HSV ‐1 ICP 27 targets the TBK 1‐activated STING signalsome to inhibit virus‐induced type I IFN expression
2016; Springer Nature; Volume: 35; Issue: 13 Linguagem: Inglês
10.15252/embj.201593458
ISSN1460-2075
AutoresMaria H Christensen, Søren B. Jensen, Juho J. Miettinen, Stefanie Luecke, Thaneas Prabakaran, Line S. Reinert, Thomas C. Mettenleiter, Zhijian J. Chen, David M. Knipe, Rozanne M. Sandri-Goldin, Lynn W. Enquist, Rune Hartmann, Trine H. Mogensen, Stephen A. Rice, Tuula A. Nyman, Sampsa Matikainen, Søren R. Paludan,
Tópico(s)Immune Cell Function and Interaction
ResumoArticle27 May 2016free access Source DataTransparent process HSV-1 ICP27 targets the TBK1-activated STING signalsome to inhibit virus-induced type I IFN expression Maria H Christensen Maria H Christensen Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Søren B Jensen Søren B Jensen Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Juho J Miettinen Juho J Miettinen University of Helsinki, Helsinki, Finland Search for more papers by this author Stefanie Luecke Stefanie Luecke Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Thaneas Prabakaran Thaneas Prabakaran Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Line S Reinert Line S Reinert Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Thomas Mettenleiter Thomas Mettenleiter Friedrich-Loeffler-Institut, Insel Riems, Germany Search for more papers by this author Zhijian J Chen Zhijian J Chen Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author David M Knipe David M Knipe Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rozanne M Sandri-Goldin Rozanne M Sandri-Goldin Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA Search for more papers by this author Lynn W Enquist Lynn W Enquist Princeton University, Princeton, NJ, USA Search for more papers by this author Rune Hartmann Rune Hartmann orcid.org/0000-0003-1159-066X Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Department of Molecular Biology and Genetics, Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Trine H Mogensen Trine H Mogensen Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Aarhus University Hospital Skejby, Aarhus, Denmark Search for more papers by this author Stephen A Rice Stephen A Rice Department of Microbiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Tuula A Nyman Tuula A Nyman University of Helsinki, Helsinki, Finland Search for more papers by this author Sampsa Matikainen Sampsa Matikainen Finnish Institute of Occupational Health, Helsinki, Finland Search for more papers by this author Søren R Paludan Corresponding Author Søren R Paludan orcid.org/0000-0001-9180-4060 Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Maria H Christensen Maria H Christensen Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Søren B Jensen Søren B Jensen Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Juho J Miettinen Juho J Miettinen University of Helsinki, Helsinki, Finland Search for more papers by this author Stefanie Luecke Stefanie Luecke Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Thaneas Prabakaran Thaneas Prabakaran Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Line S Reinert Line S Reinert Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Thomas Mettenleiter Thomas Mettenleiter Friedrich-Loeffler-Institut, Insel Riems, Germany Search for more papers by this author Zhijian J Chen Zhijian J Chen Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author David M Knipe David M Knipe Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA Search for more papers by this author Rozanne M Sandri-Goldin Rozanne M Sandri-Goldin Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA Search for more papers by this author Lynn W Enquist Lynn W Enquist Princeton University, Princeton, NJ, USA Search for more papers by this author Rune Hartmann Rune Hartmann orcid.org/0000-0003-1159-066X Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Department of Molecular Biology and Genetics, Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark Search for more papers by this author Trine H Mogensen Trine H Mogensen Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Aarhus University Hospital Skejby, Aarhus, Denmark Search for more papers by this author Stephen A Rice Stephen A Rice Department of Microbiology, University of Minnesota Medical School, Minneapolis, MN, USA Search for more papers by this author Tuula A Nyman Tuula A Nyman University of Helsinki, Helsinki, Finland Search for more papers by this author Sampsa Matikainen