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Inhibition of enterovirus 71 entry by transcription factor XBP1
Biochemical and Biophysical Research Communications 420 (2012) 882–887
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibition of enterovirus 71 entry by transcription factor XBP1 Jia-Rong Jheng 1, Chiou-Yan Lin 1, Jim-Tong Horng ⇑,1, Kean Seng Lau Department of Biochemistry and Research Center for Emerging Viral Infections, Chang Gung University, 259 Wen-Hwa First Road, Kweishan, Taoyuan 333, Taiwan
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Article history: Received 6 March 2012 Available online 24 March 2012 Keywords: Enterovirus 71 Endoplasmic reticulum stress Inositol-requiring enzyme 1 Unfolded protein response Translational attenuation X-box-binding protein 1
a b s t r a c t Inositol-requiring enzyme 1 (IRE1) plays an important role in the endoplasmic reticulum (ER), or unfolded protein, stress response by activating its downstream transcription factor X-box-binding protein 1 (XBP1). We demonstrated previously that enterovirus 71 (EV71) upregulated XBP1 mRNA levels but did not activate spliced XBP1 (XBP1s) mRNA or its downstream target genes, EDEM and chaperones. In this study, we investigated further this regulatory mechanism and found that IRE1 was phosphorylated and activated after EV71 infection, whereas its downstream XBP1s protein level decreased. We also found that XBP1s was not cleaved directly by 2Apro, but that cleavage of eukaryotic translation initiation factor 4G by the EV71 2Apro protein may contribute to the decrease in XBP1s expression. Knockdown of XBP1 increased viral protein expression, and the synthesis of EV71 viral protein and the production of EV71 viral particles were inhibited in XBP1-overexpressing RD cells. When incubated with replication-deficient and UV-irradiated EV71, XBP1-overexpressing RD cells exhibited reduced viral RNA levels, suggesting that the inhibition of XBP1s by viral infection may underlie viral entry, which is required for viral replication. Our findings are the first indication of the ability of XBP1 to inhibit viral entry, possibly via its transcriptional activity in regulating molecules in the endocytic machinery. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Three major transmembrane ER stress sensor proteins have been identified and characterized comprehensively: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and IRE1. These proteins manage three arms of the unfolded protein response (UPR) signaling pathway and activate different subsets of genes to manage stress. Activation of PERK initiates the phosphorylation of eukaryotic initiation factor 2a (eIF2a), which causes the inhibition of global translation [1]. ATF6 belongs to a highly conserved family of regulated intramembrane proteolysis transcription factors, which are cleaved to release cytosolic domains that transit to the nucleus to regulate the transcription of a collection of ER stress target genes, including BiP, by binding to a consensus ER stress response element (ERSE) sequence in the promoter region of these proteins [2,3]. IRE1 undergoes oligomerization to activate its kinase and endoribonuclease (RNase) activities to transmit the UPR signals [4]. The RNase of IRE1 splices mRNA encoding the transcription factor XBP1, leading to a frameshift splicing and subsequent translation of the active spliced XBP1 protein (XBP1s) [5]. XBP1 was originally identified as a basic leucine zipper transcription factor in B cells and is required for plasma cell differentiation ⇑ Corresponding author. Fax: +886 3 211 8407. 1
E-mail address: [email protected] (J.-T. Horng). These authors contributed equally to this project.
0006-291X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.bbrc.2012.03.094
[6]. By binding the ERSE promoter region, the spliced transcription factor XBP1s can sequentially stimulate chaperones to promote the folding capacity in the ER [7,8]. The clearing of incorrectly folded proteins retained in the ER is now known to occur through their retrotranslocation into the cytosol in a process involving ER-associated degradation (ERAD), which is also induced by XBP1 [4]. EV71 is a positive-stranded RNA virus that belongs to the group of human enteroviruses whose members include poliovirus and coxsackievirus. EV71 has a genome of about 7.5 kb comprising an open reading frame flanked by a 50 untranslated region (UTR) and a 30 poly(A) tail. The 50 UTR of the viral genome is an internal ribosome entry site (IRES), which directs translation of the viral RNA in a cap-independent fashion to synthesize a polyprotein that can be cleaved by viral proteases 2Apro and 3Cpro to produce mature and functional structural and nonstructural proteins [9]. 2Apro is also responsible for the cleavage of eukaryotic translation initial factor G (eIF4G), which in turn inhibits the cap-dependent translational inhibition of global cellular proteins [10]. Numerous studies suggest a relationship between the ER stress response and viral infection, and many viruses can regulate UPR to benefit their replication or to trigger an immune response [11]. We showed previously that EV71 infection activates and modifies the UPR signaling pathways to benefit viral replication [12]. XBP1 mRNA was upregulated, but no IRE1-mediated splicing was detected. In the present study, we examined further the role of IRE–XBP1 of UPR during EV71 replication.
