of years of co-evolution, herpesviruses have developed effective strategies to moderate immune control for securing lifelong persistence in their respective hosts. In the case of CMV, viral immune evasion of natural killer (NK) cell- and T cell-mediated responses has been identified and well characterized (Lemmermann et al, 2012; Lisnic et al, 2015). However, the mechanism by which CMV evades innate immune control following PRR signaling remains poorly understood. MCMV is a well-established model to study the delicate balance between CMV and its host. So far, no MCMV protein has been identified to specifically target the cGAS-STING pathway, and no study has described the in vivo influence of a beta-herpesviral cGAS-STING modulator. Here, we describe m152 as the first MCMV protein to specifically engage the adaptor protein STING within the first few hours of infection. m152, which is an ER-resident type I transmembrane protein, has been previously reported to efficiently thwart both NK- and T cell-dependent immune responses by preventing cell surface expression of the NKG2D ligand retinoic acid early inducible gene-1 (RAE-1) and major histocompatibility complex class I molecules (MHC class I), respectively (Ziegler et al, 1997; Krmpotic et al, 1999; Lodoen et al, 2003; Fink et al, 2013). We now show that m152 additionally modulates the cGAS-STING pathway, independently of its effect on NK- and T cell-mediated responses. At a very early time point after MCMV infection, m152 perturbs the translocation of activated STING from the ER to the Golgi compartment and thereby inhibits the type I IFN response to MCMV infection. Interestingly, m152 has no effect on STING-mediated NF-κB activation, which suggests that STING may activate NF-κB signaling prior to trafficking. We observed both in vitro and in vivo that the inhibitory effect of m152 generates a permissive environment resulting in enhanced viral transcription. However, the absence of STING does not create an advantage for MCMV replication in the first hours of infection, which suggests that STING may have a pro-viral role. We made use of the ability of m152 to selectively delay STING translocation from the ER to the Golgi to show that STING activates NF-κB signaling already from the ER and that this response is indeed beneficial for early MCMV transcription. This study highlights a dual role for STING in the context of MCMV infection, as well as the resourcefulness of MCMV in encoding a single viral protein targeting three major immune responses to foster an optimal environment for establishing a successful infection in the host. Results The MCMV m152 protein specifically downmodulates STING-dependent type I IFN induction Recently, it was shown that the initial type I IFN response upon MCMV infection depends on the key adaptor protein STING (Lio et al, 2016), which mediates signaling downstream of cytosolic DNA sensing. Since MCMV has evolved a plethora of evasion strategies to modulate innate and adaptive immune responses, we hypothesized that MCMV would have evolved a mechanism to counteract the STING-mediated innate immune response. To address this, we developed an unbiased luciferase-based reporter assay in 293T cells to screen for modulators of IFNβ transcription encoded by MCMV. As 293T cells do not express endogenous cGAS or STING, we reconstituted the pathway by transiently expressing a murine Cherry-STING fusion protein and induced signaling by co-expression of cGAS-GFP. To monitor IFNβ induction, a reporter plasmid composed of the murine IFNβ promoter upstream of the firefly luciferase gene (IFNβ-Luc) was co-transfected. In total, 173 MCMV open reading frames (ORFs) (Munks et al, 2006) were tested for their ability to inhibit the cGAS-STING pathway (Appendix Fig S1). Among the MCMV proteins tested, the MCMV type I transmembrane protein m152 significantly inhibited IFNβ promoter activity downstream of cGAS-STING signaling to a similar extent as the known IRF3 antagonist KSHV ORF36 (Hwang et al, 2009) compared to either empty vector (ev) or the cellular type I transmembrane protein CD4 (Fig 1A). To investigate whether m152 targets multiple PRR-mediated signaling pathways, we co-expressed RIG-I N, a constitutively active form of the cytosolic RNA sensor RIG-I. As expected, influenza NS1, an antagonist of RIG-I signaling, markedly inhibited IFNβ promoter induction, whereas m152, as for the control CD4, had no effect on RIG-I signaling (Fig 1B). Upon overexpression of TBK1 or expression of a constitutively active form of IRF3 (IRF3-5D) (Lin et al, 1998), m152 expression had no effect on type I IFN induction (Fig 1C and D). Additionally, to exclude an effect of m152 on interferon-α/β receptor (IFNAR) signaling, 293T cells were co-transfected with an ISG56 promoter firefly luciferase reporter construct and IFNAR signaling was activated by the addition of recombinant IFNβ. MCMV M27, a known modulator downstream of IFNAR-dependent signaling (Zimmermann et al, 2005), inhibited ISG56 promoter induction, whereas m152 did not have an effect (Fig 1E). Thus far, these results suggest that the MCMV m152 protein specifically targets STING-dependent signaling prior to the activation of the kinase TBK1 and the transcription factor IRF3. Figure 1. The MCMV m152 protein specifically targets STING-dependent signaling A. 293T cells were co-transfected with expression plasmids for Cherry-STING, the murine IFNβ-luciferase reporter (IFNβ-Luc), a Renilla luciferase normalization control (pRL-TK), and the indicated expression plasmids or empty vector (ev). Cells were additionally co-transfected with expression plasmids for cGAS-GFP (stimulated) or IRES-GFP (unstimulated). 