Sec62 Suppresses Foot-And-Mouth Disease Virus Proliferation by Promotion of IRE1 −Α RIG-I Antiviral Signaling

Sec62 Suppresses Foot-and-Mouth Disease Virus Proliferation by Promotion of IRE1 −α RIG-I Antiviral Signaling

This information is current as Shichong Han, Lejiao Mao, Ying Liao, Shiqi Sun, Zhihui of October 7, 2021. Zhang, Yaxia Mo, Haiyun Liu, Xiaoying Zhi, Shunmei Lin, Ho Seong Seo and Huichen Guo J Immunol 2019; 203:429-440; Prepublished online 5 June 2019; doi: 10.4049/jimmunol.1801546 http://www.jimmunol.org/content/203/2/429 Downloaded from

Supplementary http://www.jimmunol.org/content/suppl/2019/06/04/jimmunol.180154

Material 6.DCSupplemental http://www.jimmunol.org/ References This article cites 54 articles, 16 of which you can access for free at: http://www.jimmunol.org/content/203/2/429.full#ref-list-1

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Sec62 Suppresses Foot-and-Mouth Disease Virus Proliferation by Promotion of IRE1a–RIG-I Antiviral Signaling

Shichong Han,*,1 Lejiao Mao,*,1 Ying Liao,†,1 Shiqi Sun,* Zhihui Zhang,* Yaxia Mo,* Haiyun Liu,* Xiaoying Zhi,* Shunmei Lin,‡ Ho Seong Seo,‡ and Huichen Guo*

Foot-and-mouth disease virus (FMDV) is highly infectious and causes a major plague in animal farming. Unfolded protein response is one of the major cellular responses to pathogenic infections, which performs a crucial role in cell survival, apoptosis, and antiviral innate immune response. In this study, we showed that FMDV infection activated two unfolded protein response branches (PERK- eIF2a and ATF6 signaling) in both baby hamster kidney cells (BHK-21) and porcine kidney (PK-15) cells, whereas it suppressed the IRE1a-XBP1 signaling by decreasing IRE1a level. Further study revealed IRE1a signaling as an important antiviral innate

immune mechanism against FMDV. Sec62, the transport protein, was greatly decreased at the late stages of FMDV infection. By Downloaded from overexpression and knockdown study, we also found that the expression of Sec62 was positively involved in the levels of IRE1a and RIG-I and subsequent activation of downstream antiviral signaling pathways in FMDV-infected PK-15 cells. Taken together, our study demonstrates that Sec62 is an important antiviral factor that upregulates IRE1a–RIG-I–dependent antiviral innate immune responses, and FMDV evades antiviral host defense mechanism by downregulating Sec62-IRE1a/RIG-I. The Journal of Immunology, 2019, 203: 429–440. http://www.jimmunol.org/ oot-and-mouth disease virus (FMDV) is a positive-sense change that allows the viral RNA to be transferred across the ssRNA virus from the genus Aphthovirus in the family plasma or endosomal membrane into cytoplasm (11, 12) where the F Picornaviridae (1, 2). It is nonenveloped and consists of viral genomic RNA is translated into viral proteins. The RNA a densely packed icosahedral protein shell that is ∼30 nm in di- genome replication machinery is then assembled by these proteins ameter (3). FMDV is known to cause foot-and-mouth disease on the endoplasmic reticulum (ER)–derived membrane leaflet. ER (FMD) in cloven-hoofed animals, which leads to severe economic membranes are also involved in the formation of viral vesicles in loss in animal farming. Currently, the outbreaks of FMD are poliovirus infection. The ER, one of the most important organ- controlled by combined efforts of mass vaccination and animal elles, forms the cell interconnect tubular network (13). It has culling (4). Understanding the viral pathogenesis of FMDV and multiple functions, including protein synthesis, folding, post- by guest on October 7, 2021 host antiviral defense mechanisms against it would provide the- translational modification, and storage of Ca2+. ER stress is in- oretical basis for the improvement of FMD prevention and control duced by numerous stimuli, such as pathogen infection, amino strategies (5). starvation, heat shock, excessive consumption of ER membrane, Genome replication of positive-strand RNA viruses in eukary- and Ca2+ flux. When cells cannot keep the balance between the otes always takes place on the intracellular membrane system protein-folding capacity and cellular protein requirement (14, 15), (6, 7). For example, poliovirus and coxsackie virus B3 use the misfolded proteins accumulate in the ER lumen and trigger the membranes of early secretory pathway for their replication (8, 9). unfolded protein response (UPR) to relieve the ER stress and re- After viruses bind to specific host receptors at the cell surface or within cover the balance (16). However, if the homeostasis cannot be endocytic vesicles (10), viral capsid undergoes a conformational restored by UPR, cell death is triggered (17). UPR also plays an