Sampsa Matikainen Finnish Institute of Occupational Health, Helsinki, Finland Search for more papers by this author Søren R Paludan Corresponding Author Søren R Paludan orcid.org/0000-0001-9180-4060 Department of Biomedicine, University of Aarhus, Aarhus, Denmark Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark Search for more papers by this author Author Information Maria H Christensen1,2, Søren B Jensen1,2, Juho J Miettinen3, Stefanie Luecke1,2, Thaneas Prabakaran1,2, Line S Reinert1,2, Thomas Mettenleiter4, Zhijian J Chen5,6, David M Knipe7, Rozanne M Sandri-Goldin8, Lynn W Enquist9, Rune Hartmann1,2,10, Trine H Mogensen1,2,11, Stephen A Rice12, Tuula A Nyman3, Sampsa Matikainen13 and Søren R Paludan 1,2 1Department of Biomedicine, University of Aarhus, Aarhus, Denmark 2Aarhus Research Center for Innate Immunology, University of Aarhus, Aarhus, Denmark 3University of Helsinki, Helsinki, Finland 4Friedrich-Loeffler-Institut, Insel Riems, Germany 5Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX, USA 6Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX, USA 7Department of Microbiology and Immunobiology, Harvard Medical School, Boston, MA, USA 8Department of Microbiology & Molecular Genetics, University of California, Irvine, CA, USA 9Princeton University, Princeton, NJ, USA 10Department of Molecular Biology and Genetics, Aarhus Research Center for Innate Immunity, Aarhus University, Aarhus, Denmark 11Aarhus University Hospital Skejby, Aarhus, Denmark 12Department of Microbiology, University of Minnesota Medical School, Minneapolis, MN, USA 13Finnish Institute of Occupational Health, Helsinki, Finland *Corresponding author. Tel: +45 87167843; E-mail: [email protected] The EMBO Journal (2016)35:1385-1399https://doi.org/10.15252/embj.201593458 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 Herpes simplex virus (HSV) 1 stimulates type I IFN expression through the cGAS–STING–TBK1 signaling axis. Macrophages have recently been proposed to be an essential source of IFN during viral infection. However, it is not known how HSV-1 inhibits IFN expression in this cell type. Here, we show that HSV-1 inhibits type I IFN induction through the cGAS–STING–TBK1 pathway in human macrophages, in a manner dependent on the conserved herpesvirus protein ICP27. This viral protein was expressed de novo in macrophages with early nuclear localization followed by later translocation to the cytoplasm where ICP27 prevented activation of IRF3. ICP27 interacted with TBK1 and STING in a manner that was dependent on TBK1 activity and the RGG motif in ICP27. Thus, HSV-1 inhibits expression of type I IFN in human macrophages through ICP27-dependent targeting of the TBK1-activated STING signalsome. Synopsis Type I interferon (IFN) is important for control of infection with viruses, including herpes simplex virus (HSV)-1. DNA viruses, such as HSV-1, induce IFN expression after sensing of viral DNA by cGAS and signaling through the STING–TBK1–IRF3 signaling pathway. Here, we demonstrate that the HSV-1 protein ICP27 targets the STING-TBK1 signalsome and inhibits IRF3 activation, thus preventing IFN expression. ICP27 prevents activation of IRF3 but not TBK1. ICP27 is translocated to the cytoplasm at late time points of infection where is co-localizes with TBK1 and STING. ICP27 associates with STING and TBK1 in a manner dependent on expression of both host proteins. ICP27 from the Simplexvirus genera of Alphaherpesvirinae inhibits IFN production in a manner dependent on the RGG box. Introduction Herpes simplex virus 1 (HSV-1) is a human pathogenic herpesvirus belonging to the subfamily of Alphaherpesvirinae. HSV-1 is widely distributed in human populations and capable of triggering a broad range of pathologies, ranging from the benign cold sores to fatal encephalitis. Primary HSV-1 infections are initiated by lytic viral replication in mucosal epithelial cells, and from this site, the virus enters into sensory neurons, where a lifelong latent–recurrent infection is established (Roizman et al, 2013). It is well established that the immune status of the host is a central determinant of the clinical outcome of HSV-1 infections (Paludan et al, 2011). The innate immune system is the first line of defense against pathogens, including HSV-1 (Paludan et al, 2011). This arm of the immune system exerts antiviral activity primarily through type I interferons (IFN)s and natural killer cells. In the case of HSV-1 infections, recent work has established that impaired ability