J.-R. Jheng et al. / Biochemical and Biophysical Research Communications 420 (2012) 882–887
2. Materials and methods 2.1. Cell culture and viral infection Rhabdomyosarcoma (RD) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under humidified 5% CO2 in air. EV71 BrCr was obtained from the ATCC (accession no. VR 784). Viral propagation and titer determination by plaque assay were performed in RD cells, and the titer was recorded as the number of plaque-forming units per milliliter [13]. 2.2. Antibodies and reagents Rabbit polyclonal antibodies to 3A were raised against GST-3A [14]. Rabbit polyclonal antibodies against eIF4G and lamin A/C, and mouse monoclonal anti-GST were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal anti-phospho-IRE1 and anti-XBP1 were from Abcam (Ab48187, Cambridge, UK) and Anaspec (#54578, San Jose, CA, USA), respectively. Antibody to GAPDH was from Abnova (Taoyuan, Taiwan). Anti-His tag was from Serotec (#MCA1396B, Oxford, UK). Plasmids encoding XBP1s, 2Apro, 3Cpro, and 2Apro(C110A) were cloned into pcDNA3. 1-mycHis() (Invitrogen, Carlsbad, CA). GST-2Apro and GST-2Apro (C110A) were cloned into pGEX-2T and pGEX-5X-1 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), respectively. EV71 50 UTR and 5 ERSE were cloned into pcDNA3.1-mycHis-B(+)-Fluc, in which Fluc was PCR amplified from pGL3 and inserted into pcDNA3. 1/myc-His-B(+) to generate pCMV-50 UTR-Fluc (4643-IRES-Fluc) and pCMV-5ERSE-Fluc, respectively. Stealth RNAi™ siRNA of XBP1 (siXBP1, #HSS111391) and scrambled negative controls Lo GC (#12935200) were from Invitrogen. 2.3. UV irradiation and viral RNA determination UV irradiation of EV71 was performed as described previously, and the remaining titer was measured by plaque assay [15]. RD cells transfected with XPB1 plasmid or its vector control were incubated with UV-treated virus (UV-EV71), the cells were collected after 0, 0.5, and 1 h, and the viral RNA was quantified using quantitative real-time PCR (qPCR). To quantify the changes in gene expression, the DCt method was used to calculate relative fold changes normalized against the GAPDH control. 2.4. Western blotting and nucleus/cytoplasm fractionation assays The cells were harvested at the times indicated, and lysates were prepared with lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, and 1 mM MgCl2) containing protease inhibitor cocktail (Roche). Equal amounts of cellular protein were analyzed by SDS–PAGE, the bands were transferred to a polyvinylidene fluoride membrane, and the proteins on the membrane were detected with enhanced chemiluminescence reagents (Millipore). RD cells were mock infected or infected with EV71 at a multiplicity of infection (MOI) of 10. The cells were harvested at the times indicated, and the nuclei and cytoplasm were separated as described [12]. 2.5. In vitro protease cleavage assay Wild-type and the catalytic-inactive mutant (C110A: cysteine mutated to alanine at amino acid 110) of 2Apro of EV71 were cloned into pGEX vectors, and these GST-fusion proteins were purified according to the manufacturer’s instruction (GE Healthcare BioSciences AB). The purified GST-fusion proteins were dialyzed in buffer (25 mM Tris–HCl, pH 7.5, 50 mM NaCl, 5 mM DTT, 0.5 mM
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EDTA, and 25% glycerol) before use. RD cells were treated with or without 2 lg of thapsigargin (Tg) for 4 h and were lysed with lysis buffer without proteinase inhibitors. The cell lysates were centrifuged at 17,900g for 10 min at 4 °C, and the supernatant was collected for the cleavage assay. Thirty micrograms of supernatant was incubated with 30 lg of GST-2A or GST-2Apro(C110A) in a total volume of 100 ll, at 37 °C for 4 h, and then analyzed by SDS–PAGE and Western blotting. 2.6. Reporter assays Each well containing 3 104 RD cells in a 48-well plate was transfected with Lipofectamine with 0.4 lg of firefly luciferase reporter gene and 0.04 lg of pTK-Rluc encoding Renilla luciferase as an internal control. The luciferase activities were measured using a dual-luciferase reporter kit (Promega, Madison, WI, USA). The results were normalized to the Renilla luciferase activity of the internal control. 2.7. Data analysis The data were analyzed using two-tailed Student’s t test and are expressed as the mean ± SEM. P < 0.05 was considered significant. 3. Results To understand better the regulatory mechanism of the EV71-induced IRE1-XBP1 signaling pathway, we examined whether the upstream IRE was activated by EV71. RD cells were infected with EV71, and the cells were harvested at the designated times postinfection (p.i.). EV71 phosphorylated IRE1 from 7 h p.i., and the phosphorylation level correlated with the viral translation, as indicated by the level of viral 3A protein, from 5 h p.i. (Fig. 1A). In response to ER stress, the RNase function of IRE1 is activated to initiate the nonconventional splicing of transcription factor XBP1 mRNA to produce XBP1s, which then increases the folding capacity for targeting misfolded proteins for degradation [5,16]. We demonstrated previously that EV71 increases the XBP1 mRNA level but that spliced XBP1 mRNA and downstream target genes, EDEM and chaperones, are not produced [12]. We next asked whether the regulation of XBP1 is responsible for the decrease in XBP1 expression. In a time-course experiment, the expression of XBP1s did not increase in the nucleus where it occurs normally, but was reduced by viral infection (lanes 2, 4, and 6, Fig. 1B). Nuclear XBP1s expression was increased by DTT treatment, indicating that the reduction in XBP1s was specific to EV71 infection (lane 7, Fig. 1B). To understand whether the reduced XBP1 expression resulted from viral replication, we used UV inactivation of EV71 to inhibit viral replication but not viral entry [17]. RD cells were infected with UV-irradiated virus, which abolished the titer completely (data not shown); cells were harvested at 6 h p.i. and analyzed by immunoblotting analysis (Fig. 1C). Similar to the mock-infected cells, UV-treated EV71 showed similar intensity of XBP1s expression compared with the mock-treated virus, indicating that the reduction in XBP1s required viral replication (lanes 2 and 3, Fig. 1C). Enterovirus infection attenuates cellular protein synthesis [10]. We could not exclude the possibility that the reduction in XBP1s expression was mediated by virally encoded proteases 2Apro and 3Cpro. We transfected plasmids encoding EV71 2Apro and 3Cpro into RD cells and examined their effects on endogenous XBP1s induced by Tg (Fig. 2A). EV71 infection reduced the level of endogenous XBP1s in cells treated with or without Tg (lanes 3 and 4, Fig. 2A). Cells transfected with plasmids encoding 2Apro but not 3Cpro showed a reduced level of XBP1s, although this reduction was less than that induced by the virus (lanes 5 and 6, Fig. 2A).
