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1Department of Immunobiology, Yale University, New Haven, CT 06520, USA. 2De-partment of Laboratory Medicine, Yale University, New Haven, CT 06520, USA.3Department of Laboratory Medicine, University of Washington, Seattle, WA98195, USA. 4Division of Cell Medicine, Department of Life Science, Medical Re-search Institute, Kanazawa Medical University, Uchinada, Ishikawa, Japan. 5Divi-sion of Gastroenterology and Hepatology, Department of Medicine, Universityof Washington, Seattle, WA 98195, USA. 6Department of Microbial Pathogenesis,Yale University, New Haven, CT 06520, USA. 7Department of Comparative Medi-cine, Yale University, New Haven, CT 06520, USA. 8Howard Hughes Medical Insti-tute, Chevy Chase, MD 20814, USA.*Corresponding author. Email: [email protected] (A.I.); [email protected] (S.L.F.)
Fink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
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IRE1a promotes viral infection by conferring resistanceto apoptosisSusan L. Fink,1,2,3* Teshika R. Jayewickreme,1 Ryan D. Molony,1 Takao Iwawaki,4
Charles S. Landis,5 Brett D. Lindenbach,6,7 Akiko Iwasaki1,8*
The unfolded protein response (UPR) is an ancient cellular pathway that detects and alleviates protein-foldingstresses. The UPR components X-box binding protein 1 (XBP1) and inositol-requiring enzyme 1a (IRE1a) promotetype I interferon (IFN) responses. We found that Xbp1-deficient mouse embryonic fibroblasts and macrophageshad impaired antiviral resistance. However, this was not because of a defect in type I IFN responses but rather aninability of Xbp1-deficient cells to undergo viral-induced apoptosis. The ability to undergo apoptosis limited infectionin wild-type cells. Xbp1-deficient cells were generally resistant to the intrinsic pathway of apoptosis through anindirectmechanism involving activation of the nuclease IRE1a. We observed an IRE1a-dependent reduction in theabundance of the proapoptoticmicroRNAmiR-125a and a corresponding increase in the amounts of themembersof the antiapoptotic Bcl-2 family. The activation of IRE1a by the hepatitis C virus (HCV) protein NS4B in XBP1-proficientcells also conferred apoptosis resistance and promoted viral replication. Furthermore, we found evidence of IRE1aactivation and decreased miR-125a abundance in liver biopsies from patients infected with HCV compared to thosein the livers of healthy controls. Our results reveal a prosurvival role for IRE1a in virally infected cells and suggest apossible target for IFN-independent antiviral therapy.
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INTRODUCTIONGreat advances have been made in our understanding of the moleculardefinitions of pattern recognition receptors (PRRs) and the pathogen-associated molecular patterns (PAMPs) that cells use to distinguishviruses from self (1, 2). PRR engagement results in the transcription ofthe genes that encode the type I interferons (IFNs) IFN-a and IFN-b,which bind to the IFN-a/b receptor to induce the expression ofhundreds of IFN-stimulated genes (ISGs). ISGs act in concert to blockfurther viral replication and spread, as well as to support the activationof adaptive antiviral immunity (3). However,many viruses have evolvedevasion mechanisms to limit PRR recognition and signal transduction,andPRR-independentmechanisms for innate sensing of viral infectionsremain unclear.
Endoplasmic reticulum (ER) stress occurs during infection by vari-ous viruses, presumably due to the overwhelming synthesis of viral pro-teins (4). The unfolded protein response (UPR) is a ubiquitous cellularpathway to detect and alleviate ER stress. The UPR is initiated by threesensors that residewithin the ER: protein kinase receptor–like ERkinase(PERK), activating transcription factor 6 (ATF6), and inositol-requiringenzyme 1 (IRE1) (5–7). IRE1, a highly conserved UPR sensor, oligo-merizes and autophosphorylates in response to ER stress, which activatesits cytosolic ribonuclease (RNase) domain and initiates a nonconven-tional mRNA splicing reaction of Xbp1 mRNA (8). Once processed,the splicedXbp1mRNAencodes a transcription factor, which controlsthe expression of target genes. IRE1a targets other specific mRNAs,leading to their degradation in a process termed regulated IRE1-
dependent decay (RIDD) (9, 10). ER stress synergistically enhancescytokine and IFN responses to PRR engagement through IRE1aand X-box binding protein 1 (XBP1) (11–13). Specific activation ofIRE1a also occurs during innate immune recognition of PAMPs byToll-like receptors (TLRs) (11). In this setting, XBP1 promotes theproduction of inflammatory cytokines and IFN-b. Moreover, IRE1agenerates ligands for RIG-I–like receptors (RLRs) during the UPR(14), which are degraded by SKIV2L RNA exosomes to prevent in-appropriate activation of type I IFN responses (15). These observationsprompted us to investigate the possible role of XBP1 in innate immuneresponses to viral infections, with the hypothesis that XBP1 could pro-mote IFN-mediated viral resistance.
Here, we describe an unexpected role for XBP1 in antiviral resistance,not through enhancement of the IFN response but rather by modu-lating susceptibility to host cell apoptosis. Xbp1-deficient cells wereresistant to apoptosis during infection with vesicular stomatitis virus(VSV) and herpes simplex virus (HSV), and failure to undergo celldeath resulted in increased viral replication. Xbp1 deficiency resultsin activation of its upstream enzyme IRE1a, which degrades specificcytosolic RNA targets (16, 17). We found that apoptosis resistance intheXbp1-deficient cells required IRE1a and its degradationofmicroRNA(miRNA),miR-125a. Notably, the hepatitis C virus (HCV) nonstructuralprotein 4B (NS4B), which stimulates IRE1a activation (18), promotedthe survival of infected cells and viral replication.Moreover, liver biopsiesfrom patients infected with HCV showed IRE1a activation and reducedmiR-125a abundance compared to healthy controls. These findingshighlight the role of UPR effectors in regulating IFN-independentmechanisms of innate antiviral resistance through the induction ofapoptosis to limit viral infection.
RESULTSXbp1 deficiency impairs control of viral infectionTo determine the effect of Xbp1 deficiency on host defense against viralreplication, we infected Xbp1−/− mouse embryonic fibroblasts (MEFs)with an RNA virus, VSV, and a DNA virus, HSV. XBP1 deficiency was
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achieved through an insertion of the neomycin resistance gene intoparts of exons 1 and 2, as well as the intervening intron (19). Thisinsertion still enables Xbp1 mRNA splicing but results in a frameshiftof the remaining amino acids to prevent protein production. Comparedto wild-type (WT) MEFs, VSV replication was enhanced in Xbp1−/−
MEFs as determined by measuring VSV-G–green fluorescent protein(GFP) relative abundance by flow cytometry (Fig. 1, A and B) and byplaque assays of released virus from MEF culture medium (Fig. 1C).Similarly, Xbp1−/− MEFs also supported increased replication of HSV-1–GFP as indicated by measurement of GFP abundance (Fig. 1, Dand E) and viral titer in the supernatant (Fig. 1F).