*World Organisation for Animal Health–China National Foot-and-Mouth Disease data; and H.G., Y.L., L.M., and S.H. prepared the manuscript. All authors reviewed Reference Laboratory, State Key Laboratory of Veterinary Etiological Biology, the results and approved the final version of the manuscript. Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Address correspondence and reprint requests to Dr. Huichen Guo, World Organisa- Lanzhou 730046, Gansu, People’s Republic of China; †Department of Avian Dis- tion for Animal Health–China National Foot-and-Mouth Disease Reference Labora- eases, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural tory, State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Sciences, Shanghai 200241, People’s Republic of China; and ‡Biotechnology Divi- Research Institute, Chinese Academy of Agricultural Sciences, Xujiaping 1, Lanzhou sion, Korea Atomic Energy Research Institute, Jeongeup 56212, Republic of Korea 730046, Gansu, People’s Republic of China. E-mail address: [email protected] 1S.H., L.M., and Y.L. contributed equally to this work. The online version of this article contains supplemental material. ORCIDs: 0000-0001-6044-4971 (L.M.); 0000-0003-4975-739X (S.S.); 0000-0002- Abbreviations used in this article: ATF4, activating transcription factor 4; ATF6, 7103-1344 (H.S.S.). activating transcription factor 6; ATF6-N, N-terminal ATF6 fragment; BHK-21, baby Received for publication November 27, 2018. Accepted for publication May 6, 2019. hamster kidney; BiP, IgH chain binding protein; eIF2a, eukaryotic translation initiation factor 2a; ER, endoplasmic reticulum; FMD, foot-and-mouth disease; FMDV, This work was supported by grants from the National Natural Science Foundation of foot-and-mouth disease virus; h.p.i., hour postinfection; IRE1a, inositol-requiring China (31672592 and 31811540395), the National Key Research and Development enzyme 1a; MOI, multiplicity of infection; NC, negative control; PERK, protein Program (2017YFD0500900, 2017YFD0501100, and 2016YFE0204100), Central kinase R–like ER kinase; PK-15, porcine kidney; RIDD, regulated IRE1-dependent Public Interest Scientific Institution Basal Research Fund (1610312016002, decay; siRNA, small interfering RNA; TCID , 50% tissue culture-infective dose; 1610312018003, and Y2017JC57), and the Elite Youth Program of the Chinese 50 TG, thapsigargin; UPR, unfolded protein response; XBP1, X-box binding protein-1; Academy of Agricultural Sciences. XBP1s, spliced XBP1. H.G. and L.M. designed the research; S.H., L.M., and Y.L. carried out the experi- mental work; S.S. and Z.Z. constructed the recombinant plasmids; Y.M. and H.L. Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 performed real-time qPCR; X.Z. performed the ELISA; S.L. and H.S.S. analyzed the www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801546 430 Sec62 PROTEIN IS INVOLVED IN ANTI-FMDV IMMUNE RESPONSE essential role in viral replication and pathogenesis, which has (no. 4947), phospho-IRF7 (no. 5184), TRAF3 (no. 4729), TRAF6 become an attractive strategy for the design and development of (no. 8028), and phospho-TBK1 (no. 5483) were purchased from Cell broad-spectrum antiviral agents. Increasing evidence indicates Signaling Technology (Danvers, MA). Abs against c-Myc (M4439) and Flag (MA1-91878) and HRP-, FITC-, or TRITC-conjugated secondary that viruses selectively modulate the UPR to take advantage of Abs were purchased from Sigma. Ab against phospho-IRE1a (NB100- the beneficial effects and inhibit the detrimental effects on viral 2323) was obtained from Novus Biologicals (Littleton, CO). Abs against replication. A number of viruses were found to induce UPR, ATF6 (ab122897) and Sec62 (ab140644) were purchased from Abcam b including viruses in the Picornaviridae family, such as entero- (Cambridge, MA). Abs against -actin (sc-47778), Lamin B1 (sc-377000), a-Tubulin (sc-398103), p65 (sc-8008), and IkB-a (sc-371) were purchased virus 71 (18) and coxsackie virus B3 (19). However, there is from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal pig serum no study focusing on the characterization of UPR on FMDV against FMDV was obtained from the World Organization for Animal infection. Health/China National Foot-and-Mouth Disease Reference Laboratory. UPR is initiated by three ER stress sensors: protein kinase R–like Thapsigargin (TG) (T9033) was purchased from Sigma and dissolved in ER kinase (PERK), activating transcription factor 6 (ATF6), and DMSO. Cells were treated with 100 nM TG for 5 or 10 h to induce ER stress, and DMSO treatment was set as NC. inositol-requiring enzyme 1a (IRE1a). The ER chaperone IgH chain binding protein (BiP), a principal regulator of the UPR, Plasmids binds to these sensors and keep9s them inactive under normal By using conventional RT-PCR, the full-length cDNAs of porcine PERK, conditions (20–22). However, when unfolded and misfolded ATF6, IRE1a, and Sec62 were amplified from PK-15 cells, and the cDNAs p9roteins accumulate in the ER lumen, BiP is dissociated from of hamster PERK, ATF6, IRE1a, and Sec62 were amplified from BHK-21 these sensors, which leads to the activation of these sensors and cells. Porcine Sec62 cDNA was inserted between BamH I and Xho I re- striction sites in pCMV-3Tag-4 vector (Agilent Technologies, Santa Clara, subsequent initiation of UPR signaling (20–23). Sec62 is one of