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Fig. 2. The reduction in XBP1s is not caused by direct cleavage by 2A but by translational shutoff mediated by eIF4G cleavage. (A) RD cells were transfected with plasmids encoding viral proteases 2A and 3C for 24 h or infected with EV71 for 5 h. Anti-His tag and anti-3A antibodies were used to monitor the expression of 2A-his and 3C-his and viral infection, respectively. (B) Cell lysates prepared from RD cells pretreated with or without Tg were incubated with 30 lg of recombinant wild-type or mutant 2A for 4 h at 37 °C. ‘‘bf’’ is the dialysis buffer that was used as a negative control, and ‘‘PC’’ is the positive control, which comprised lysates prepared from RD cells transfected with 2A.
Fig. 1. EV71 induces phosphorylation of IRE1 and reduction of XBP1s. (A) RD cells were preadsorbed with EV71 (MOI10) on ice between 1 and 0 h p.i. The medium containing the unbound virus was replaced with DMEM containing 2% FBS and the temperature was increased to 37 °C. The infection protocol is sketched in the upper panel. The cells were harvested at the times indicated for western blotting using specific antibodies against p-IRE1 and 3A. The 3A antibodies recognized 3A and its precursor protein 3AB. Membranes were also probed with an antibody against GAPDH to control the loading of lanes. DTT (5 mM)- and Tg (2 lM)-treated cell lysates were used as a positive control for IRE1 phosphorylation under conditions of definitive UPR induction. These are representative of three independent assays. (B) RD cells were infected with EV71 (MOI 10) and the cells were collected to produce nuclear and cytoplasm fractions at 3, 6, and 6 h p.i. Equal amounts of lysate of the nuclear or cytosolic fraction were subjected to Western blotting. The nuclear fraction of cells treated with 2.5 mM DTT for 6 h were used as a control for the expression of XBP1s. Lamin A/C was a loading control for the nuclear fraction. (C) RD cells were infected with either mock-treated or UV-inactivated virus. Six hours after infection, the nuclear fraction was analyzed by Western blotting.
We next determined whether XBP1is the direct substrate of 2Apro protease by incubating recombinant wild-type or mutant GST-2Apro with cell lysates obtained from cells treated with or without Tg (Fig. 2B). Mutation of C110S in 2Apro causes defected autocleavage catalytic activity of 2Apro released from viral polyprotein in cis and complete loss of proteolytic activity toward eIF4G in trans [18]. Catalytic cleavage of eIF4G by wild-type2Apro, but not its catalytic mutant variant C110A, was detected, suggesting specificity of 2Apro (upper panel, Fig. 2B). XBP1s was upregulated in Tg-treated cells (lanes 4–6, Fig. 2B). Parallel examination of its cleavage by 2Apro and its mutant showed that XBP1s was not the direct substrate of 2Apro (lanes 4–6, Fig. 2B). Together, these data suggest that the inhibition of XBP1s expression level is mediated by translational attenuation induced by proteolytic cleavage of eIF4G, but not of XBP1s by viral proteases. To test the hypothesis that IRE-XBP1-mediated ER stress inhibits viral replication, we measured viral replication in XBP1-transfected cells. We constructed a chimeric protein in which the transcriptionally active XBP1s was cloned into pcDNA3.1-mycHis. This chimeric protein has the characteristic of the wild-type protein because it is expressed correctly and can activate a luciferase reporter plasmid
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driven by the ERSE promoter (Fig. S1). RD cells were transfected with the XBP1s overnight and then infected with EV71, and the cells were collected at times p.i. for western blotting. The viral protein expression as indicated by 3A became visible from 7 h p.i.; at this time, less viral protein was translated in XBP1s-transfected compared with the vector control-transfected cells (lane 8 versus 9, Fig. 3A). However, this inhibition disappeared at 9 h p.i., suggesting that XBP1s had been compensated or the viral translation reached a plateau (lanes 11 and 12, Fig. 3A). The viral titer in XBP1s-overexpressing cells decreased significantly (Fig. 3B). We also analyzed viral replication using RNA interference to knock down XBP1s. siRNA targeting XBP1s (siXBP1) was transfected into RD cells (Fig. 3C and D). Compared with mock-transfected cells, cells transfected with siXBP1s showed a marked reduction in XBP1 level (Fig. 3D). By contrast, the GAPDH level was not affected under the same conditions. Viral protein synthesis increased in XBP1s-knockdown cells at 5 h p.i. (lanes 1 and 2), although the viral protein level reached a plateau afterward (Fig. 3C). These data suggest that the virus caused XBP1 disappearance, which facilitated its replication. To examine if the possible inhibitory effect of XBP1 on viral translation was associated with viral IRES activity, IRES activity of EV71 was measured by luciferase driven by IRES. The reporter plasmid (CMV-EV71 50 UTR-Fluc) containing the EV71 4643IRES was placed upstream of the Fluc open reading frames (upper panel, Fig. S2). At 24 h after transfection with XBP1s or the corresponding vector control, the reporter containing EV71 IRES was then transfected alone with the internal control plasmid pTK-Rluc, and the cells were harvested for dual-luciferase reporter assay 8 h posttransfection. The transfection of XBP1 showed little effect on EV71 IRES activity, as indicated by the luciferase activity in the cells (bottom panel, Fig. S2), suggesting that XBP1 did not target viral IRES activity. To examine whether XBP1 inhibits viral entry, we used UV-irradiated EV71, which lack replication activity but can bind to and enter host cells [17]. EV71 at an MOI of 100 was inactivated by UV irradiation, and the remaining titers of UVEV71 were inhibited completely as detected by plaque assay (data not shown). UV-EV71 was added to control vector-transfected or XBP1-transfected RD cells, and the cells were harvested after 0, 0.5, and 1 h. The viral RNA was measured as an index of viral uptake (Fig. 4). The XBP1-transfected cells showed no signal of viral uptake; the vector-transfected cells showed a significant increase in virus uptake in a time-dependent manner, suggesting that XBP1 inhibits viral entry (Fig. 4).