To determine whether the impaired viral control in Xbp1−/− MEFsresulted from deficient IFN responses, we measured the expression ofgenes encoding type I IFNs and of an ISG,Mx1, in MEFs infected withVSV. Unexpectedly, we observed enhanced induction of Ifna4 andIfnb1 in Xbp1−/− MEFs infected with VSV compared to WT MEFs(fig. S1, A and B). Induction of Mx1 also increased in Xbp1−/− MEFs(fig. S1C). Moreover, Xbp1−/−MEFs were not impaired in IFN respon-siveness because pretreatment with IFN-b preventedVSV replication inXbp1−/− MEFs (fig. S1D). In contrast and consistent with previous re-ports (12, 13), the Xbp1-deficient MEF response to transfection withpolyinosinic:polycytidylic acid [poly(I:C)] [an MDA5 (melanomadifferentiation–associated protein 5) agonist] was impaired (fig. S1E),suggesting that the enhanced IFN response to VSV was specific to rep-licating virus. Together, these findings suggest that Xbp1 contributes toprotective antiviral responses independently of type I IFNs.
Fink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
Xbp1 deficiency confers resistance to virus-triggeredcell deathViral infection often culminates in the death of infected host cells. Todetermine whether the phenotype we observed in the Xbp1-deficientMEFs was due to a difference in the death of infected cells, we evaluatedcell death and the abundance of virally encoded GFP after infection.During VSV infection, we found thatmost of theWT cells (~85%)weredead 24 hours after infection. In contrast, most Xbp1−/− MEFs (~74%)were resistant to cell death and accumulated higher amounts of viralprotein as determined by measurement of GFP abundance (Fig. 2, Aand B). Similarly, whereas a large proportion of HSV-infectedWT cellsunderwent cell death, infectedXbp1−/−MEFswere resistant to death (Fig.2, C and D). To determine whether acute ablation of Xbp1 expression
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Fig. 1. Xbp1 deficiency enhances the susceptibility of MEFs to HSV and VSV.(A to F) WT and Xbp1−/− MEFs were infected with VSV-GFP at a multiplicity ofinfection (MOI) of 1 [(A) to (C)] or with HSV-1–GFP at an MOI of 10 [(D) to (F)].Twenty-four hours later, the extent of infection was determined by measuringthe relative abundance of GFP by flow cytometry. Data are from one experimentrepresentative of three independent experiments (A and D). The mean fluores-cence intensity (MFI) of GFP in the indicated cells was then determined. Data aremeans ± SD of three independent experiments (B and E). Viral titers in the cellculture medium were measured by plaque assay at 48 (C) and 72 (F) hours afterinfection. PFU, plaque-forming units. Data are means ± SD of three independentexperiments (C and F). *P < 0.05; ***P < 0.001 compared to WT, unpaired t test.
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Fig. 2. Xbp1-deficient cells are resistant to cell death during infection with VSVand HSV. (A to D) WT and Xbp1−/−MEFs were left uninfected (mock) or were infectedwith VSV-GFP [(A) and (B)] or HSV-1–GFP [(C) and (D)] for 24 hours. Cell deathwas thenassessed with a membrane-impermeant, amine-reactive fluorescent dye, which wasmeasured by flow cytometry. Data are from one experiment and are representativeof three experiments (A and C). The percentages of dead cells were then determined.Data are means ± SD of three independent experiments (B and D). (E and F) BMDMswere cultured from Xbp1flox/flox ESR Cre+ (Xbp1D) or Cre− littermate (WT) mice in thepresence of tamoxifen. Cells were infected with VSV-GFP at the indicated MOI for24 hours. Viability was then assessed by measuring 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction. Data aremeans ± SD of three replicates and are representative of three experiments (E and F).**P < 0.01 compared to WT, unpaired t test.
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would have a similar effect, we treated WT MEFs with smallinterfering RNA (siRNA) targeting Xbp1. Xbp1 knockdown stronglysuppressed VSV-induced cell death and enhanced production ofvirally encoded GFP (fig. S2, A and B), consistent with the results ofexperiments with Xbp1−/− MEFs. Reconstitution of Xbp1−/− MEFswith plasmid-encoded Xbp1 restored VSV-induced cell death andrestricted the production of virally encoded GFP (fig. S2C). To deter-mine whether these findings extended to additional cell types, wecultured bone marrow–derived macrophages (BMDMs) from micewith a tamoxifen-inducible conditional Xbp1 deletion (Xbp1flox/flox
ESR Cre) (20). Xbp1D BMDMs were resistant to death during VSVinfection (Fig. 2, E and F, and fig. S2D), indicating that Xbp1 geneticdeficiency results in protection from cell death in fibroblasts andmacrophages. These results suggest that there should not be anXbp1-dependent antiviral phenotype against viruses that do nottrigger the death of infected cells. We found that infection with aVSV-Gpseudotyped lentivirus encodingGFPdid not result in host celldeath (fig. S3A). In this case, we did not observe enhanced productionof virally encoded GFP in Xbp1−/− MEFs (fig. S3, A and B). Thesefindings suggest that impaired control of VSV and HSV infection byXbp1−/− MEFs was directly related to their resistance to cell death.
Apoptosis limits the replication of VSV and HSVThe end point of cell death can result from numerous upstreamsignaling pathways. VSV infection induces apoptotic cell death inJurkat cells (21) and MCF-7 breast adenocarcinoma cells (22). WTMEFs infected with VSV demonstrated active caspase-3 (Fig. 3A), in-dicating apoptotic cell death in these cells. In contrast, Xbp1−/−MEFsfailed to activate caspase-3 during VSV infection (Fig. 3A). Similarly,HSV-infected WT MEFs contained active caspase-3, and Xbp1−/−
MEFs were resistant to the activation of this apoptotic effector (Fig.3B). These findings were not limited to MEFs because the activationof caspase-3 and caspase-7 also occurred in VSV-infected BMDMs andXbp1D BMDMs were resistant to VSV-induced caspase-3 activation(Fig. 3C).