CA) to obtain the Myc-tagged expression construct (porcine Myc-Sec62). Downloaded from the well-characterized components of the translocation machinery All other cDNAs were inserted into pCMV-N-Flag vector (Beyotime for importing proteins into the ER, which also includes Sec61, Biotechnology, Shanghai, China) to generate the Flag-tagged expression Sec63, SIL1, GRP170, and BiP (24–26). Sec62 was known as an constructs as follows: a hamster Flag-Sec62 (EcoR I/Xho I), a hamster Flag-PERK (EcoR I/Xho I), a porcine Flag-ATF6 (EcoR I/Xho I), a porcine autophagy receptor, which delivers the ER-selective components Flag-PERK (Sal I/Xba I), a hamster Flag-IRE1a (Sal I/Xba I), a hamster to autophagosome–lysosome for degradation (27, 28). This pro- Flag-ATF6 (Xho I/Spe I), and a porcine Flag-IRE1a (Xho I/Spe I). All tein, therefore, might be a critical molecular component in plasmid constructs were analyzed and verified through DNA sequencing. maintenance and recovery of ER homeostasis (26, 28). We pre- The luciferase reporter plasmids pGL4–NF-kB–luc, pGL4-IRF3-luc, http://www.jimmunol.org/ b viously reported that Sec62 is upregulated at 4–6 h postinfection pGL4–IFN- –luc, and pRL-TK were kindly provided by Prof. S. Xiao (30). The promoter sequences of NF-kB, IRF3, and IFN-b referred to the (h.p.i.) during FMDV infection, suggesting that Sec62 may be porcine genome. involved in FMDV growth/replication (29). Hence, it will be interesting to investigate the role of Sec62 in UPR and FMDV Western blot analysis proliferation. Cell lysates were prepared by adding 1 3 SDS-PAGE sample buffer to In this study, we used two different types of FMDV-permissive the cells and boiled at 100˚C for 5 min. Proteins were separated by SDS- cells, baby hamster kidney (BHK-21) cells and porcine kidney PAGE and transferred to polyvinylidenedifluoride (PVDF) membrane (Amersham, Piscataway, NJ). Membrane was blocked with 5% fat-free (PK-15) cells, to address whether FMDV induces UPR and the role milk at room temperature for 1 h and incubated with primary Ab over- by guest on October 7, 2021 of Sec62 in FMDV proliferation. Our study found that FMDV night at 4˚C, followed by incubation with HRP-conjugated secondary Ab infection activated PERK–eukaryotic translation initiation factor for 1 h. Signals were detected with ECL Plus Western blot detection re- 2a (eIF2a) and ATF6-signaling branches of UPR, whereas the agents (PerkinElmer, Waltham, MA). IRE1a–X-box binding protein-1 (XBP1) signaling was sup- Confocal microscopy pressed. Further study revealed that the expression of Sec62 was PK-15 and BHK-21 cells grown on coverslips were infected with FMDV at upregulated at the early times of infection but downregulated MOI of 5. After 5 h.p.i., cells were fixed with 4% paraformaldehyde and thereafter. Sec62 suppressed FMDV proliferation by upregulating permeabilized using 0.1% Triton X-100 in PBS. Cells were then blocked IRE1a–RIG-I–mediated antiviral innate immune responses. Al- with 5% BSA in PBS for 1 h, followed by overnight incubation with primary together, these results will be useful to understand FMDV path- Ab diluted in PBS containing 5% BSA (1:200) at 4˚C. Cells were washed ogenesis and host antiviral defense mechanisms against FMDV. three times with PBS, followed by 1 h incubation with FITC- or TRITC- conjugated secondary Ab diluted in 5% BSA (1:400) at room temperature. Following washes with PBS three times, nuclei were stained with DAPI (Beyotime) for 15 min at room temperature. Cells were visualized using a Materials and Methods laser-scanning confocal microscope (LSCM, Leica SP8; Leica Micro- Cells and viruses systems; Solms, Germany) at wavelengths of 405, 488, and 561 nm. PK-15 and BHK-21 cells were maintained in DMEM (Life Technologies, Nucleus/cytosol fractionation assay Carlsbad, CA) supplemented with 10% FBS (Sigma, St. Louis, MO) and 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen, Carlsbad, Cells were collected and subjected to nucleus and cytosol fractionation CA) at 37˚C with 5% CO2. FMDV serotype O strain O/BY/CHA/2010 using Nucleus/Cytosol Fractionation Kit (BioVision, Milpitas, CA), (GenBank accession no. JN998085.1) was obtained from the World Or- according to the protocol recommended by the manufacturer. The cy- ganization for Animal Health/China National Foot-and-Mouth Disease toplasmic and nuclear fractions were then subjected to Western blot Reference Laboratory (Lanzhou, Gansu, P.R. China). Propagation of analysis with indicated Abs. a-Tubulin and Lamin B1 were used as a FMDV was carried out in BHK-21 cells, and viral titers were determined cytoplasmic and a nuclear protein marker, respectively. by 50% tissue culture-infective dose (TCID50) assay using BHK-21 cells. FMDV infection was performed at a multiplicity of infection (MOI) of RNA extraction, RT-PCR, quantitative real-time 1. Mock-inoculated cells were included in a parallel experiment as nega- RT-PCR analysis tive control (NC). Total RNA isolated by TRIzol reagent (Invitrogen) was used to synthesize Abs and chemicals cDNA with Moloney murine leukemia virus reverse transcriptase (Invi- trogen) using oligo-dT, according to the manufacturer’s instructions. Abs against BiP (no. 3183), PERK (no. 3192), phospho-PERK (no. 3179), cDNAs were then amplified with Ex Taq polymerase (TaKaRa, Dalian, eIF2a (no. 2103), phospho-eIF2a (no. 3597), activating transcription China) by using specific primers (Table I). PCR products were resolved in factor 4 (ATF4) (no. 11815), CHOP (no. 5554), IRE1a (no. 3294), MAVS 1% agarose gel prestained with GelRed Nucleic Acid Gel Stain (Biotium, (no. 4983), RIG-I (no. 3743), phospho-IKKa/IKKb (no. 2078), phospho- Fremont, CA). To resolve the unspliced and spliced forms of XBP1, PCR IkBa (no. 9246), phospho-p65 (no. 3033), IRF3 (no. 4302), phospho-IRF3 products were resolved using 2.5% agarose gel. The Journal of Immunology 431