4. Discussion In this study, we examined the role of the IRE1-XBP1 pathway in viral replication, and we conclude that XBP1 plays a critical role in EV71 replication at the entry step based on the following findings: (1) IRE1 was activated but no XBP1 splicing was detected during infection; (2) expression of XBP1 downregulated viral replication; (3) XBP1 was subject to translational shutoff by EV71-induced eIF4G cleavage; and (4) the uptake of UV-irradiated virus decreased in XBP1-overexpressed cells. We demonstrated previously that EV71 does not change the EDEM mRNA level driven by unfolded protein response element during the course of infection [12]. The resulting inhibition of the expression of genes such as EDEM may be beneficial to viral infection by inhibiting viral protein degradation by the ERAD mechanism. The gene for XBP1 contains an ERSE element and can be transactivated by ATF6 and XBP1 itself [5,19]. However, using a reporter assay, we reported that the EV71 did not activate the ERSE of XBP1 [12]. In our current study, we found further evidence that the translation of XBP1s is inhibited by attenuation of EV71-induced eIF4G-cleavage (Fig. 2).
Fig. 3. Expression of XBP1s inhibits viral replication. (A) Viral protein synthesis was inhibited by the overexpression of XBP1s. RD cells were transfected with XBP1s or a control vector (4 lg each) for 24 h. The cells were infected with EV71 (MOI 10) and were harvested at the times indicated and subjected to Western blotting using specific antibodies against 3A. (B) Viral titer in RD cells transiently transfected with XBP1s plasmids was measured by plaque assay. After transfection for 24 h, the cells were infected with EV71 (MOI 10), and the amount of virus present in the cells and culture medium was measured by plaque assay. The average plaque numbers of cells transfected with XBP1s plasmid were normalized to that of vector control, arbitrarily set to 1. This is an average of three independent experiments. ⁄p < 0.005. (C and D) Time-course analysis of viral protein expression in siXBP1-transfected RD cells. Cells were transfected with scrambled siRNA or siXBP1 for 3 days. The cells were infected with EV71 (MOI 10) and harvested at the times indicated, and 50 lg aliquots of each cell lysate were used for Western blotting. The silencing effect by siXBP1by immunoblotting is shown in (D).
Picornaviral proteinases catalyze the degradation of cellular targets, which shuts off global host protein synthesis. However, viral RNA undergoes cap-independent IRES translation, which is
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Fig. 4. XBP1-overexpressing RD cells show reduced viral entry of UV-irradiated EV71. EV71 (MOI 100) was irradiated by UV and added to cells transfected with XBP1 or a control vector, and the cells were harvested after 0, 0.5, and 1.0 h. Intracellular viral RNA level was measured by qPCR. To adjust for differences in viral binding in the two cell types, the ratio of viral RNA to the internal control was normalized to the RNA level at 0 h, arbitrarily set to 1. ⁄p < 0.05.
not affected by the virus itself. We demonstrated recently that EV71 infection upregulates several ER-resident chaperone genes, including BiP and calreticulin, both of which contain ERSE [20,21]. These chaperone proteins were upregulated by EV71, but this upregulation was not caused by an increase in RNA levels [12]. Chaperone genes such as BiP may contain an IRES structure, whose activity increases under stress [22] and which may escape cap-dependent translational shutoff. We cannot exclude the possibility that EV71 increases the stability of existing chaperone genes to facilitate the folding and assembly of rapidly synthesized viral proteins. The inhibition of XBP1 by viral infection might be explained by the notion that XBP1 expression is unfavorable for viral replication. This speculation is supported by the inhibition of viral replication by the overexpression of XBP1 (Fig. 3A and B). Conversely, the reduced XBP1 expression by siRNA is advantageous to viral replication because it increases viral translation (Fig. 3C and D). Modulation of XBP1 signaling events by other viruses has also been observed. In HCMV-infected cells or in cells carrying HCV replicons, the transcriptional activity of XBP1 is inhibited despite the presence of significant levels of spliced XBP1 [23,24]. The suppression of IRE-XBP1 may promote HCV replication because the IRES-dependent translational activity of HCV increases in IRE–/–MEF cells with a defective IRE-XPB1 pathway [24]. We found that XBP1 can inhibit EV71 entry, possibly by altering XBP1s transcriptional activity to regulate the expression of viral uptake-related proteins. It would be tempting to study these proteins in XBP1-overexpressing RD cells by transcriptomic or proteomic approaches. Acknowledgments This study was supported by Chang Gung Memorial Hospital (Grant numbers CMRPD 180072 and 190192) and by National Science Council (99-2321-B-001-035, 98-2311-B-182-003-MY3).