to stimulate type I IFN expression strongly enhances susceptibility to herpes simplex encephalitis (Zhang et al, 2007; Perez de Diego et al, 2010; Sancho-Shimizu et al, 2011; Herman et al, 2012; Andersen et al, 2015). Crucial for the activation of the antiviral program is the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs) (Akira et al, 2006; Paludan & Bowie, 2013). Both RNA and DNA have been reported to stimulate IFN production during HSV-1 infections. The dsRNA-sensing TLR3 pathway is central for mounting full IFN responses in different cell types in the CNS, and genetic defects in this pathway lead to susceptibility to herpes simplex encephalitis (Zhang et al, 2007; Perez de Diego et al, 2010; Sancho-Shimizu et al, 2011; Herman et al, 2012; Andersen et al, 2015). Herpesviral DNA and mitochondrial DNA released in response to infection-induced stress are detected by the nucleotidyltransferase cGAS in a sequence-independent fashion (Sun et al, 2013; Wu et al, 2013; Rongvaux et al, 2014; West et al, 2015). DNA sensing stimulates signaling dependent on the adaptor protein stimulator of interferon genes (STING) (Ishikawa et al, 2009). Experiments in mice have shown that the lack of cGAS or STING renders mice highly susceptible to HSV-1 infections (Ishikawa et al, 2009; Li et al, 2013b). At the mechanistic level, DNA sensing by cGAS leads to production of cyclic GMP-AMP (cGAMP), which docks on STING to induce a conformational change allowing recruitment of the kinase TANK-binding kinase 1 (TBK1). This in turn allows recruitment of the transcription factor IFN regulatory factor 3 (IRF3) to the phosphorylated surface of STING, where IRF3 is phosphorylated and activated in a TBK1-dependent manner, to stimulate IFN-β gene transcription (Tanaka & Chen, 2012). Interestingly, the phosphorylation of the adaptor protein creates a positively charged surface to which IRF3 can bind. This is a property conserved among all of the three main IFN-stimulating adaptors in innate immunity, namely STING, TRIF, and MAVS (Liu et al, 2015). Because viruses have co-evolved with their hosts, they have gained mechanisms to evade and suppress host immune responses. Different proteins of HSV-1 are described to target immunological pathways, including the MHC pathway by the infected cell protein (ICP) 47 (York et al, 1994; Oldham et al, 2016). With respect to viral evasion of the type I IFN pathway, most work has concentrated on the viral E3 ubiquitin ligase-infected cell protein (ICP)0. HSV-1 deficient for ICP0 induces enhanced type I IFN levels in in vitro cell systems, compared to wild-type (WT) virus. At the mechanistic level, ICP0 has been reported to target the DNA sensor IFI16 for degradation (Orzalli et al, 2012), but also to employ other mechanisms (Cuchet-Lourenco et al, 2013). Other HSV-1 proteins reported to interfere with induction of type I IFN expression include VP16, Us11, ICP34.5, and ICP27 (Melchjorsen et al, 2006; Verpooten et al, 2009; Xing et al, 2012, 2013). Moreover, Vhs, ICP0, and ICP27 have also been reported to inhibit type I IFN-stimulated signaling through the Jak-STAT pathway (Mossman et al, 2000; Suzutani et al, 2000; Johnson et al, 2008). ICP27 is a multifunctional protein that is essential for HSV-1 replication (Rice & Knipe, 1990). The essential function of the protein is supported by the fact that ICP27 is conserved among all herpesviruses (Sandri-Goldin, 2011). The described functions of ICP27 in the viral replication cycle include transport of intronless HSV-1 mRNA transcripts from the nucleus to the cytosol in a manner where ICP27 favors the transport of viral transcripts over host mRNAs (Koffa et al, 2001; Chen et al, 2005; Tian et al, 2013). However, the mechanistic basis for inhibition of innate immune responses by ICP27 remains poorly defined. In recent years, it has emerged that macrophages constitute a central source of type I IFN during infections with viruses, including HSV-1, and that IFN from this source contributes significantly to antiviral immunity (Eloranta & Alm, 1999; Kumagai et al, 2007; Goritzka et al, 2015). Despite this, there is no mechanistic information on how HSV-1 evades the type 1 IFN response in human macrophages. In this work, we report that although only very limited productive replication occurs in HSV-1-infected human macrophages, ICP27 is expressed and the protein is essential for counteracting the production of type I IFNs. ICP27, which is highly conserved within the Simplexvirus genera of the