20 hours post-transfection, cells were lysed and a dual-luciferase assay was performed. B. An expression plasmid for RIG-I N (stimulated) or ev (unstimulated) was co-transfected with IFNβ-Luc, pRL-TK and the indicated expression plasmids in 293T cells and analyzed as in (A). C. An expression plasmid for TBK1 (stimulated) or ev (unstimulated) was co-transfected together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). D. 293T cells were co-transfected with a plasmid expressing constitutively active IRF3 (IRF3-5D; stimulated) or IRES-GFP (unstimulated) together with IFNβ-Luc, pRL-TK and the indicated expression plasmids and analyzed as in (A). E. The ISG56-luciferase reporter, pRL-TK, and the indicated expression plasmids were co-transfected in 293T cells. 24 hours post-transfection, cells were stimulated with 0.1 ng/μl human IFNβ or mock stimulated and analyzed 16 h later as described in (A). F, G. iMEFgt/gt stably expressing Cherry-STING and either ev or V5-tagged m152 were stimulated with 5 μg/ml ISD (F), 10 μg/ml poly(I:C) (G), or mock stimulated with Lipofectamine. 4 hours post-stimulation, RNA was extracted to determine IFNβ mRNA transcripts by qRT–PCR. H–K. iBMDM stably expressing ev or m152-V5 were stimulated in duplicates with 10 μg/ml cGAMP (H), 5 μg/ml ISD (I), Newcastle disease virus (NDV) infection (J), or 1 μM CpG DNA (K). 6 (H) or 16 (I-K) hours later, secreted IFNβ (H-J) or TNFα (K) levels were determined by ELISA. Data information: (A-G) Data are combined from three independent experiments. (H-K) Experiments were performed three (H, I, K) or two (J) times independently and one representative experiment is shown. Student's t-test (unpaired, two-tailed), n.s. not significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data are shown as mean ± SD. Download figure Download PowerPoint To validate our results in a more physiological setting, we examined the effect of m152 on IFNβ transcription in murine embryonic fibroblasts (MEF). For this, we utilized immortalized goldenticket MEF (iMEFgt/gt), which do not express endogenous STING due to an I199N missense mutation in STING (Sauer et al, 2011; Appendix Fig S2A). We reconstituted STING expression in these cells by transducing them with a murine Cherry-STING fusion construct resulting in iMEFgt/gt Cherry-STING. These cells were additionally transduced to stably express V5-tagged m152, and the expression was verified by immunoblotting (Appendix Fig S2A). Upon stimulation with interferon-stimulatory DNA (ISD), we observed an inhibition of IFNβ transcription in m152 expressing cells (Fig 1F). In contrast, upon stimulation with the RLR ligand poly(I:C), the presence of m152 did not affect IFNβ transcription (Fig 1G). Since the cGAS-STING signaling pathway is crucial to mount a potent type I IFN response upon MCMV infection in macrophages (Chan et al, 2017), we addressed whether m152 expression reduces IFNβ secretion in this cell type. We generated immortalized bone marrow-derived macrophages (iBMDM) stably expressing V5-tagged m152 (Appendix Fig S2B). As expected, upon stimulation with 2′3′-cGAMP or ISD, lower levels of secreted IFNβ were detected in the presence of m152 (Fig 1H and I). In contrast, m152 had no effect on Newcastle disease virus (NDV)-induced RIG-I signaling (Yoneyama et al, 2004) (Fig 1J), nor did it affect CpG-induced TLR9 signaling (Fig 1K). Collectively, these data show that m152 selectively targets cGAS-STING signaling upstream of the TBK1-IRF3 axis, but does not affect RIG-I-, TLR9-, or IFNAR-mediated signaling. m152 co-localizes and interacts with STING in resting and stimulated cells In unstimulated cells, STING is localized in the ER (Ouyang et al, 2012). Upon activation, STING translocates from the ER to the Golgi compartment, which is a crucial prerequisite for downstream signaling leading to the induction of the type I IFN response mediated by TBK1 and IRF3 (Liu et al, 2015). MCMV m152 is likewise described as an ER-resident protein (Ziegler et al, 1997) and as we have observed that m152 targets STING-mediated signaling, we sought to examine whether m152 translocates together with STING. For this, we first transfected HeLa cells with expression constructs for murine Cherry-STING and V5-tagged m152. As expected, STING and m152 co-localized in the ER in unstimulated