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Inhibition of enterovirus 71 entry by transcription factor XBP1 Jia-Rong Jheng 1, Chiou-Yan Lin 1, Jim-Tong Horng ⇑,1, Kean Seng Lau Department of Biochemistry and Research Center for Emerging Viral Infections, Chang Gung University, 259 Wen-Hwa First Road, Kweishan, Taoyuan 333, Taiwan
a r t i c l e
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Article history: Received 6 March 2012 Available online 24 March 2012 Keywords: Enterovirus 71 Endoplasmic reticulum stress Inositol-requiring enzyme 1 Unfolded protein response Translational attenuation X-box-binding protein 1
a b s t r a c t Inositol-requiring enzyme 1 (IRE1) plays an important role in the endoplasmic reticulum (ER), or unfolded protein, stress response by activating its downstream transcription factor X-box-binding protein 1 (XBP1). We demonstrated previously that enterovirus 71 (EV71) upregulated XBP1 mRNA levels but did not activate spliced XBP1 (XBP1s) mRNA or its downstream target genes, EDEM and chaperones. In this study, we investigated further this regulatory mechanism and found that IRE1 was phosphorylated and activated after EV71 infection, whereas its downstream XBP1s protein level decreased. We also found that XBP1s was not cleaved directly by 2Apro, but that cleavage of eukaryotic translation initiation factor 4G by the EV71 2Apro protein may contribute to the decrease in XBP1s expression. Knockdown of XBP1 increased viral protein expression, and the synthesis of EV71 viral protein and the production of EV71 viral particles were inhibited in XBP1-overexpressing RD cells. When incubated with replication-deficient and UV-irradiated EV71, XBP1-overexpressing RD cells exhibited reduced viral RNA levels, suggesting that the inhibition of XBP1s by viral infection may underlie viral entry, which is required for viral replication. Our findings are the first indication of the ability of XBP1 to inhibit viral entry, possibly via its transcriptional activity in regulating molecules in the endocytic machinery. Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction Three major transmembrane ER stress sensor proteins have been identified and characterized comprehensively: PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and IRE1. These proteins manage three arms of the unfolded protein response (UPR) signaling pathway and activate different subsets of genes to manage stress. Activation of PERK initiates the phosphorylation of eukaryotic initiation factor 2a (eIF2a), which causes the inhibition of global translation [1]. ATF6 belongs to a highly conserved family of regulated intramembrane proteolysis transcription factors, which are cleaved to release cytosolic domains that transit to the nucleus to regulate the transcription of a collection of ER stress target genes, including BiP, by binding to a consensus ER stress response element (ERSE) sequence in the promoter region of these proteins [2,3]. IRE1 undergoes oligomerization to activate its kinase and endoribonuclease (RNase) activities to transmit the UPR signals [4]. The RNase of IRE1 splices mRNA encoding the transcription factor XBP1, leading to a frameshift splicing and subsequent translation of the active spliced XBP1 protein (XBP1s) [5]. XBP1 was originally identified as a basic leucine zipper transcription factor in B cells and is required for plasma cell differentiation ⇑ Corresponding author. Fax: +886 3 211 8407. 1
E-mail address: [email protected] (J.-T. Horng). These authors contributed equally to this project.
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[6]. By binding the ERSE promoter region, the spliced transcription factor XBP1s can sequentially stimulate chaperones to promote the folding capacity in the ER [7,8]. The clearing of incorrectly folded proteins retained in the ER is now known to occur through their retrotranslocation into the cytosol in a process involving ER-associated degradation (ERAD), which is also induced by XBP1 [4]. EV71 is a positive-stranded RNA virus that belongs to the group of human enteroviruses whose members include poliovirus and coxsackievirus. EV71 has a genome of about 7.5 kb comprising an open reading frame flanked by a 50 untranslated region (UTR) and a 30 poly(A) tail. The 50 UTR of the viral genome is an internal ribosome entry site (IRES), which directs translation of the viral RNA in a cap-independent fashion to synthesize a polyprotein that can be cleaved by viral proteases 2Apro and 3Cpro to produce mature and functional structural and nonstructural proteins [9]. 2Apro is also responsible for the cleavage of eukaryotic translation initial factor G (eIF4G), which in turn inhibits the cap-dependent translational inhibition of global cellular proteins [10]. Numerous studies suggest a relationship between the ER stress response and viral infection, and many viruses can regulate UPR to benefit their replication or to trigger an immune response [11]. We showed previously that EV71 infection activates and modifies the UPR signaling pathways to benefit viral replication [12]. XBP1 mRNA was upregulated, but no IRE1-mediated splicing was detected. In the present study, we examined further the role of IRE–XBP1 of UPR during EV71 replication.
