A Novel Mitochondrial MAVS/Caspase-8 Platform Links RNA Virus−Induced Innate Antiviral Signaling to Bax/Bak-Independent Apoptosis This information is current as of October 2, 2021. Souhayla El Maadidi, Laura Faletti, Birgit Berg, Christin Wenzl, Katrin Wieland, Zhijian J. Chen, Ulrich Maurer and Christoph Borner J Immunol 2014; 192:1171-1183; Prepublished online 3 January 2014; Downloaded from doi: 10.4049/jimmunol.1300842 http://www.jimmunol.org/content/192/3/1171 Supplementary http://www.jimmunol.org/content/suppl/2014/01/03/jimmunol.130084 http://www.jimmunol.org/ Material 2.DCSupplemental References This article cites 54 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/192/3/1171.full#ref-list-1 Why The JI? Submit online. by guest on October 2, 2021 • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology A Novel Mitochondrial MAVS/Caspase-8 Platform Links RNA Virus–Induced Innate Antiviral Signaling to Bax/Bak-Independent Apoptosis Souhayla El Maadidi,*,† Laura Faletti,*,† Birgit Berg,*,†,1 Christin Wenzl,*,†,2 Katrin Wieland,* Zhijian J. Chen,‡ Ulrich Maurer,*,x,{ and Christoph Borner*,x,{ Semliki Forest virus (SFV) requires RNA replication and Bax/Bak for efficient apoptosis induction. However, cells lacking Bax/Bak continue to die in a caspase-dependent manner. In this study, we show in both mouse and human cells that this Bax/Bak-independent pathway involves dsRNA-induced innate immune signaling via mitochondrial antiviral signaling (MAVS) and caspase-8. Bax/Bak- deficient or Bcl-2– or Bcl-xL–overexpressing cells lacking MAVS or caspase-8 expression are resistant to SFV-induced apoptosis. The signaling pathway triggered by SFV does neither involve death receptors nor the classical MAVS effectors TNFR-associated factor-2, IRF-3/7, or IFN-b but the physical interaction of MAVS with caspase-8 on mitochondria in a FADD-independent manner. Downloaded from Consistently, caspase-8 and -3 activation are reduced in MAVS-deficient cells. Thus, after RNA virus infection MAVS does not only elicit a type I antiviral response but also recruits caspase-8 to mitochondria to mediate caspase-3 activation and apoptosis in a Bax/Bak-independent manner. The Journal of Immunology, 2014, 192: 1171–1183. emliki Forest virus (SFV) is an enveloped, single-stranded, anticancer therapy and vaccination (5). It is therefore crucial to and positive-sense RNA virus belonging to the Alphavirus elucidate the exact molecular mechanism by which SFV induces http://www.jimmunol.org/ S genus of the Togaviridae family. Its pathogenicity in humans apoptosis in host cells. and animals varies from asymptomatic to fatal, causing encephalitis We previously showed that apoptosis induced by SFV is RNA or epidemic polyarthritis, depending on the virulence of the strain replication dependent and involves the intrinsic mitochondrial (1, 2). The virus has a broad host range, replicates to high titers, and signaling pathway, in particular Bak (4). Bak and Bax are required induces apoptosis of numerous mammalian cells (3, 4). Importantly, for mitochondrial outer membrane permeabilization (MOMP) and its strong death- and type I IFN–inducing capacity has spurred the the subsequent release of cytochrome c, which then activates caspase- development of various SFV vectors that are currently used for 3 via the Apaf-1/caspase-9 apoptosome (6). Effective MOMP de- pends on so-called BH3-only proteins that sense the apoptotic by guest on October 2, 2021 stimulus and either directly bind to and activate Bax/Bak or in- *Institute of Molecular Medicine and Cell Research, Albert Ludwigs University † Freiburg, D-79104 Freiburg, Germany; Faculty of Biology, Albert Ludwigs Univer- teract with the Bcl-2–like survival factors Bcl-2, Bcl-xL, or Mcl-1 sity Freiburg, D-79104 Freiburg, Germany; ‡Department of Molecular Biology, to release Bax/Bak from inhibitory constraints (6). Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390; xSpemann Graduate School of Biology and Medicine, Albert Interestingly, cells infected with SFV or other alphaviruses such Ludwigs University Freiburg, D-79104 Freiburg, Germany; and {Centre for Biolog- as Sindbis continue to die in an apoptotic manner when Bax/Bak ical Signaling Studies, D-79104 Freiburg, Germany are depleted (4) or when Bcl-2 is overexpressed (3, 7, 8). This 1Current address: greenovation Biotech, Freiburg, Germany. behavior is typical for apoptosis induced by death receptor sig- 2Current address: U3Pharma, Martinsried, Germany. naling (9). However, we were unable to inhibit SFV-induced ap- Received for publication March 29, 2013. Accepted for publication November 25, optosis by blocking TNF-a, Fas ligand (FasL), TRAIL, and/or 2013. their