cells (Fig 2A, upper panel). Upon overexpression of cGAS-GFP to activate STING-dependent signaling, we observed that STING translocated to the perinuclear region as described previously (Ishikawa et al, 2009) (Fig 2A, lower panel). Notably, m152 co-localized with STING in the perinuclear region. This shows that, like STING, m152 translocates upon stimulation of the cGAS-STING pathway. Figure 2. STING and the MCMV m152 protein co-localize and interact under unstimulated and stimulated conditions A, B. (A) HeLa cells were co-transfected with expression plasmids for Cherry-STING, V5-tagged m152, and either ev (unstimulated) or cGAS-GFP (stimulated). (B) iMEFgt/gt were co-transfected with expression plasmids for V5-tagged m152 together with either Cherry-STING or ev in combination with cGAS-GFP (stimulated) or ev (unstimulated). Twenty-four hours post-transfection, cells were fixed for immunolabeling with an anti-V5 antibody. White boxes indicate the region shown at a higher magnification. Scale bar represents 10 μm. C. Lysates of Cherry-STING and either CD4-V5 or m152-V5 expressing 293T cells were subjected to immunoprecipitation (IP) with an anti-V5 antibody. Input and IP samples were analyzed by IB with the indicated antibodies. D. iMEF stably expressing ev or m152-V5 were left unstimulated or stimulated with 10 μg/ml ISD and lysed 90 min later. m152 was immunoprecipitated with an anti-V5 antibody, and samples were analyzed by IB with V5, STING, and phospho-TBK1 (pTBK1)-specific antibodies. Data information: IB shown are representative of three (C) or two (D) independent experiments. Source data are available online for this figure. Source Data for Figure 2 [embj2018100983-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint Subsequently, we wanted to address whether the translocation of m152 upon cGAS activation is STING-dependent. When transfecting iMEFgt/gt with an expression plasmid for V5-tagged m152 alone, m152 was detected in the ER (Fig 2B, first panel). When we reconstituted STING expression, m152 and STING co-localized in the ER and upon overexpression of cGAS, both proteins translocated to the perinuclear region (Fig 2B, second and third panel), consistent with our results in HeLa cells. Notably, upon co-transfection with cGAS and in the absence of STING, m152 remained in the ER and did not translocate to the perinuclear region (Fig 2B, last panel), suggesting that the translocation of m152 is dependent on STING. Since m152 and STING translocate simultaneously upon stimulation, we sought to examine whether these proteins interact with each other. Upon overexpression in 293T cells, STING co-immunoprecipitated with m152, but not with the control protein CD4 (Fig 2C). To show that m152 interacts with endogenous STING, we generated iMEF stably expressing V5-tagged m152 (Appendix Fig S2C) and either left them unstimulated or stimulated with ISD. Endogenous STING co-immunoprecipitated with m152 in unstimulated as well as stimulated cells (Fig 2D), correlating with our observation that m152 and STING co-localize regardless of stimulation status. The luminal N-terminal domain of m152 directs its interaction with STING Next, we aimed to identify the domain of m152 which is essential for its interaction with STING and its effect on STING-mediated signaling. We constructed a series of chimeric m152 proteins by exchanging target domains singly or in combination with the corresponding region in CD4 (Fig 3A, (1)-(6)), a well-characterized cellular type I transmembrane protein which was previously used for the generation of chimeric proteins (Ziegler et al, 2000; Barton et al, 2006). All generated m152 mutants were expressed in 293T cells, and their localization was determined in unstimulated and stimulated conditions. Excepting the CD4-m152SPCTD (5) mutant, which only partially localizes with STING, wild-type m152 and all m152 mutants (1–4 and 6) co-localize with STING in unstimulated and stimulated cells (Fig EV1A). Next, we analyzed the m152-CD4 chimeric proteins in the established IFNβ luciferase reporter assay described previously. We observed that all m152 chimeras that still contained the luminal domain of m152 (1–4) retained the ability to inhibit signaling downstream of cGAS-STING (Fig 3B). Both m152-CD4 chimeric proteins where the N-terminal domain of m152 was replaced with that of CD4, namely CD4-m152SPCTD (5) and CD4-m152SPTMCTD (6), lost the ability to inhibit STING-mediated signaling (Fig 3B), showing that the N-terminal domain of m152 is responsible for its effect on STING signaling. Co-IP experiments in 293T cells confirmed that the N-terminal domain of m152 mediates its interaction with STING, whereas the transmembrane and C-terminal domain of m152 are inconsequential (Fig 3C). We also constructed an m152 mutant lacking N-linked glycosylation (Fink et al, 2013) (Fig 3A, (7)) and observed that post-translational N-linked glycosylation of m152 does not contribute to the impeding effect of m152 on STING-dependent