mRNA levels were quantified by an Applied Biosystems 7500 Real- experiments. Statistical analysis was carried out by unpaired student Time PCR system using a two-step amplification program with SYBR t test. The statistical significance was established at p , 0.05 (*) and Green PCR Master Mix (TaKaRa). The PCR was set up under the fol- p , 0.01 (**). lowing thermal cycling conditions: 95˚C for 5 min, followed by 45 cycles, with 1 cycle consisting of 95˚C for 15 s and 60˚C for 1 min. Values obtained for each mRNA were normalized to the GAPDH Results mRNA. mRNA levels were quantified by comparing the value of ex- FMDV induces ER stress and activates PERK-eIF2a pathway perimental samples to that of mock-infected samples or vector-transfected samples. All samples were run in triplicate, and the experiment was re- FMDV is a fast-replication RNA virus, as the viral protein peated three times. expression can be detected as early as 4 h.p.i. by Western blot analysis, and the cytopathic effect appears at 6 h.p.i. in FMDV- Plasmid transfection infected PK-15 and BHK-21 cells (data not shown). This PK-15 or BHK-21 cells were seeded in six-well plates or 24-well plates and biological characteristic suggests that cumulative viral protein grown to ∼80% confluency. Cells were transfected with the indicated synthesis could induce UPR. During ER stress, BiP binds to the plasmids using Lipofectamine 2000 (Invitrogen) according to the manu- overloaded or misfolded proteins present in the ER lumen and facturer’s manual. Twenty-four hours posttransfection, cells were infected with FMDV. Cells were harvested at the indicated times postinfection and dissociates from the three ER stress sensors PERK, ATF6, then subjected to Western blot analysis, quantitative real-time RT-PCR, or and IRE1a, effectively activating their respective UPR pathways luciferase reporter assay. The supernatants of culture medium were col- (16, 20, 21). Thus, BiP is the essential regulator of the UPR lected to determine viral titers by TCID50 assay. pathways; moreover, its synthesis is induced under conditions of RNA interference ER stress (32). To evaluate the induction of ER stress during FMDV infection, we first examined the induction of Bip protein. Downloaded from Small interfering RNAs (siRNAs) targeting to Sec62, ATF6, PERK, and PK-15 and BHK-21 cells were infected with FMDV for 0, 2, 4, 6, IRE1a and NC siRNA were synthesized by Gene Pharma (Shanghai, China). The sequences of siRNAs used in this study were as follows: porcine Sec62 8, and 10 h and subjected to Western blot analysis by using Bip siRNA, 59-CCAGCACAGUAGUGGAAAU-39, mouse Sec62 siRNA, 59- Ab. Cells treated with TG (ER stress inducer) were included in a GGAGGAUGACGACAAAGAU-39; porcine ATF6 siRNA, 59-GCAGAA- parallel experiment as positive control, and mock infection was CCUCAACCACUUU-39, mouse ATF6 siRNA, 59-GCCACCAGAAGUA- also included as NC. As shown in Fig. 1, TG treatment greatly UGGGUU-39; porcine PERK siRNA, 59-GCAGAUCACUAGUGAUUAU- 9 9 9 induced the expression of Bip in both PK-15 and BHK-21 cells; 3 , mouse PERK siRNA, 5 -CCUUGGUUUCAUCUAGCCU-3 ; porcine http://www.jimmunol.org/ IRE1a siRNA, 59-GGAGGUUAUCGACCUGGUU-39, mouse IRE1a similar to the positive control, the expression of BiP was increased siRNA, 59-GCGAGAAGCAGCAGACUUU-39; NC siRNA, 59-UUCUC- in a time-dependent manner following FMDV infection in both CGAACGUGUCACGU-39. Cells grown to 70% confluency were trans- cell types, especially in the rapid growth phase, when lots of viral fected with siRNA using Lipofectamine RNAiMAX (Invitrogen) proteins were accumulated (4–10 h.p.i.). Consistent with this according to the manufacturer instructions. Thirty-six hours posttransfection, cells were infected with FMDV. Cells were harvested at finding, mRNA level of BiP was also significantly increased by the indicated times postinfection and then subjected to downstream FMDV in a time-dependent manner in both PK-15 and BHK-21 applications. cells (Supplemental Fig. 1A). This result suggests that FMDV infection induces UPR during its rapid replication stage. TCID50 assay