Herpesvirinae, translocates from the nucleus to the cytoplasm during infection, where it interacts with the activated TBK1–STING signalsome, thus inhibiting IRF3 activation through the cGAS–STING pathway. Our work reveals a novel mechanism of viral inhibition of IFN expression. Moreover, the work demonstrates that viruses must have a broad repertoire of operative immune evasion strategies to counteract host responses even in cell types not supporting productive replication. Results HSV-1 harbors a mechanism to inhibit type I IFN expression, which is dependent on viral gene expression but independent of ICP0 To evaluate the abilities of different HSV-1 strains to stimulate type I IFN expression, we infected PMA-differentiated macrophage-like THP1 cells with four different strains of HSV-1. Accumulation of type I IFN bioactivity was measured in supernatants harvested at different time points. As shown in Fig 1A, all strains tested induced a modest but significant IFN response at 12 h post-infection. In supernatants from cells infected with the KOS (or KOS1.1), 17+, and McKrae strains, the IFN bioactivity peaked at the 12 h time point. However, infection with the F strain led to a pronounced further increase in stimulation of IFN bioactivity between 12 and 18 h of infection. The viruses did not affect viability of the cells within the time frame of the experiment (data not shown). These data led us to hypothesize that HSV-1 harbors a mechanism to inhibit type I IFN expression, which is dependent on viral gene expression, and not activated by the F strain. It should be noted that alternative interpretations of these data cannot formally be excluded, including differential accumulation of non-productive viral DNA replication intermediates by the HSV-1 strains being the cause of differential IFN responses. To start testing the hypothesis of viral evasion of IFN expression, we first evaluated whether inhibition of viral replication could augment induction of IFN production by the KOS strain. Interestingly, the blockage of viral replication by UV treatment of virus or acyclovir enabled the KOS strain to induce significantly more IFN than the replication competent virus, whereas the replication capacity of the F strain did not impact on the IFN-inducing potential (Fig 1B and C, and Appendix Fig S1A and data not shown). Figure 1. HSV-1 harbors a mechanism to inhibit type I IFN expression, which is dependent on viral gene expression but independent of ICP0 THP1 cells were infected with the shown strains of HSV-1 (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 h for measurement of type I IFN bioactivity. THP1 cells were treated with 0.1 μg/ml of acyclovir (ACV) and infected with the KOS and F HSV-1 strains (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. THP1 cells were treated with infectious or UV-inactivated HSV-1 (strain KOS). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. MDMs were infected with ICP0-deficient or revertant HSV-1 (strain KOS, MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. THP1-derived cells deficient for cGAS or STING were infected with the shown strains of HSV-1 (MOI 3) or stimulated with poly(I:C) (2 μg/ml). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. Data information: Data are presented as means of triplicates ± SD; symbols for P-values: **0.001 < P < 0.01; ***P < 0.001; ns, not significant. Download figure Download PowerPoint HSV-1 ICP0 is an ubiquitin E3 ligase, which has been reported to be central for viral evasion of type I IFN responses in human fibroblasts (Mossman et al, 2001; Eidson et al, 2002; Orzalli et al, 2012, 2013). We confirmed the previously published data by the finding that supernatants from human foreskin fibroblasts (HFF)s infected with HSV-1 7134 ICP0-deficient KOS-derived HSV-1 did indeed lead to strongly elevated IFN induction (Appendix Fig S1B). However, there is no information on the role of ICP0 in the inhibition of innate responses in human macrophages. Therefore, we infected primary human monocyte-derived macrophages (MDM)s with the HSV-1 7134 ICP0-deficient mutant virus as well as the 7134R rescued virus. In contrast to what was seen in HFFs, lack of ICP0 did not affect the ability of the virus to induce type I IFN production (Fig 1D). Likewise, ICP0 deficiency did not affect HSV-1-induced expression of IFN-α or IFN-β mRNA in peripheral blood mononuclear cells (Appendix Fig S1C and D). Finally, we were interested in evaluating whether the elevated IFN induction by the HSV-1F strain