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2. Materials and methods 2.1. Cell culture and viral infection Rhabdomyosarcoma (RD) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) under humidified 5% CO2 in air. EV71 BrCr was obtained from the ATCC (accession no. VR 784). Viral propagation and titer determination by plaque assay were performed in RD cells, and the titer was recorded as the number of plaque-forming units per milliliter [13]. 2.2. Antibodies and reagents Rabbit polyclonal antibodies to 3A were raised against GST-3A [14]. Rabbit polyclonal antibodies against eIF4G and lamin A/C, and mouse monoclonal anti-GST were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal anti-phospho-IRE1 and anti-XBP1 were from Abcam (Ab48187, Cambridge, UK) and Anaspec (#54578, San Jose, CA, USA), respectively. Antibody to GAPDH was from Abnova (Taoyuan, Taiwan). Anti-His tag was from Serotec (#MCA1396B, Oxford, UK). Plasmids encoding XBP1s, 2Apro, 3Cpro, and 2Apro(C110A) were cloned into pcDNA3. 1-mycHis() (Invitrogen, Carlsbad, CA). GST-2Apro and GST-2Apro (C110A) were cloned into pGEX-2T and pGEX-5X-1 (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), respectively. EV71 50 UTR and 5 ERSE were cloned into pcDNA3.1-mycHis-B(+)-Fluc, in which Fluc was PCR amplified from pGL3 and inserted into pcDNA3. 1/myc-His-B(+) to generate pCMV-50 UTR-Fluc (4643-IRES-Fluc) and pCMV-5ERSE-Fluc, respectively. Stealth RNAi™ siRNA of XBP1 (siXBP1, #HSS111391) and scrambled negative controls Lo GC (#12935200) were from Invitrogen. 2.3. UV irradiation and viral RNA determination UV irradiation of EV71 was performed as described previously, and the remaining titer was measured by plaque assay [15]. RD cells transfected with XPB1 plasmid or its vector control were incubated with UV-treated virus (UV-EV71), the cells were collected after 0, 0.5, and 1 h, and the viral RNA was quantified using quantitative real-time PCR (qPCR). To quantify the changes in gene expression, the DCt method was used to calculate relative fold changes normalized against the GAPDH control. 2.4. Western blotting and nucleus/cytoplasm fractionation assays The cells were harvested at the times indicated, and lysates were prepared with lysis buffer (10 mM Tris, pH 7.4, 10 mM NaCl, and 1 mM MgCl2) containing protease inhibitor cocktail (Roche). Equal amounts of cellular protein were analyzed by SDS–PAGE, the bands were transferred to a polyvinylidene fluoride membrane, and the proteins on the membrane were detected with enhanced chemiluminescence reagents (Millipore). RD cells were mock infected or infected with EV71 at a multiplicity of infection (MOI) of 10. The cells were harvested at the times indicated, and the nuclei and cytoplasm were separated as described [12]. 2.5. In vitro protease cleavage assay Wild-type and the catalytic-inactive mutant (C110A: cysteine mutated to alanine at amino acid 110) of 2Apro of EV71 were cloned into pGEX vectors, and these GST-fusion proteins were purified according to the manufacturer’s instruction (GE Healthcare BioSciences AB). The purified GST-fusion proteins were dialyzed in buffer (25 mM Tris–HCl, pH 7.5, 50 mM NaCl, 5 mM DTT, 0.5 mM
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EDTA, and 25% glycerol) before use. RD cells were treated with or without 2 lg of thapsigargin (Tg) for 4 h and were lysed with lysis buffer without proteinase inhibitors. The cell lysates were centrifuged at 17,900g for 10 min at 4 °C, and the supernatant was collected for the cleavage assay. Thirty micrograms of supernatant was incubated with 30 lg of GST-2A or GST-2Apro(C110A) in a total volume of 100 ll, at 37 °C for 4 h, and then analyzed by SDS–PAGE and Western blotting. 2.6. Reporter assays Each well containing 3 104 RD cells in a 48-well plate was transfected with Lipofectamine with 0.4 lg of firefly luciferase reporter gene and 0.04 lg of pTK-Rluc encoding Renilla luciferase as an internal control. The luciferase activities were measured using a dual-luciferase reporter kit (Promega, Madison, WI, USA). The results were normalized to the Renilla luciferase activity of the internal control. 2.7. Data analysis The data were analyzed using two-tailed Student’s t test and are expressed as the mean ± SEM. P < 0.05 was considered significant. 3. Results To understand better the regulatory mechanism of the EV71-induced IRE1-XBP1 signaling pathway, we examined whether the upstream IRE was activated by EV71. RD cells were infected with EV71, and the cells were harvested at the designated times postinfection (p.i.). EV71 phosphorylated IRE1 from 7 h p.i., and the phosphorylation level correlated with the viral translation, as indicated by the level of viral 3A protein, from 5 h p.i. (Fig. 1A). In response to ER stress, the RNase function of IRE1 is activated to initiate the nonconventional splicing of transcription factor XBP1 mRNA to produce XBP1s, which then increases the folding capacity for targeting misfolded proteins for degradation [5,16]. We demonstrated previously that EV71 increases the XBP1 mRNA level but that spliced XBP1 mRNA and downstream target genes, EDEM and chaperones, are not produced [12]. We next asked whether the regulation of XBP1 is responsible for the decrease in XBP1 expression. In a time-course experiment, the expression of XBP1s did not increase in the nucleus where it occurs normally, but was reduced by viral infection (lanes 2, 4, and 6, Fig. 1B). Nuclear XBP1s expression was increased by DTT treatment, indicating that the reduction in XBP1s was specific to EV71 infection (lane 7, Fig. 1B). To understand whether the reduced XBP1 expression resulted from viral replication, we used UV inactivation of EV71 to inhibit viral replication but not viral entry [17]. RD cells were infected with UV-irradiated virus, which abolished the titer completely (data not shown); cells were harvested at 6 h p.i. and analyzed by immunoblotting analysis (Fig. 1C). Similar to the mock-infected cells, UV-treated EV71 showed similar intensity of XBP1s expression compared with the mock-treated virus, indicating that the reduction in XBP1s required viral replication (lanes 2 and 3, Fig. 1C). Enterovirus infection attenuates cellular protein synthesis [10]. We could not exclude the possibility that the reduction in XBP1s expression was mediated by virally encoded proteases 2Apro and 3Cpro. We transfected plasmids encoding EV71 2Apro and 3Cpro into RD cells and examined their effects on endogenous XBP1s induced by Tg (Fig. 2A). EV71 infection reduced the level of endogenous XBP1s in cells treated with or without Tg (lanes 3 and 4, Fig. 2A). Cells transfected with plasmids encoding 2Apro but not 3Cpro showed a reduced level of XBP1s, although this reduction was less than that induced by the virus (lanes 5 and 6, Fig. 2A).