Some viruses induce apoptosis as a means of viral transmission andavoidance of the immune system (23). In other cases, apoptosis isbeneficial for the host and limits viral replication. We observed de-creased abundance of virally encoded GFP in the population of deadcells during HSV infection of WT MEFs (Fig. 2C), suggesting that ap-optosis may limit viral replication. To test this hypothesis, we added acaspase inhibitor, carbobenzoxy-valyl-alanyl-aspartyl-[O-methyl]-fluoromethylketone (zVAD), to infected cells. We found that zVAD
Fink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
prevented the death of VSV-infected MEFs (Fig. 3D) and led to theincreased abundance of virally encoded GFP (Fig. 3, D and E), pheno-copying the result obtained from experiments with Xbp1−/− MEFs.Similarly, inhibition of apoptosis with zVAD increased the abundanceof virally encodedGFP inHSV-infectedMEFs, to an extent similar tothat attained in Xbp1−/−MEFs (Fig. 3F). In addition, overproductionof the antiapoptotic protein BCL2 limited the death of VSV-infectedcells and led to the increased abundance of virally encoded GFP (fig.S3C). Together, these findings suggest that Xbp1-deficient cells areresistant to virus-induced apoptosis and that apoptosis directly limitsviral replication.
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Fig. 3. Apoptosis induced by VSV and HSV limits viral infection. (A to B) WTand Xbp1−/− MEFs were left uninfected (mock) or were infected with VSV-GFP(A) or HSV-1–GFP (B) for 24 hours. Cells were then stained with an antibodyspecific for active (cleaved) caspase-3, which wasmeasured by flow cytometry. Dataare from one experiment and are representative of three experiments. (C) BMDMswere cultured from Xbp1flox/flox ESR Cre+ (Xbp1D) or Cre− littermate (WT) mice in thepresence of tamoxifen. Cells were infected with VSV-GFP at the indicated MOI for7 hours. Caspase-3 activity was then assessed by measuring fluorometric sub-strate cleavage and is shown relative to that in uninfected cells. Data are means ±SD of three replicates and are representative of three experiments. (D to F) MEFswere infected in the presence of zVAD to inhibit caspase activity. Twenty-four hoursafter infection, cell death was assessed with a membrane-impermeant, amine-reactive fluorescent dye, which was measured by flow cytometry. The extent ofinfection was determined by measuring the relative abundance of GFP by flow cy-tometry. Data are fromone experiment and are representative of three independentexperiments. *P < 0.01 compared to WT, unpaired t test.
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Resistance to virally induced apoptosis in Xpb1−/− cells isindependent of Beclin 1 and CHOPER stress is associatedwith autophagy,which regulates cell survival (24).In particular, XBP1 promotes transcription of the gene encoding theautophagy component, Beclin 1 (25). Consistent with these data, wefound decreased Beclin 1 in Xbp1-deficient cells (fig. S4A). However,knockdown of Beclin 1 with Beclin 1–specific siRNA did not affectVSV infection or the induction of cell death in either WT or Xbp1-deficient MEFs (fig. S4B). Our finding that Xbp1-deficient cells wereresistant to VSV- and HSV-induced apoptosis suggests the possibilitythat apoptosis during viral infection may directly result from XBP1-mediated transcriptional activity. During ER stress, XBP1 partially con-tributes to the production of the apoptosis mediator CHOP (C/EBPhomologous protein) (26), an induction process that may play a role invirus-induced apoptosis. As a functional control, transfection withChop-specific siRNA prevented the death of MEFs treated with theER stress–inducing agents tunicamycin and thapsigargin (fig. S5, A andB). In contrast, Chop knockdown did not prevent the death of VSV- orHSV-infected MEFs (fig. S5B). Further arguing against a direct role forXBP1-mediated transcriptional activity in virally induced cell death, wedid not observe Xbp1 splicing (fig. S6A) or induction of the expressionof the UPR-responsive genes Hspa5 [encoding BiP (binding immuno-globulin protein)] and Chop during VSV infection (fig. S6, B and C),consistent with published observations for VSV (27) and HSV (28, 29).
Xbp1-deficient cells are resistant to the intrinsic pathwayof apoptosisBecause we did not find evidence for XBP1-mediated transcriptionalactivity in promoting apoptosis during infection, we hypothesized thatXbp1-deficient cells may be inherently resistant to apoptosis in general.We therefore treated Xbp1−/−MEFs with a panel of apoptosis-inducingstimuli. Staurosporine and gliotoxin stimulate the intrinsic or mito-chondrial pathway of apoptosis (30–33), whereas ligation of the tumornecrosis factor (TNF) and Fas receptors initiates the extrinsic apoptoticpathway (34). Xbp1−/− MEFs were specifically resistant to stimuli thatinduced the intrinsic pathway of apoptosis, both as demonstrated byincreased viability (Fig. 4A) and impaired caspase-3 activation (Fig.4B). Consistent with previous studies demonstrating a protective roleof Xbp1 during ER stress (26, 35–37), Xbp1−/− MEFs were slightlymore susceptible to death induced by tunicamycin (Fig. 4A). In con-trast, there was no difference betweenWTandXbp1−/−MEFs in necrot-ic death induced by a high concentration of cycloheximide (Fig. 4A).These findings were further verified in experiments with Xbp1DBMDMs, which demonstrated resistance to staurosporine and the se-lective Bcl-2 inhibitor ABT-737 (38) but not to TNF-induced apoptosis(Fig. 4C). Thus, Xbp1 genetic deficiency results in specific protectionfrom the intrinsic pathway of apoptosis.
The resistance of Xbp1-deficient cells to apoptosis resultsfrom the activation of IRE1aXbp1 deficiency results in activation of its upstream enzyme IRE1a,which degrades specific cytosolic RNA targets (16, 17). Although theycannotmakeXBP1s protein,Xbp1-deficient cells transcribemRNA thatcontains the IRE1a cleavage sites. Consistent with previous reports, weobserved IRE1a activation in Xbp1-deficient cells, indicated by Xbp1mRNA splicing (fig. S7A). The magnitude of IRE1a activation inXbp1-deficient cells was not as robust as the response to canonicalUPR stimulation with thapsigargin, nor were classical RIDD substratesdiminished (fig. S7B), consistent with other studies (39).