Once UPR is activated, PERK protein dissociates from BiP and by guest on October 7, 2021 Recombinant plasmid- or siRNA-transfected PK-15 and BHK-21 cells were consequently activates itself by oligomerization and autophos- infected with FMDV. At specific time points of postinfection, culture 3 phorylation of the free luminal domain. The activated PERK causes medium of the cells were collected and centrifuged at 5000 g to remove a cell debris. Serial 10-fold dilutions of viral culture supernatant were translational attenuation by directly phosphorylating the eIF2 and prepared in FBS-free DMEM and added into 96-well plates. Eight rep- finally prevents further translational loading of the ER lumen licates were set for each dilution. A total of 100 ml of virus diluent was (16, 20, 33). Meanwhile, the phospho-eIF2a preferentially initiates added into each well. Subsequently, 100 ml of BHK-21 cell suspension in the translation of ATF4 because of the unique mRNA structure of 3 6 DMEM with 10% FBS was added into each well at 1.5 10 cells/ml ATF4 (34). ATF4 then migrates to the nucleus and promotes the and mixed. After incubation for 72 h at 37˚C under 5% CO the number 2, expression of UPR-stimulated genes, including proapoptotic pro- of wells with or without cytopathic effect was counted. The TCID50 was calculated by the Reed–Muench method (31). Each test was performed tein CHOP (35, 36). Thus, we examined whether the PERK-eIF2a three times. signaling is activated in response to FMDV infection. As we ex- Luciferase reporter assay pected, in mock-infected PK-15 and BHK-21 cells, there was no increase of phospho-PERK, phospho-eIF2a, ATF4, and CHOP PK-15 cells in 24-well plates were cotransfected with 0.02 mg of pRL-TK along with the time course. However, in FMDV-infected cells, the plasmid (as an internal control) and 0.1 mg of pGL4–NF-kB–luc, pGL4- a IRF3-luc, or pGL4–IFN-b–luc and the indicated expression plasmids. At phospho-PERK and phospho-eIF2 were detected at 4–10 h.p.i., 24 h posttransfection, cells were infected with FMDV and incubated for similar to those in cells treated with TG. Subsequently, the ex- 5 h. Cell lysates were prepared and analyzed for firefly and Renilla lu- pression of ATF4 and CHOP were increased (Fig. 1). The mRNA ciferase activity using a dual-luciferase reporter assay system (Promega, levels of ATF4 and CHOP were also increased following FMDV Madison, WI) according to the manufacturer’s instructions. Firefly lucif- infection (Supplemental Fig. 1B, 1C). These results demonstrate erase activity was normalized to Renilla luciferase activity. Data were a represented as means of at least three independent experiments with three that FMDV infection activates PERK-eIF2 signaling branch in biological replicates. both PK-15 and BHK-21 cells. ELISA FMDV infection induces ATF6 cleavage and nuclear translocation The levels of IFN-b and IL-6 in the cell culture medium were detected by using porcine IFN-b QuantiKine ELISA kit (R&D Systems, Minneapolis, ATF6 is the second sensor of UPR, which exists in the ER as a MN) and porcine IL-6 QuantiKine ELISA kit (Novatein Biosciences, 90-kDa glycosylated protein precursor. In response to ER stress, Woburn, MA) according to the manufacturer’s instructions. ATF6 is released from BiP and translocates to the Golgi apparatus. Statistical analysis ATF6 is then cleaved into a 50-kDa N-terminal fragment, which The band intensities of specific proteins were determined by densi- enters into nucleus as an active transcription factor, binds to the tometry using ImageJ software. Results were accounted for at least three promoter region together with an ER stress response element, independent experiments and presented as the mean 6 SD of triplicate and finally induces the expression of ER chaperones (22, 37). To 432 Sec62 PROTEIN IS INVOLVED IN ANTI-FMDV IMMUNE RESPONSE