receptors with neutralizing Abs (4). Nevertheless, caspase-8, This work was supported by Spemann Graduate School of Biology and Medicine a crucial mediator of death receptor signaling clearly was processed Grant GSC-4 and Centre for Biological Signalling Studies Grant EXC-294 (to C.B. and U.M., both funded by the Excellence Initiative of the German Federal and State and activated in SFV-infected cells (4, 10), and previous studies Governments, Germany). L.F. was supported by the Virtual Liver Network, spon- showed that the caspase-8 inhibitors cytokine response modifier sored by the German Federal Ministry of Education and Research, and Z.J.C. was A or FADD-like IL-1 b–converting enzyme-inhibitory protein (FLIP) supported by National Institutes of Health Grant AI-093967. were able to interfere with alphavirus-induced apoptosis (11, 12). Address correspondence and reprint requests to Dr. Christoph Borner, Institute of Molecular Medicine and Cell Research, Albert Ludwigs University Freiburg, Stefan This pointed to a caspase-8–dependent but Bax/Bak- and death Meier Strasse 17, D-79104 Freiburg, Germany. E-mail address: christoph.borner@ receptor–independent apoptosis signaling pathway triggered by SFV. uniklinik-freiburg.de RNA viruses induce an antiviral type I IFN response in infected The online version of this article contains supplemental material. host cells (13). After receptor-mediated endocytosis, the viruses Abbreviations used in this article: DISC, death-inducing signaling complex; eIF2a, release their genomic RNA into the cytoplasm where it replicates, eukaryotic initiation factor 2 a; FasL, Fas ligand; FLIP, FADD-like IL-1 b–convert- producing dsRNA as a byproduct (14). Cytoplasmic viral dsRNA ing enzyme-inhibitory protein; HCV, hepatitis C virus; IFNAR, IFN-a/b receptor; MAVS, mitochondrial antiviral signaling; MDA-5, melanoma differentiation Ag-5; is sensed by a class of ubiquitous cytoplasmic RNA helicases, reti- MEF, mouse embryonic fibroblast; MOI, multiplicity of infection; MOMP, mitochon- noic acid inducible gene-I (RIG-I) (15), and melanoma differentia- drial outer membrane permeabilization; OAS, oligoadenylate synthetase; PARP, poly (ADP-ribose) polymerase; PI, propidium iodide; PKR, protein kinase R; PLA, prox- tion Ag-5 (MDA-5) (16), which trigger an antiviral signaling imity ligation assay; RIG-I, retinoic acid inducible gene-I; SFV, Semliki Forest virus; cascade via their common adaptor called mitochondrial antiviral sh-Ctrl, scrambled control; shRNA, short hairpin RNA; TRAF, TNFR-associated signaling (MAVS) (also called IFN-b stimulator 1, virus-induced factor; TX, Triton X-100; WT, wild-type. signaling adapter, or caspase activation and recruitment domain Copyright Ó 2014 by The American Association of Immunologists, Inc. 0022-1767/14/$16.00 adaptor inducing IFN-b) (17–20). This leads to the production www.jimmunol.org/cgi/doi/10.4049/jimmunol.1300842 1172 SFV APOPTOSIS BY MAVS/CASPASE-8 INDEPENDENT OF Bax/Bak of type I IFNs (IFN-a/b) via NF-kB and IRF-3/-7 transcription were grown in high-glucose DMEM (4.5 g/l glucose) supplemented with factors (13). IFN-a/b then induce, via a common cell surface IFN- 10% FCS. MEFs were obtained from different sources (see Acknowl- a/b receptor (IFNAR) and the JAK-STAT signaling pathway, the edgments); the other cells were purchased from American Tissue Cell Collection. expression of IFN-inducible, dsRNA-activated protein kinase R (PKR) and 29-59-oligoadenylate synthetase (2-5-OAS) (21, 22). Both SFV production and titration enzymes are strongly activated by dsRNA (23, 24). Although PKR The virulent SFV prototype strain L10 was used in this study. Adherent blocks protein synthesis by phosphorylating eukaryotic initiation mosquito A. albopictus cells were infected with 20 multiplicity of infection factor 2 a (eIF2a), the 29-59-oligoadenylates generated by OAS ac- (MOI) SFV in L15 medium supplemented with 10% FCS and 4% Difco tivate the latent endonuclease RNase L to degrade viral RNAs (24). Bacto phosphate tryptose broth. After incubating at 28˚C for 24 h, the viral Although overexpression of PKR was shown to induce apoptosis supernatant was harvested, centrifuged at 3000 3 g for 10 min, and stored 2 (25, 26) and PKR2/2 mouse embryonic fibroblasts (MEFs) were at 80˚C. Virus titers from the culture medium of infected MEFs (10 MOI) were determined by the plaque assay. Briefly, monolayers of Vero resistant to cell death induced by dsRNA and LPS (27), the role of cells with .90% confluency were infected with 10-fold serial dilutions of PKR in the context of RNA virus–induced apoptosis has remained virus in high-glucose DMEM supplemented with 2% FCS and 20 mM enigmatic (23).