Fink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
To determine whether IRE1a was involved in resistance to apopto-sis, we knocked down Ire1awith siRNA (fig. S7C). As a functional con-trol, Ire1a-specific siRNA efficiently prevented Xbp1 mRNA splicing(fig. S7D). IRE1a knockdown in Xbp1−/− MEFs reversed resistance toVSV-induced cell death (Fig. 5A and fig. S7E). Expression of human
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Fig. 4. Xbp1-deficient cells are resistant to the intrinsic pathway of apopto-sis. (A and B) WT and Xbp1−/− MEFs were treated with staurosporine (sts), glio-toxin (glio), TNF and low-dose cycloheximide (TNF), Fas antibody and low-dosecycloheximide (Fas), and tunicamycin (TM) to induce the UPR, or high-dose cyclo-heximide (CHX). Twenty-four hours later, viability was assessed by measuring MTSreduction (A). Seven hours after treatment, caspase-3 activity was assessed bymeasuring fluorometric substrate cleavage and is shown relative to WT cells(B). Data are means ± SD of three replicates and are representative of threeexperiments. (C) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1D) orCre− littermate (WT) mice in the presence of tamoxifen and treated with the in-dicated inducers of cell death as described in (A). Twenty-four hours later, viabilitywas assessed by measuring MTS reduction. Data are means ± SD of three repli-cates and are representative of three experiments. *P < 0.05; **P < 0.001 com-pared to WT, unpaired t test.
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IRE1a, which was resistant to mouse Ire1a-specific siRNA, preventedthis reversal (fig. S7F). IRE1a knockdown alone was minimally cyto-toxic but restored caspase-3 activation in Xbp1−/− MEFs in responseto staurosporine (Fig. 5B) and sensitized these cells to staurosporine-induced cell death (Fig. 5C). To determine whether the RNase activityof IRE1a mediated resistance to apoptosis, we treated Xbp1-deficientcells with the selective IRE1a nuclease inhibitor 4m8C (8-formyl-7-hydroxy-4-methylcoumarin) (40). We found that 4m8C reversed the
Fink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
resistance of Xbp1-deficient cells toapoptosis both during infectionwith VSV and during treatmentwith staurosporine (Fig. 5D). As anegative control, the structurally sim-ilar compound AMC (7-amino-4-methylcoumarin) had no effect atan equimolar concentration (fig.S2D). These findings indicate thatthe RNase activity of IRE1a contributesto resistance to the intrinsic pathwayof apoptosis observed in the settingof Xbp1 deficiency. We consistentlyobserved Xbp1 mRNA splicing to asmall extent in WT cells (fig. S7, Aand C), suggesting that IRE1a hassome basal activity (41), which couldregulate apoptotic responses in WTcells. Consistent with this, IRE1aknockdown in WT MEFs was nottoxic alone (Fig. 5, B, C, and E) butsensitized cells to VSV-induced celldeath and limited viral replication(Fig. 5, E and F).
IRE1a targets the proapoptoticmiRNA miR-125aStudies of coding genes targeted byIRE1a for RIDD have not revealedobvious candidates to explain our ob-served IRE1a-mediated resistance toapoptosis (9, 42). Thus, we focusedon miRNAs, which are also targetedby RIDD (43). To this end, we per-formed miRNA profiling of Xbp1-deficient cells and found fourmiRNAsthat were decreased in abundance inXbp1-deficient cells with active IRE1a(Fig. 6A). The miRNA miR-125awas represented twice among thesedifferentially expressed miRNAs.Quantitative polymerase chain re-action (PCR) analysis confirmed thedecrease in miR-125a abundance inXbp1-deficient cells, which was re-stored by reconstitution of the cellswith plasmid-encoded Xbp1 (fig.S8A). The miRNA miR-125a sensi-tizes cells to apoptosis and is thoughtto negatively regulate antiapoptoticBcl-2 family members, including B
cell lymphoma–extra large (Bcl-xL) and myeloid cell leukemia1 (Mcl-1) (44–46). In accordance with decreased miR-125a, we foundan increase in the abundances of antiapoptotic Bcl-xL andMcl-1 pro-teins in Xbp1-deficient cells (Fig. 6B). To test whether these effectswere dependent on IRE1a, we crossed Xbp1flox/flox × ESR Cre+ miceto Ern1flox/flox mice. BMDMs obtained from Xbp1flox/flox × Ern1flox/flox ×ESR Cre+ mice were treated with tamoxifen to generate XBP1D IRE1aDcells. We found that the protein amounts of both Bcl-xL and Mcl-1 in
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Fig. 5. The resistance of Xbp1-deficient cells to apoptosis results from the activation of IRE1a. (A to F) Analysis ofthe effects of IRE1a knockdown on cell death. (A, E, and F) WT and Xbp1−/− MEFs were transfected with siRNA targetingIre1a or control siRNA (ctrl siRNA). Cells were then left uninfected (mock) or infected with VSV-GFP for 24 hours. Celldeath was then assessed with a membrane-impermeant, amine-reactive fluorescent dye, and the relative abundance ofGFP was measured by flow cytometry. Data are from one experiment and are representative of three (A) or two (E and F)independent experiments. (B and C) The indicated siRNA-transfected MEFs were left untreated (mock) or treated withstaurosporine. Seven hours later, caspase-3 activity was assessed by measuring fluorometric substrate cleavage and isshown relative to that in untreated WT cells (B). Twenty-four hours after treatment, viability was assessed by measuringMTS reduction (C). (D) BMDMs were cultured from Xbp1flox/flox ESR Cre+ (Xbp1D) or Cre− littermate (WT) mice in thepresence of tamoxifen and the IRE1a inhibitor 4m8C. Cells were then infected with VSV-GFP at an MOI of 2 or weretreated with staurosporine. Viability was assessed 24 hours later by measuring MTS reduction. Data are means ± SDof three replicates and are representative of three experiments (B, C, and D). *P < 0.01; **P < 0.001, unpaired t test.
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XBP1D IRE1aD BMDMs were reduced compared to those in XBP1Dcells, albeit not to the amount observed in the WT cells (Fig. 6B).These results indicated that antiapoptotic Bcl-xL and Mcl-1 proteinswere increased in abundance inXbp1-deficient cells, in amanner largelydependent on IRE1a. Consistent with our observation of sensitizationto apoptosis by IRE1a knockdown in WT cells (Fig. 5E), IRE1aD cellshad increased miR-125a abundance (fig. S8B). Inhibition of the RNaseactivity of IRE1a with the selective IRE1a nuclease inhibitor 4m8C wassufficient to increase the abundance of miR-125a (fig. S8B).
Finally, we wished to examine the extent to which miR-125a degra-dation by IRE1a was responsible for the prosurvival phenotype of theXbp1-deficient cells. Reconstitution of miR-125a with an miRNA mi-metic was sufficient to restore caspase-3 activation in Xbp1−/−MEFsin response to staurosporine (Fig. 6C) and to sensitizeXbp1-deficientcells to the intrinsic pathway of apoptosis (Fig. 6D and fig. S8C). Re-
) 6 June 2017
constitution with miR-125a also reversed the resistance of Xbp1−/−
MEFs to VSV-induced cell death (Fig. 6E). Finally, neutralizing miR-125a in WT cells with an miRNA hairpin inhibitor resulted in anantiapoptotic state resembling that observed in Xbp1-deficient cells(fig. S8D). Together, these findings suggest that the IRE1a-dependentdecrease in miR-125a abundance contributes to the resistance toapoptosis.