proceeded through the same innate immune pathways as for the other strains tested. Therefore, CRISPR/Cas9-generated THP1-derived cells lacking cGAS or STING were infected with different virus strains and IFN bioactivity in the supernatants was measured. For all virus strains, the stimulated IFN bioactivity was abrogated in cells lacking cGAS or STING (Fig 1E). Collectively, these data demonstrate that HSV-1 harbors a mechanism to inhibit type I IFN expression, which is dependent on viral gene expression, but independent of ICP0. This viral immune evasion strategy targets component(s) in the cGAS–STING pathway. Expression of HSV-1 ICP27 in macrophages correlates inversely with induction of type I IFN Given the dependence on viral replication products for inhibition of type I IFN production, we used quantitative MS-based proteomics to map the HSV-1 proteome in the cytoplasm of infected MDMs. Altogether, we identified 17 of the 77 annotated HSV-1 proteins. Among the viral proteins identified, 10 were virion proteins and 2 were involved in viral assembly and egress. In addition, we identified three proteins with functions in viral genome replication, and finally, the 2 proteins ICP4 and ICP27 involved in early viral gene expression (Fig 2A and Table S1). While ICP4 is mainly described to regulate viral transcription, ICP27 is a multifunctional protein with many identified protein interaction partners (Sandri-Goldin, 2011). The expression of ICP27 in HSV-1-infected macrophages was confirmed by Western blotting on lysates from MDMs, and interestingly, we observed lower expression of ICP27 in cells infected with the F strain as compared to the KOS strain at 12 hpi (Fig 2B). Despite the detectable expression of viral proteins in MDMs, the virus was not able to produce progeny virus in this cell type (Fig 2C), potentially suggesting alternative functions for HSV-1 proteins in MDMs. We therefore compared expression of ICP27 in THP1 cells after infection with the four HSV-1 strains evaluated for IFN stimulation in Fig 1A. Interestingly, the KOS and McKrae strains, which induced less type I IFN after 18 h of infection, stimulated strong ICP27 expression, whereas infection with the 17+ strain led to a modest but clear expression of ICP27 (Fig 2D). In contrast to this, the F strain did not express ICP27 in the THP1 cells. Thus, ICP27 is part of a small subset of non-structural HSV-1 proteins expressed during infection in macrophages, and its expression correlated inversely with expression of type I IFNs. Figure 2. HSV-1 does not replicate productively in macrophages, but ICP27 expression correlates inversely with type I IFN production MDMs were infected for 18 h with HSV-1 (KOS, MOI 1). Cytosolic viral proteins were detected by mass spectrometry using iTRAQ labeling. Identified viral proteins are shown. MDMs were infected with the KOS or F strains of HSV-1 (MOI 3). Cell lysates were harvested 12 hpi, and levels of ICP27 and β-actin were determined by Western blotting. HFFs and MDMs were infected with HSV-1 (KOS, MOI 0.1, and 1.0). Culture medium was replaced 3 hpi and isolated 24 hpi for viral plaque assay. Data are presented as means of triplicates ± SD. THP1 cells were infected with the indicated strains of HSV-1 (MOI 3) for 12 h. Cell lysates were isolated, and levels of ICP27 and β-actin were determined by Western blotting. Source data are available online for this figure. Source Data for Figure 2 [embj201593458-sup-0002-SDataFig2.pdf] Download figure Download PowerPoint HSV-1 ICP27 is a negative regulator of IFN induction through the cGAS–STING pathway in macrophages It has been reported that the strain F has 961-bp changes relative to the strain 17+, resulting in 310 amino acid differences between strains F and 17+, affecting 63 out of 77 viral proteins (Szpara et al, 2010). Thus, given the difference between HSV-1 strains, we further wanted to test ICP27 as a candidate immune evasion protein. First, to evaluate the role of ICP27 in modulation of the type I IFN response in human macrophages, we measured type I IFN bioactivity in supernatants from THP1 cells infected with either WT or ICP27-deficient KOS. At 12 h post-infection, both WT and ICP27-deficient virus induced modest but significant IFN production, with the ICP27-deficient strain inducing slightly more type I IFN than the WT strain (Fig 3A). However, while the IFN levels in supernatants from cells infected with the WT strain decreased after the 12-h time point, the IFN levels in supernatant of