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Fig. 2. The reduction in XBP1s is not caused by direct cleavage by 2A but by translational shutoff mediated by eIF4G cleavage. (A) RD cells were transfected with plasmids encoding viral proteases 2A and 3C for 24 h or infected with EV71 for 5 h. Anti-His tag and anti-3A antibodies were used to monitor the expression of 2A-his and 3C-his and viral infection, respectively. (B) Cell lysates prepared from RD cells pretreated with or without Tg were incubated with 30 lg of recombinant wild-type or mutant 2A for 4 h at 37 °C. ‘‘bf’’ is the dialysis buffer that was used as a negative control, and ‘‘PC’’ is the positive control, which comprised lysates prepared from RD cells transfected with 2A.
Fig. 1. EV71 induces phosphorylation of IRE1 and reduction of XBP1s. (A) RD cells were preadsorbed with EV71 (MOI10) on ice between 1 and 0 h p.i. The medium containing the unbound virus was replaced with DMEM containing 2% FBS and the temperature was increased to 37 °C. The infection protocol is sketched in the upper panel. The cells were harvested at the times indicated for western blotting using specific antibodies against p-IRE1 and 3A. The 3A antibodies recognized 3A and its precursor protein 3AB. Membranes were also probed with an antibody against GAPDH to control the loading of lanes. DTT (5 mM)- and Tg (2 lM)-treated cell lysates were used as a positive control for IRE1 phosphorylation under conditions of definitive UPR induction. These are representative of three independent assays. (B) RD cells were infected with EV71 (MOI 10) and the cells were collected to produce nuclear and cytoplasm fractions at 3, 6, and 6 h p.i. Equal amounts of lysate of the nuclear or cytosolic fraction were subjected to Western blotting. The nuclear fraction of cells treated with 2.5 mM DTT for 6 h were used as a control for the expression of XBP1s. Lamin A/C was a loading control for the nuclear fraction. (C) RD cells were infected with either mock-treated or UV-inactivated virus. Six hours after infection, the nuclear fraction was analyzed by Western blotting.
We next determined whether XBP1is the direct substrate of 2Apro protease by incubating recombinant wild-type or mutant GST-2Apro with cell lysates obtained from cells treated with or without Tg (Fig. 2B). Mutation of C110S in 2Apro causes defected autocleavage catalytic activity of 2Apro released from viral polyprotein in cis and complete loss of proteolytic activity toward eIF4G in trans [18]. Catalytic cleavage of eIF4G by wild-type2Apro, but not its catalytic mutant variant C110A, was detected, suggesting specificity of 2Apro (upper panel, Fig. 2B). XBP1s was upregulated in Tg-treated cells (lanes 4–6, Fig. 2B). Parallel examination of its cleavage by 2Apro and its mutant showed that XBP1s was not the direct substrate of 2Apro (lanes 4–6, Fig. 2B). Together, these data suggest that the inhibition of XBP1s expression level is mediated by translational attenuation induced by proteolytic cleavage of eIF4G, but not of XBP1s by viral proteases. To test the hypothesis that IRE-XBP1-mediated ER stress inhibits viral replication, we measured viral replication in XBP1-transfected cells. We constructed a chimeric protein in which the transcriptionally active XBP1s was cloned into pcDNA3.1-mycHis. This chimeric protein has the characteristic of the wild-type protein because it is expressed correctly and can activate a luciferase reporter plasmid
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driven by the ERSE promoter (Fig. S1). RD cells were transfected with the XBP1s overnight and then infected with EV71, and the cells were collected at times p.i. for western blotting. The viral protein expression as indicated by 3A became visible from 7 h p.i.; at this time, less viral protein was translated in XBP1s-transfected compared with the vector control-transfected cells (lane 8 versus 9, Fig. 3A). However, this inhibition disappeared at 9 h p.i., suggesting that XBP1s had been compensated or the viral translation reached a plateau (lanes 11 and 12, Fig. 3A). The viral titer in XBP1s-overexpressing cells decreased significantly (Fig. 3B). We also analyzed viral replication using RNA interference to knock down XBP1s. siRNA targeting XBP1s (siXBP1) was transfected into RD cells (Fig. 3C and D). Compared with mock-transfected cells, cells transfected with siXBP1s showed a marked reduction in XBP1 level (Fig. 3D). By contrast, the GAPDH level was not affected under the same conditions. Viral protein synthesis increased in XBP1s-knockdown cells at 5 h p.i. (lanes 1 and 2), although the viral protein level reached a plateau afterward (Fig. 3C). These data suggest that the virus caused XBP1 disappearance, which facilitated its replication. To examine if the possible inhibitory effect of XBP1 on viral translation was associated with viral IRES activity, IRES activity of EV71 was measured by luciferase driven by IRES. The reporter plasmid (CMV-EV71 50 UTR-Fluc) containing the EV71 4643IRES was placed upstream of the Fluc open reading frames (upper panel, Fig. S2). At 24 h after transfection with XBP1s or the corresponding vector control, the reporter containing EV71 IRES was then transfected alone with the internal control plasmid pTK-Rluc, and the cells were harvested for dual-luciferase reporter assay 8 h posttransfection. The transfection of XBP1 showed little effect on EV71 IRES activity, as indicated by the luciferase activity in the cells (bottom panel, Fig. S2), suggesting that XBP1 did not target viral IRES activity. To examine whether XBP1 inhibits viral entry, we used UV-irradiated EV71, which lack replication activity but can bind to and enter host cells [17]. EV71 at an MOI of 100 was inactivated by UV irradiation, and the remaining titers of UVEV71 were inhibited completely as detected by plaque assay (data not shown). UV-EV71 was added to control vector-transfected or XBP1-transfected RD cells, and the cells were harvested after 0, 0.5, and 1 h. The viral RNA was measured as an index of viral uptake (Fig. 4). The XBP1-transfected cells showed no signal of viral uptake; the vector-transfected cells showed a significant increase in virus uptake in a time-dependent manner, suggesting that XBP1 inhibits viral entry (Fig. 4).