IRE1a activation during HCV infection mediates resistanceto apoptosisSome viruses encode genes that promote IRE1a activation. HCVNS4Bactivates IRE1a (18), and IRE1a activation is also seen inHCV-infectedcells (47). Curiously, in cells expressing NS4B, IRE1a splices Xbp1mRNA, but XBP1 targets are not transcribed (18), suggesting that thevirus uses IRE1a for another reason. Furthermore, HCV is suggested to
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Fig. 6. IRE1a mediates reduction in proapoptotic miR-125a. (A) BMDMs werecultured from Xbp1flox/flox ESR Cre+ (Xbp1Dor Cre− littermate (WT) mice in the presenceof tamoxifen. Volcano plot demonstratingthe distribution of miRNAs between WTand Xbp1D BMDMs measured using theNanoString nCounter assay. Data are fromoneexperimentwithquadruplicate samples(B) BMDMs were cultured from Xbp1flox/flo
ESR Cre+ (Xbp1D), Xbp1flox/flox Ern1 flox/flox ESRCre+ (Xbp1D Ire1aD), or Cre− littermate (WTmice in the presence of tamoxifen. The relative abundances of Bcl-xL, Mcl-1, and b-actinin the cell lysates were determined by Western blotting and densitometry. The ratio oBcl-xL orMcl-1 tob-actin is shown, normalizedto that in WT cells. Data are means ± SD othree independent experiments. a.u., arbitraryunits. (C and D) WT and Xbp1−/− MEFs weretransfected with negative control miRNA mimetic (miR-ctrl) or a miR-125a mimetic. Cellwere left untreated (mock) or treated withstaurosporine. Sevenhours later, caspase-3activity was assessed bymeasuring fluorometricsubstrate cleavage and is shown relative tothat in untreated WT cells (C). Twenty-fouhours after treatment, viability was assessedby measuring MTS reduction (D). Data aremeans ± SD of three replicates and are representative of two experiments. (E) The indicated miRNA-transfected MEFs were infectedwithVSV-GFP for 24hours. Cell deathwas thenassessed with a membrane-impermeantamine-reactive fluorescent dye, which wameasured by flow cytometry. The extent of infection was determined by measuring the relative abundance of GFP by flow cytometryData are from one experiment and are representative of two independent experiments*P < 0.01; **P < 0.001, unpaired t test.
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cause resistance to the intrinsic path-way of apoptosis (48, 49), althoughthe mechanism of this effect remainsunknown.
Consistent with previous reports(18), we detected IRE1a activationas indicated by spliced XBP1 mRNAin cells transiently transfected with aplasmid encoding NS4B (Fig. 7A).The abundance of miR-125a was de-creased (fig. S8E), and NS4B expres-sion induced the IRE1a-dependentresistance to staurosporine (Fig. 7, Band C). We infected Huh-7.5 humanhepatoma cells with trans-packagedHCV replicons encoding Gaussialuciferase (Gluc), which enabled usto quantitate HCV replication overtime (50). IRE1a inhibition alonewas not cytotoxic, but it sensitizedHCV-infected cells to death (Fig. 7D).Furthermore, inhibition of IRE1adecreased the secretion of virally en-coded luciferase, a marker of viralreplication (Fig. 7E). These findingssuggest that IRE1a activation duringHCV infectionmaypromote viral rep-lication by inhibiting the death of theinfected cells. To determine whetherthese findings extended to humanHCV infection, we quantified splicedXBP1mRNA in the human liver tissueof patients infected with HCV. WedetectedHCV-associated IRE1a acti-vation, as indicated by an increase inspliced XBP1 mRNA abundance inliver tissue from HCV-infected pa-tients compared to that in the livertissue of HCV-negative controls (Fig.7F). In addition to IRE1a activation,HCV-infected patients also exhibitedstatistically significantly reducedmiR-125a abundance (Fig. 7G), sug-gesting that IRE1a activation duringhuman HCV infection may conferresistance to apoptosis.
DISCUSSIONHere, we examined the IRE1a-XBP1branch of the UPR in innate antiviraldefense.We uncovered an unexpectedrole for Xbp1 and IRE1a in modulat-ing susceptibility to the intrinsic
pathway of apoptosis, which is induced during VSV andHSV infectionand plays a critical role in limiting viral replication. Xbp1−/−MEFs wereprotected from virally induced cell death and, as a consequence, sus-tained more viral replication despite an increased IFN response com-pared to that of WT cells. In Xbp1−/− cells with active IRE1a, theFink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
abundance of the proapoptotic miRNA miR-125a was decreased,conferring resistance to the intrinsic apoptotic pathway. IRE1a activa-tion by HCV NS4B in WT cells also conferred resistance to apoptosisand promoted viral replication. Therefore, Xbp1-deficient cells withactive IRE1a gained apoptosis resistance, which suggests that IRE1a
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Fig. 7. IRE1a-mediated apoptosis resistance induced by HCV NS4B. (A) HeLa cells were transfected with constructsexpressing GFP alone (vector) or together with HCV NS4B. RNA was isolated 72 hours later. XBP1 mRNA maturation fromthe unspliced (u) to spliced (s) form was analyzed by reverse transcription PCR (RT-PCR). Data are from one experimentand are representative of two independent experiments. (B and C) Transfected cells were treated with IRE1a inhibitor 2for 24 hours, and staurosporine was then added. Twenty hours later, cell death was assessed with a membrane-impermeant,amine-reactive fluorescent dye, which was measured by flow cytometry. Data are from one experiment and are representativeof three independent experiments (B). The percentage of dead cells among transfected GFP-positive cells was calculated (C).Data are means ± SD of three independent experiments. (D and E) Huh-7.5 cells were infected with trans-packaged HCVencoding luciferase. Forty-eight hours later, viability was determined by measuring cellular adenosine triphosphate (D).Secretion of virally encoded luciferase was measured 24 and 48 hours after infection (E). Data are means ± SD of threereplicates and are representative of two experiments. (F and G) RNA was isolated from the liver tissue of HCV-infectedpatients (n = 11) and HCV-negative controls (n = 6). Expression of spliced XBP1 and miR-125a (relative to an internalcontrol) was determined by quantitative RT-PCR. Data are means ± SEM. *P < 0.05; **P < 0.01, unpaired t test. ***P < 0.01,Mann-Whitney test.