signaling (Fig EV1B). In addition, we mutated the stalk region of m152, which is required for its binding to and effect on MHC class I (Janssen et al, 2016) (Fig 3A, (8)+(9)), and can show that this region is dispensable for its effect on STING signaling and interaction with STING (Fig 3D and E). These m152 mutants (7–9) also still co-localized with STING (Fig EV1A). Figure 3. The N-terminal domain of m152 directs the interaction with STING Schematic representation of wild-type m152, wild-type CD4, CD4-m152 chimeric constructs, m152 glycosylation mutants, and m152 stalk mutants used in this study. CD4-m152 chimeras (1–6): The relevant domain of m152 was replaced singly or in combination with the respective domain of murine CD4. m152 glycosylation mutant (7): asparagine (N) at position 83, 230, and 263 was mutated to glutamine (Q). m152 stalk mutants (8-9): m152-Δstalk has a deletion of amino acids 300-326 (8), and for m152-GSstalk, a glycine-serine linker ((G4S)5) was inserted to replace the stalk region (9). SP = signal peptide, NTD = N-terminal domain, TM = transmembrane domain, CTD = C-terminal domain. Branched symbols represent the three glycosylation sites of m152. Cherry-STING, IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated), or IRES-GFP (unstimulated) and indicated expression plasmids as shown in (A) were transiently expressed in 293T cells and a dual-luciferase assay was performed. Data are combined from two out of three independent experiments and shown as mean ± SD. 293T cells were co-transfected with expression plasmids for Cherry-STING and indicated chimeras as shown in (A). An anti-V5 IP was performed, and samples were analyzed by IB with indicated antibodies. IB shown is representative of three independent experiments. Cherry-STING, IFNβ-Luc, pRL-TK, and either CD4, m152, m152-Δstalk, or m152-GSstalk were transiently expressed in 293T cells. For stimulation, samples were co-transfected with cGAS-GFP, and unstimulated samples with IRES-GFP. Lysates were analyzed as described in (B). Data are combined from two out of three independent experiments and shown as mean ± SD. 293T cells were co-transfected with expression plasmids for Cherry-STING and either CD4, m152, m152-Δstalk, or m152-GSstalk. An anti-V5 IP was performed, and samples were analyzed by IB with indicated antibodies. Immunoblot shown is representative of three independent experiments. Data information: Student's t-test (unpaired, two-tailed), n.s. not significant, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure. Source Data for Figure 3 [embj2018100983-sup-0004-SDataFig3.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Subcellular localization of m152 mutant proteins in 293T cells 293T cells were co-transfected with expression plasmids for Cherry-STING, V5-tagged m152, or the respective m152 mutant (as described in Fig 3A) and either ev (unstimulated) or cGAS-GFP (stimulated). Twenty-four hours post-transfection, cells were fixed for immunolabeling with an anti-V5 antibody. Scale bar represents 10 μm. 293T cells were co-transfected with expression plasmids for Cherry-STING, IFNβ-Luc, pRL-TK and either CD4, m152 or the m152-N83Q-N230Q-N263Q mutant. For stimulation, samples were co-transfected with cGAS-GFP whereas unstimulated samples were co-transfected with IRES-GFP. A dual-luciferase assay was performed. Data are combined from three independent experiments and shown as mean ± SD. Download figure Download PowerPoint The luminal loop regions of STING are the sites of interaction with m152 CMV and their respective hosts share a dynamic co-evolution spanning millions of years. While the m152 protein is highly conserved among various MCMV strains, there is no known homologue in HCMV. Interestingly, we observed that m152 selectively inhibited IFNβ promoter activity downstream of murine STING (mSTING), but not human STING (hSTING) (Fig 4A). Figure 4. Binding of m152 to both ER-luminal loop regions of murine STING is a prerequisite for its antagonistic activity A. 293T cells were co-transfected with expression plasmids for IFNβ-Luc, pRL-TK, cGAS-GFP (stimulated), or IRES-GFP (unstimulated) and indicated expression plasmids together with murine Cherry-STING (left panel) or human STING (right panel). Lysates were analyzed by dual-luciferase assay. Data are combined from two out of three independent experiments and shown as mean ± SD. B. Schematic representation of the predicted m152 (red) and STING (gray) topology in the ER membrane. (1) and (2) specify the ER-luminal loop regions of STING with the sequence alignments from murine and human STING shown below. In murine STING, N41 in loop (1) was mutated to E41 and loop (2) was exchanged with loop (2) of human STING (hL2). The resultant constructs were designated mSTING N41E, mSTING hL2, and mSTING N41E-hL2. In human STING, mutations were introduced vice versa, resulting in hSTING E41N, hSTING mL2, and hSTING E41N-mL2. C. IFNβ-Luc, pRL-TK
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