cells infected with ICP27-deficient virus increased very strongly in this time interval (Fig 3A). This was also seen when measuring levels of the IFN-stimulated gene CXCL10 (Appendix Fig S2A). Similar to the observation in THP1 cells, ICP27-deficient HSV-1 also stimulated type I IFN bioactivity to much higher levels than WT virus in MDMs (Fig 3B). The WT and ICP27-deficient viruses did not affect cell viability within the time frame of the experiments (data not shown). The potent induction of type I IFNs by the ICP27-deficient mutant was not limited to human macrophages, because it was also seen in HFFs and murine macrophages (Appendix Fig S2B and C). The observed elevated IFN induction by ICP27-deficient HSV-1 was unlikely to be explained merely by the impairment of the replication cycle, since both ICP0-deficient and ICP27-deficient viruses failed to induce expression of the major capsid protein VP5 (Appendix Fig S2D), but only the latter virus induced elevated levels of IFN. To examine whether the elevated IFN response induced by the ICP27-deficient virus proceeded through the cGAS–STING pathway, THP1-derived KO cell lines were infected with HSV-1 KOS or ΔICP27 virus. Measurement of IFN bioactivity in the supernatants revealed that the elevated IFN production in cells infected with the ΔICP27 virus was dependent on cGAS and STING (Fig 3C), thus suggesting that ICP27 is required for targeting of the cGAS–STING pathway in the context of HSV-1 infection. To investigate whether ICP27 is sufficient to inhibit the cGAS–STING pathway, HEK293T cells were transfected with STING, ICP27, and an ISRE-driven luciferase promoter. STING transfection alone stimulated luciferase expression, but ICP27 strongly inhibited this response (Fig 3D). Figure 3. ICP27 inhibits the cGAS–STING pathway in macrophages THP1 cells were infected with HSV-1 KOS or an ICP27-deficient mutant (ΔICP27) on KOS genetic background (MOI 3). Supernatants were harvested from untreated cultures or cells infected for 12 or 18 h for measurement of type I IFN bioactivity. MDMs were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. THP1-derived cells deficient for cGAS or STING were infected with HSV-1 KOS or ΔICP27 (MOI 3). Supernatants were harvested 18 hpi for measurement of type I IFN bioactivity. HEK293T cells were transfected with 12 ng STING plasmid DNA, 5 or 25 ng ICP27 plasmid DNA, and reporter gene constructs as indicated (2 × 104 cells per well). Reporter gene activity was measured in lysates isolated 24 h post-transfection. THP1 cells were infected with HSV-1 KOS or ΔICP27 (MOI 3) in the presence or absence of acyclovir (ACV). Supernatants were isolated 18 hpi for measurement of type I IFN bioactivity. Data information: Data are presented as means of triplicates ± SD; symbols for P-values: ***P < 0.001; ns, not significant. Download figure Download PowerPoint ICP27 is an immediate-early protein, but is also produced at late time points during infection, and reported to be packed into viral particles as a tegument protein (Maringer & Elliott, 2010). Western blotting of viral stocks confirmed the presence of ICP27 in virions (Appendix Fig S2E). Therefore, to examine whether ICP27 was responsible for the replication-dependent nature of the evasion of IFN responses, we treated cells with acyclovir prior to infection with WT or ΔICP27 HSV-1. Acyclovir treatment inhibited late but not early ICP27 expression in THP1 cells (Appendix Fig S2F), thus suggesting early ICP27 expression in macrophages to occur as an immediate-early or gene, and the late ICP27 expression to exhibit features of a late gene. As also observed in Fig 1B, inhibition of viral replication enabled WT HSV-1 to induce high levels of IFN (Fig 3E). By contrast, for the ΔICP27 virus, which induced much more IFN than the WT virus, IFN levels were not affected by acyclovir treatment. Collectively, these data suggest that de novo produced ICP27 targets the STING pathway in immortalized and primary cells to inhibit production of type I IFN. ICP27 inhibits the cGAS–STING pathway downstream of TBK1 phosphorylation but upstream of IRF3 phosphorylation The ICP27 protein is reported to have specific functions in both nuclear and cytosolic compartments, enabled through a shuttling mechanism, which is independent of other HSV-I proteins (Mears & Rice, 1998). To start characterization of the mechanism through which ICP27 inhibits the cGAS–STING pathway, we first determined the subcel
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