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knockdown or inhibition is a model for the loss of apoptosis resistance.Finally, we observed IRE1a activation and substantially less miR-125a abundance in liver biopsies from HCV-infected patients, sug-gesting the in vivo relevance of the survival strategy used by HCV.These results highlight a previously unappreciated role of the IRE1a-XBP1 axis in the regulation of apoptosis and its consequences in viralsusceptibility.
Previous studies showed that after the engagement of TLRs andRLRs, XBP1 plays an important role in enhancing cytokine and IFNproduction in macrophages and dendritic cells (11–13). Given theobservation that transfection with poly(I:C) induced less IFN produc-tion in Xbp1−/− MEFs compared to that in WT MEFs, our results areconsistent with these previous findings that Xbp1 promotes RLRsignaling for IFN production in MEFs. However, despite this impair-ment in IFN production downstream of RLRs, infected Xbp1-deficientMEFs produced large amounts of IFN.We speculate that the enhancedIFN response observed in Xbp1−/− MEFs may result from both pro-longed cellular survival and an accumulation of viral PAMPs. Apoptoticcaspases can cleave and inactivate signaling proteins important for theIFN response, suggesting that the apoptotic process directly antagonizesthe IFN response (51–54). In addition, IRE1a can cleave host RNA forRLR stimulation (15). Therefore, our results highlight a distinct conse-quence of IRE1a activation, wherebyXbp1 deficiency results in a robustprosurvival response, leading to prolonged RLR stimulation that miti-gates impairment inRLR signaling to generate enhanced IFN responses.The prosurvival signals induced through IRE1a activation are so dom-inant that they overcome ISG-mediated antiviral functions and enablevirus replication.
In addition to protection from virus-induced death, we found thatXbp1 deficiency conferred resistance to the intrinsic pathway of apopto-sis stimulated by various chemical inducers. IRE1a activation inXbp1-deficient cells contributed to this apoptosis resistance becausesiRNA-mediated knockdown of Ire1a expression or inhibition ofIRE1a nuclease function rendered the Xbp1-deficient cells susceptibleto apoptosis. RIDD targets both coding and noncoding RNAs, includ-ing miRNAs (42). We found that IRE1a reduced the amount of miR-125a, leading to the enhanced expression of its target genes. The targetsinclude genes encoding prosurvivalmembers of the Bcl-2 family (44–46).miRNA controls target genes at the transcriptional and transla-tional levels (55). We found that the prosurvival proteins Bcl-xLand Mcl-1 were present in increased amounts. The biological targetsand functions of the other miRNAs identified in this study, namelymiR-1224 andmiR-804, have not yet beenwell described. The relevanceof these other miRNAs in IRE1a-dependent phenotypes will be inves-tigated in future studies.
Our study revealed an unexpected role for IRE1a in controlling ap-optosis. Many studies of the UPR have been performed with high con-centrationsof pharmacological inducers ofER stress, such as tunicamycinand thapsigargin, which inevitably lead to cell death. In these experimen-tal settings of irremediable ER stress, variousmechanisms have been pro-posed for inducing apoptosis (56). The net effect of IRE1a activity inpromoting cell death compared to cell survival has been controversial,with some studies suggesting that IRE1a promotes cell survival (57–59),and others suggesting that IRE1a promotes cell death (60–62). Specif-ically, IRE1a was proposed to induce apoptosis by degrading miRNAsthat lead to increased caspase-2 abundance (60), although there areconflicting data challenging this observation (63). IRE1a has beenlinked to apoptosis in cells irreversibly damaged by the activation ofthe JNK (c-Jun N-terminal kinase)–ASK1 (apoptosis signal–regulating
Fink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
kinase 1) pathway (64, 65). IRE1a appears to have opposing roles inregulating apoptosis: prosurvival in the absence of irreversible UPRbut proapoptotic under irreversible UPR. This double-edged natureof IRE1amay be important to consider for the use of IRE1a inhibitorsin the treatment of various human diseases (66).
We speculate that IRE1amay represent an ancient form of cell pro-tection that has been subverted by some viruses for their own replicativeadvantage. HCV and other members of the Flaviviridae family induceER stress and activate IRE1a. Our results suggest that HCV NS4Binduces IRE1a-dependent protection from apoptosis, which may favorthe development of chronic infection and hepatocellular carcinoma. Inthe setting of avian coronavirus infection, IRE1a also promotes cell sur-vival in association with JNK and Akt regulation (67). Influenza virusinduces only the IRE1a arm of the UPR (not ATF6 or PERK), andinhibitors of IRE1a reduce viral replication (68). Therefore, our findingspredict that the IRE1a-RIDD pathway could be exploited as a noveltarget of intervention against viral infections.
MATERIALS AND METHODSCells and virusesXbp1+/+ andXbp1−/−MEFswere gifts fromL.H.Glimcher (Weill CornellMedical College, New York, NY). H1-HeLa cells stably overexpressingthe antiapoptotic protein BCL2 have been previously described (69).MEFs and H1-HeLa cells were propagated in high-glucose Dulbecco’smodified Eagle’s medium (DMEM) (Gibco) and supplemented with10% heat-inactivated fetal bovine serum (FBS), 1%Hepes, and penicillin(100 U/ml) and streptomycin (100 mg/ml) (Gibco). Huh-7.5 cells werepropagated in high-glucose DMEM with 10% heat-inactivated FBS and1mMnonessential amino acids (Invitrogen).Xbp1flox/flox (20) (a gift fromL. H. Glimcher,Weill Cornell Medical College), Ern1flox/flox (70) [RIKENBioResource Center (BRC), Japan], and CAGGCre-ERTM (the JacksonLaboratory) mice were bred in the Yale animal facility. All proceduresperformed in this study complied with the federal guidelines and in-stitutional policies set by Yale Animal Care and Use Committee.BMDMs were prepared from 6- to 12-week-old male and female miceaccording to a previously describedmethod (71) and cultured in 0.2 mM4-hydroxytamoxifen (Sigma) during days 2 to 4 of differentiation to in-duce Cre-mediated recombination. The following genotype combina-tions of BMDMs were treated with tamoxifen to generate the cellsdescribed in Fig. 6: Xbp1flox/flox × ESR Cre+ (XBP1D), Xbp1flox/flox ×Ern1flox/flox × ESR Cre+ (XBP1D IRE1aD), or Xbp1flox/flox × ESR Cre−
(WT). VSV-G–GFPwas a gift from J. Rose (YaleUniversity, NewHaven,CT) and A. Geballe (Fred Hutchinson Cancer Research Center, Seattle,WA) and was maintained and titered in baby hamster kidney cells.HSV-1–GFP (72) was a gift fromP.Desai and S. Person (JohnsHopkinsUniversity, Baltimore, MD) and was maintained and titered in Verocells. VSV-G pseudotyped lentivirus was made by harvesting culturesupernatants of human embryonic kidney 293T cells transfected withplasmids encoding VSV-G, GFP, and Gag-Pol. Trans-packaged HCVreplicons were prepared by transfecting the JFH/Gluc replicon intoHuh-7.5[core-NS2] cells as previously described (50)
Infection and stimulation of cellsCells were incubated for 1 hour with VSV or HSV-1 in serum-free me-dium or pseudotyped virus in phosphate-buffered saline 0.1% bovineserum albumin, and then the inoculum was removed, and incubationcontinued in complete medium. Cells were incubated for 4 hourswith trans-packaged HCV, and then the inoculum was removed, and
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incubation continued in complete medium containing 60 mM IRE1Inhibitor II (Calbiochem). zVAD (Invivogen) was added at 20 to 100 mMafter removal of the viral inoculum. Poly(I:C) (1 mg/ml) was deliveredcomplexed to Lipofectamine 2000 (Invitrogen). Cells were treated with0.1 to 1 mM staurosporine (Enzo Life Sciences), 1 mM gliotoxin (Sig-ma), 10 mMABT-737 (Santa Cruz Biotechnology), TNF (50 ng/ml) +cycloheximide (0.1 mg/ml) (Abcam), antimouse CD95 (2 mg/ml) (BDPharmingen), Fas-activating antibody + cycloheximide (0.1 mg/ml),tunicamycin (10 to 100mg/ml) (Sigma), 1mMthapsigargin (Calbiochem),or cycloheximide (100 mg/ml) (high-dose CHX). Cells were treated withthe IRE1 inhibitor 4m8C (Calbiochem) or the structurally similar com-pound AMC (Sigma) at 25 mM for 3 days before infection or apoptosisinduction or 40 mM IRE1 Inhibitor II (Calbiochem) for 24 hours beforeapoptosis induction.