FIGURE 1. FMDV infection induces UPR and activates PERK-eIF2a pathway. PK-15 cells and BHK- 21 cells were infected with FMDV (MOI = 1) or mock infected and harvested at 0, 2, 4, 6, 8, and 10 h.p.i. Cells treated with TG (100 nM for 5 or 10 h) were included as positive control. BiP, PERK, phospho- PERK, eIF2a, phospho-eIF2a, ATF4, CHOP, and FMDV structural proteins were analyzed with Western blotting. b-Actin was detected as internal control. Band intensities were determined by densitometry. Phospho-PERK and phospho-eIF2a were normalized to PERK and eIF2a, respectively; BiP, ATF4, and CHOP were normalized to b-actin band. Signal value at 0 h.p.i. in mock-infected group was set as 1. The fold change of corresponding proteins at indicated time points was shown in the table. Downloaded from

determine whether FMDV infection activates ATF6, PK-15 and BHK-21 cells. The cleavage of ATF6-P into ATF6-N was increased BHK-21 cells were infected with FMDV, and Western blot anal- by FMDV infection (1.2–1.9-fold in PK-15 cells and 1.4–2.8-fold in http://www.jimmunol.org/ ysis was performed using an Ab recognizing both the full-length BHK-21 cells), which was comparable to those by TG treatment and N-terminal fragment of ATF6. As shown in Fig. 2A, both the (1.9–2-fold in PK-15 cells and 2.8–3.0-fold in BHK-21 cells). Once 90-kDa ATF6 precursor (ATF6-P) and the 50-kDa N-terminal ATF6 ATF6 is activated by cleavage, it translocates into the nucleus. We fragment (ATF6-N) were detected in mock-infected PK-15 and thus further analyzed the ratio of ATF6-N in nucleus and cytoplasm