4. Discussion In this study, we examined the role of the IRE1-XBP1 pathway in viral replication, and we conclude that XBP1 plays a critical role in EV71 replication at the entry step based on the following findings: (1) IRE1 was activated but no XBP1 splicing was detected during infection; (2) expression of XBP1 downregulated viral replication; (3) XBP1 was subject to translational shutoff by EV71-induced eIF4G cleavage; and (4) the uptake of UV-irradiated virus decreased in XBP1-overexpressed cells. We demonstrated previously that EV71 does not change the EDEM mRNA level driven by unfolded protein response element during the course of infection [12]. The resulting inhibition of the expression of genes such as EDEM may be beneficial to viral infection by inhibiting viral protein degradation by the ERAD mechanism. The gene for XBP1 contains an ERSE element and can be transactivated by ATF6 and XBP1 itself [5,19]. However, using a reporter assay, we reported that the EV71 did not activate the ERSE of XBP1 [12]. In our current study, we found further evidence that the translation of XBP1s is inhibited by attenuation of EV71-induced eIF4G-cleavage (Fig. 2).
Fig. 3. Expression of XBP1s inhibits viral replication. (A) Viral protein synthesis was inhibited by the overexpression of XBP1s. RD cells were transfected with XBP1s or a control vector (4 lg each) for 24 h. The cells were infected with EV71 (MOI 10) and were harvested at the times indicated and subjected to Western blotting using specific antibodies against 3A. (B) Viral titer in RD cells transiently transfected with XBP1s plasmids was measured by plaque assay. After transfection for 24 h, the cells were infected with EV71 (MOI 10), and the amount of virus present in the cells and culture medium was measured by plaque assay. The average plaque numbers of cells transfected with XBP1s plasmid were normalized to that of vector control, arbitrarily set to 1. This is an average of three independent experiments. ⁄p < 0.005. (C and D) Time-course analysis of viral protein expression in siXBP1-transfected RD cells. Cells were transfected with scrambled siRNA or siXBP1 for 3 days. The cells were infected with EV71 (MOI 10) and harvested at the times indicated, and 50 lg aliquots of each cell lysate were used for Western blotting. The silencing effect by siXBP1by immunoblotting is shown in (D).
Picornaviral proteinases catalyze the degradation of cellular targets, which shuts off global host protein synthesis. However, viral RNA undergoes cap-independent IRES translation, which is
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Fig. 4. XBP1-overexpressing RD cells show reduced viral entry of UV-irradiated EV71. EV71 (MOI 100) was irradiated by UV and added to cells transfected with XBP1 or a control vector, and the cells were harvested after 0, 0.5, and 1.0 h. Intracellular viral RNA level was measured by qPCR. To adjust for differences in viral binding in the two cell types, the ratio of viral RNA to the internal control was normalized to the RNA level at 0 h, arbitrarily set to 1. ⁄p < 0.05.
not affected by the virus itself. We demonstrated recently that EV71 infection upregulates several ER-resident chaperone genes, including BiP and calreticulin, both of which contain ERSE [20,21]. These chaperone proteins were upregulated by EV71, but this upregulation was not caused by an increase in RNA levels [12]. Chaperone genes such as BiP may contain an IRES structure, whose activity increases under stress [22] and which may escape cap-dependent translational shutoff. We cannot exclude the possibility that EV71 increases the stability of existing chaperone genes to facilitate the folding and assembly of rapidly synthesized viral proteins. The inhibition of XBP1 by viral infection might be explained by the notion that XBP1 expression is unfavorable for viral replication. This speculation is supported by the inhibition of viral replication by the overexpression of XBP1 (Fig. 3A and B). Conversely, the reduced XBP1 expression by siRNA is advantageous to viral replication because it increases viral translation (Fig. 3C and D). Modulation of XBP1 signaling events by other viruses has also been observed. In HCMV-infected cells or in cells carrying HCV replicons, the transcriptional activity of XBP1 is inhibited despite the presence of significant levels of spliced XBP1 [23,24]. The suppression of IRE-XBP1 may promote HCV replication because the IRES-dependent translational activity of HCV increases in IRE–/–MEF cells with a defective IRE-XPB1 pathway [24]. We found that XBP1 can inhibit EV71 entry, possibly by altering XBP1s transcriptional activity to regulate the expression of viral uptake-related proteins. It would be tempting to study these proteins in XBP1-overexpressing RD cells by transcriptomic or proteomic approaches. Acknowledgments This study was supported by Chang Gung Memorial Hospital (Grant numbers CMRPD 180072 and 190192) and by National Science Council (99-2321-B-001-035, 98-2311-B-182-003-MY3).
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