PlasmidspFLAG.XBP1u.CMV2 (Addgene plasmid #21832, from D. Ron) (73),the empty vector control c-Flag pcDNA3 (Addgeneplasmid#20011, fromS. Smale) (74), hIRE1a.pcD(Addgeneplasmid#21892, fromR.Kaufman),and hIRE1a wt (Addgene plasmid #20744, from F. Urano) (75) wereused. MEFs were transfected using TransIT-2020 (Mirus Bio). HCVNS4Bwas expressedwith an authenticN-terminal Ala residue by fusinga human ubiquitin gene to a codon-optimizedNS4B gene (76): Ubiquitinwas amplified by using primers YO-0905 (TTA ATT AAC GAG GATCCC GCC ACC ATG CAG ATC TTC GTG AAG AC) and YO-0928(TCG ATC AGG GCT GCT CTG CTG GCT CCA CCG CGG AGACGC AGC ACC); codon-optimized NS4B was amplified by usingprimers YO-0927 (GGT GCT GCG TCT CCG CGG TGG AGC CAGCAG AGC AGC CCT GAT CGA) and YO-0931 (GTT TAA ACTTAA CAA GGG ATG GGG CAG TCC T). Ubi-NS4B was thenassembled in secondary PCRs with primers YO-0905 and YO-0931,cloned into pCR2.1 (Invitrogen) for sequencing, and then subclonedinto pIRES2-EGFP (Clontech) by using the common Sac I and Pst Irestriction sites. H1-HeLa cells were transfected using TransIT-HeLa(Mirus Bio).
Expression analysisRNA isolated using the RNeasy kit (Qiagen) was used to synthesizecomplementary DNA (cDNA) using the iScript cDNA Synthesis Kit(Bio-Rad), and quantitative PCR was performed on a StratageneMx3000P or Bio-Rad CFX Connect using SYBR Green (Bio-Rad) withthe following primers (all primers listed in the 5′ to 3′ orientation):Ifna4, CTG CTA CTT GGA ATG CAA CTC (forward) and CAGTCT TGC CAG CAA GTT GG (reverse); Ifnb1, GCA CTG GGTGGA ATG AGA CTA TTG (forward) and TTC TGA GGC ATCAAC TGA CAG GTC (reverse); Mx1, AGT CCT TTC CAC AGGCAG AA (forward) and CAT TGA GAG AAA CTC ACC TAA GAAC (reverse); Xbp1s, GAG TCC GCA GCA GGT (forward) and GTGTCA GAG TCC ATG GGA (reverse); Hspa5 (Bip), TCA TCG GACGCA CTT GGA (forward) and CAA CCA CCT TGA ATG GCAAGA (reverse); Chop, GTC CCT AGC TTGGCT GACAGA (forward)and TGGAGAGCGAGGGCTTTG (reverse); Blos1, CAAGGAGCTGCA GGA GAA GA (forward) and GCC TGG TTG AAG TTC TCCAC(reverse);Pdgfrb, AACCCCCTTACAGCTGTCCT (forward) andTAATCCCGTCAGCATCTTCC (reverse); andhuman splicedXBP1,TGC TGA GTC CGC AGC AGG TG (forward) and GCT GGC AGGCTC TGG GGA AG (reverse). For analysis of Xbp1-splicing, primersflanking the spliced sequences in Xbp1 mRNA [ACA CGC TTG GGAATG GAC AC (forward) and CCA TGG GAA GAT GTT CTG GG
Fink et al., Sci. Signal. 10, eaai7814 (2017) 6 June 2017
(reverse)] were used for PCR amplification, and products were separatedby electrophoresis through a 3% agarose gel and visualized by ethidiumbromide staining.
miRNA expression analysisRNA isolated using the miRNeasy Mini Kit (Qiagen) was subjected tomiRNA profiling using the nCounter mouse miRNA expression assayversion 1.5 (NanoString) according to the manufacturer’s protocol.Data were analyzed by using the nSolver software with normalizationto the geometric mean of the top 100 miRNAs as recommended by themanufacturer. Quantitative RT-PCR with the miRCURYUniversal RTmicroRNA qPCR system (Exiqon) was used to measure miR-125a-5p.Expression was calculated relative to the manufacturer’s suggested en-dogenous control (miR-103a-3p), with equivalent results also obtainedrelative to miR-16-5p.
Assessment of cell deathCells were stained with the LIVE/DEAD Fixable Far Red Dead CellStain Kit (Molecular Probes) and analyzed by flow cytometry on aBD FACSCalibur or BD LSRFortessa. Viability was also assessed bymeasuring MTS reduction using the CellTiter 96 AQueous One Solu-tion Cell Proliferation Assay (Promega). Cells were stained with theCaspase-3, Active Form, Apoptosis Kit (BD Pharmingen) and analyzedby flow cytometry on a BD FACSCalibur. Caspase-3/7 activity wasmeasured using the SensoLyteHomogeneousRh110Caspase-3/7AssayKit (AnaSpec).