FIGURE 2. FMDV infection promotes ATF6 by guest on October 7, 2021 cleavage and nuclear translocation. (A) The cell samples in Fig. 1 were analyzed with Western blotting using Abs against ATF6 and viral proteins. b-Actin was detected as a loading control. The band intensities of ATF6-N were normalized to that of b-actin. The value at 0 h.p.i. in the mock-infected group was set as 1, and the fold change of ATF6-N at indicated time points was showninthetable.(B) PK-15 cells and BHK-21 cells were infected with FMDV or mock infected and harvested at 0, 5, and 10 h.p.i. Cells treated with TG (100 nM for 10 h) were included as positive control. Cells were lysed and fraction- ated into cytoplasm fraction and nuclei fraction. ATF6 in both fractions were assessed with Western blotting. Lamin B1 was detected as loading control of the nuclei fraction, and a-tubulin was detected as loading control of the cytoplasm fraction. The band intensities of ATF6-N were normalized to that of Lamin B1. The ratio of ATF6-N in nuclei at each time point is indicated in the table. (C)PK-15 and BHK-21 cells were infected with FMDV (MOI = 5). Mock-infected cells and TG-treated cells were included in a parallel experiment as NC and positive control. Cells were fixed at 5 h.p.i. and subjected to immunostaining. Cells were then imaged using confocal microscopy with a 1003 oil-immersion objective lens. The blue, green, and red fluorescence represent the nuclei, ATF6 protein, and FMDV, respectively. The Journal of Immunology 433 by nuclei and cytoplasm fractionation. As shown in Fig. 2B, phospho-IRE1a were kept in steady level in mock-infected cells, only 14–20% of ATF6-N was detected in the nuclei fraction whereas TG treatment greatly enhanced the levels of phospho- of mock-infected PK-15 cells, but it has been increased up to IRE1a in both cell types. Interestingly, the levels of both total 50–63% by FMDV infection, which was comparable to those in IRE1a and phospho-IRE1a were gradually decreased following TG-stimulated cells. Similar results were also observed in FMDV infection in a time-dependent manner. However, it was BHK-21 cells, as 10–11% of ATF6-N in the nuclei fraction noted that the level of IRE1a mRNA was gradually upregulated in was increased up to 56–68% by FMDV infection. To visualize response to FMDV infection (Supplemental Fig. 1D), which the translocation of ATF6 into nucleus, immunostaining was suggests that the decrease of IRE1a protein by FMDV infection is performed. As shown in Fig. 2C, ATF6 was mainly distributed in not due to transcriptional inhibition. We next measured the splicing the cytoplasm in mock-infected cells, but strong nuclear signals of of XBP1 mRNA by RT-PCR (Table I). As shown in Fig. 3B, TG ATF6 were observed in FMDV-infected cells, comparable to TG treatment induced the splicing of XBP1 mRNA, but FMDV in- treatment. Taken together, these results demonstrate that FMDV fection had no effect on the splicing of XBP1s mRNA in both cell infection activates ATF6 by cleavage and promotes its nuclear types. Taken together with findings from IRE1a experiments, translocation. these data indicate that FMDV infection suppresses IRE1a-XBP1 signaling by downregulating IRE1a. To validate the above ob- FMDV infection suppresses IRE1a-XBP1 pathway servation, the mRNA levels of XBP1s-targeted UPR genes were In response to ER stress, IRE1a, another UPR sensor, is also examined by quantitative RT-PCR (Table I). As shown in Fig. 3C, activated by homo-oligomerization and autophosphorylation, mRNA expressions of XBP1, EDEM1, P58IPK, and ERdj4 were hence obtaining kinase and endonuclease activity (16). The en- significantly increased by TG treatment, but FMDV infection had donuclease activity of IRE1a is responsible for removing an in- no effect. These results confirm that FMDV infection suppresses Downloaded from tron from the XBP1 mRNA to produce a spliced XBP1 (XBP1s) the activation of IRE1a-XBP1 pathway, probably by downregu- mRNA. The XBP1s mRNA is then translated into a functional lation of IRE1a protein. active transcription factor, which promotes the expression of genes essential for an efficient protein folding/maturation as well as the ER stress response is not favorable for FMDV proliferation components for ER-associated degradation (38–40). We next ex- Because we found that FMDV positively and negatively regulates amined whether IRE1a is phosphorylated by FMDV in PK-15 and UPR branches, we next investigated the effect of UPR on FMDV http://www.jimmunol.org/ BHK-21 cells. As shown in Fig. 3A, the levels of IRE1a and proliferation. ER stress was first induced by TG in PK-15 and by guest on October 7, 2021