Western blottingCell pellets were lysed in SDS sample buffer (Cell Signaling Technol-ogy). Proteinswere separated by SDS–polyacrylamide gel electrophoresis,transferred to polyvinylidene difluoride membranes, and incubated withantibodies against Mcl-1 (BioLegend, 613601), Bcl-xL (CellSignaling Technology, 54H6), or b-actin (Cell Signaling Technology,13E5).
Intracellular stainingCells were fixed in Cytofix/Cytoperm solution (BD) on ice, washedwithPerm/Wash buffer (BD), and stained withDyLight 550–conjugated an-tibody against Beclin 1 (Novus Biologicals, NB110-87318R) or an equalconcentration of isotype control antibody. Washed cells were analyzedby flow cytometry on a BD LSRFortessa flow cytometer.
siRNA, miR mimetics, and inhibitorsGene-specific siGENOME siRNA or siGENOME Non-TargetingsiRNA #4 (which targets firefly luciferasemRNA and has at least fourmismatches to all mouse genes) obtained fromDharmacon (ThermoFisher Scientific) was delivered complexed to Lipofectamine RNAiMAX(Invitrogen). After incubation for 48 hours, cells were replated at equaldensity before infection or apoptosis induction. miR mimetics, miRI-DIANmicroRNAhairpin inhibitor negative control #1, andmiRIDIANmiR-125a hairpin inhibitor were obtained from Dharmacon (ThermoFisher Scientific) and delivered complexed to Lipofectamine RNAiMAX(Invitrogen).
Luciferase assayConditioned cell culture medium was collected at 24 or 48 hours afterinfection, clarified by centrifugation, mixed with 1/4 volume 5× lysisbuffer (New England Biolabs), and assayed for luciferase activity(New England Biolabs).
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Liver specimensLiver samples from percutaneous biopsies of liver transplant recipientschronically infected with HCV or from HCV-negative control livertransplant recipients were obtained with the approval of the Universityof Washington Institutional Review Board. Tissue was archived inRNAlater and stored at −80°C. RNA was isolated using the miRNeasyMini Kit (Qiagen).
Statistical analysesSample size was chosen according to previous experience in similarexperiments; all samples were included in analysis. The unpairedStudent’s t test or the Mann-Whitney test was used for comparisonsbetween two groups. P values of less than 0.05 were considered statisti-cally significant.
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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/482/eaai7814/DC1Fig. S1. Increased IFN and ISG expression during VSV infection of Xbp1-deficient MEFs.Fig. S2. Knockdown of Xbp1 with siRNA mimics Xbp1 deficiency, and reconstitution with Xbp1reverses resistance to cell death.Fig. S3. Xbp1 deficiency does not increase susceptibility to a noncytotoxic virus and mimicsBCL2 overexpression.Fig. S4. Resistance to virally induced apoptosis in Xpb1-deficient cells is independent of Beclin 1.Fig. S5. Death of VSV- and HSV-infected cells does not require Chop.Fig. S6. VSV infection does not activate the UPR.Fig. S7. The resistance of Xbp1-deficient cells to apoptosis results from the activation of IRE1a.Fig. S8. The miRNA miR-125a regulates resistance to apoptosis.
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Acknowledgments: We thank H. Dong and K. Hayashi for technical support; B. Cookson andN. Kaminski for sharing laboratory facilities and equipment; and L. Glimcher, J. Rose, A. Geballe,P. Desai, S. Person, D. Ron, S. Smale, R. Kaufman, and F. Urano for providing us withvarious reagents. Funding: This work was supported by funding from the Howard HughesMedical Institute and NIH R01 AI054359 and AI064705 (to A.I.) and K08 AI119142 (to S.L.F.).S.L.F. was supported by NIH award T32 HL007974. B.D.L. was funded by R01 AI087925.Author contributions: S.L.F., T.R.J., R.D.M., T.I., C.S.L., B.D.L., and A.I. designed the experiments.S.L.F., T.R.J., R.D.M., and B.D.L. performed the experiments. S.L.F., T.R.J., R.D.M., and A.I.analyzed the data. S.L.F and A.I. wrote the manuscript. Competing interests: The authorsdeclare that they have no competing interests. Data and materials availability: The Xbp1+/+
and Xbp1−/− MEFs and Xbp1flox/flox mice require a materials transfer agreement (MTA) fromHarvard University. The Ern1flox/flox mice require an MTA from RIKEN BRC. The followingplasmids require an MTA from Addgene: pFLAG.XBP1u.CMV2, c-Flag pcDNA3, hIRE1a.pcD, andhIRE1a wt. The JFH-1 HCV replicon requires MTAs from Rockefeller University and ApathLLC (in the United States) or from the Tokyo Metropolitan Organization for Medical Researchand Toray Industries (elsewhere). The replicon was packaged by using the HCV strain J6structural genes, which were obtained under an MTA with the NIH. The Huh-7.5 cell linerequires an MTA from Washington University and Apath LLC.
Submitted 11 August 2016Accepted 19 May 2017Published 6 June 201710.1126/scisignal.aai7814
Citation: S. L. Fink, T. R. Jayewickreme, R. D. Molony, T. Iwawaki, C. S. Landis, B. D. Lindenbach,A. Iwasaki, IRE1a promotes viral infection by conferring resistance to apoptosis. Sci. Signal. 10,eaai7814 (2017).
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promotes viral infection by conferring resistance to apoptosisαIRE1
IwasakiSusan L. Fink, Teshika R. Jayewickreme, Ryan D. Molony, Takao Iwawaki, Charles S. Landis, Brett D. Lindenbach and Akiko
DOI: 10.1126/scisignal.aai7814 (482), eaai7814.10Sci. Signal.
provide a potential therapeutic target. functions to enhance cell survival in response to viral infection and mayαTogether, these data suggest that IRE1
activation and decreased miR-125a abundance.αbiopsies from HCV-infected patients also showed increased IRE1cleaved the proapoptotic microRNA miR-125a, leading to decreased apoptosis and increased viral replication. Liver
, whichαcompromised in type I IFN production or responses. Instead, these cells showed increased activation of IRE1-deficient cells had defective antiviral responses to infection by hepatitis C virus (HCV); however, the cells were notXbp1
. found that et altranscription factor XBP1, which drives the expression of genes encoding type I interferons (IFNs). Fink generates theα mRNA by the nuclease IRE1Xbp1as that occurring during viral infection. During the UPR, processing of
The unfolded protein response (UPR) alleviates the cellular stress caused by the accumulation of proteins, suchResisting death by virus
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