FIGURE 3. FMDV infection suppresses IRE1a- XBP1 pathway. (A) The cell samples in Fig. 1 were analyzed with Western blotting using Abs against phospho-IRE1a, IRE1a, and viral proteins. b-Actin was detected as a loading control. The band intensities of IRE1a and phospho-IRE1a were normalized to that of b-actin. The value at 0 h.p.i. in the mock-infected group was set as 1. The fold change of proteins at indicated time points was shown in the table. (B) PK-15 and BHK-21 cells were mock infected or infected with FMDV and harvested at indicated time points. Cells treated with TG were included as positive control. Total RNA was extracted and applied to RT- PCR using primers speciﬁc for the indicated genes. The PCR products were resolved using 1% agarose gel electrophoresis, except for XBP1u/XBP1s, for which 2.5% agarose gel was used. (C) Cells were infected with FMDV or mock infected for 4 or 8 h or treated with TG for 5 h. Total RNA was extracted and subjected to quantitative real-time RT-PCR analysis. Fold inductions of speciﬁc genes were calculated using GAPDH as internal references and normalized to the mock-infected samples at 4 h.p.i. The above data are representative of three independent experiments (**p , 0.01). 434 Sec62 PROTEIN IS INVOLVED IN ANTI-FMDV IMMUNE RESPONSE

Table I. Primers used in this study

Primer Name Sequence (59-39) Purpose pIRE1a-F 59-AGGACGTGAGTGACCGGATA-39 PCR for detecting porcine IRE1a gene pIRE1a-R 59-TCGTGGTAGTAGGGCTGGAA-39 hIRE1a-F 59-CGGGAGAACATCACTGTCCC-39 PCR for detecting hamster IRE1a gene hIRE1a-R 59-GCTCGTGCCAGTAGTAGGTC-39 pXBP1-F 59-GTTGAGAACCAGGAGTTAAGACAG-39 PCR for detecting porcine XBP1 gene pXBP1-R 59-CAGAGGGTATCTCTAAGACTAGGG-39 hXBP1-F 59-CACCTGAGCCCCGAGGAG-39 PCR for detecting hamster XBP1 gene hXBP1-R 59-TTAGTTCATTAATGGCTTCCAGC-39 3D-F 59-TTCGGCCTTTGATGCTAACCACTG-39 PCR for detecting FMDV 3D gene 3D-R 59-GCATCCCGCCCTCAACAACAAT-39 b-actin–F 59-CGGCATCCACGAAACTAC-39 PCR for detecting porcine and hamster b-actin gene b-actin–R 59-ATCTTCATCGTGCTGGGCG-39 pXBP1-F (q) 59-AGCAACAAGTGGTAGATTTG-39 qPCR for detecting porcine XBP1 gene pXBP1-R (q) 59-GTGTTTTCTCTCGTAAAAGCTGA-39 hXBP1-F (q) 59-GACAGAGAGTCAAACTAACGTGG-39 qPCR for detecting hamster XBP1 gene hXBP1-R (q) 59-GTCCAGCAGGCAAGAAGGT-39 EDEM1-F (q) 59-GGGGCATGTTCGTCTTCGG-39 qPCR for detecting porcine and hamster EDEM1 gene EDEM1-R (q) 59-CGGCAGT AGATGGGGTTGAG-39 ERdj4-F (q) 59-CTCCACAGTCAGTTTTCGTCTT-39 qPCR for detecting porcine and hamster ERdj4 gene ERdj4-R (q) 59-GGCCTTTTTGATTTGTCGCTC-39 Downloaded from pP58IPK-F (q) 59-GGTGCTGAATGTGGAGTAAAT-39 qPCR for detecting porcine P58IPK gene pP58IPK-R (q) 59-GCATGAAACTGAGATAAAGCG-39 hP58IPK-F (q) 59-TCCTGGTGGACCTGCAGTACG-39 qPCR for detecting hamster P58IPK gene hP58IPK-R (q) 59-CTGCGAGTAATTTCTTCCCC-39 pRIG-I–F (q) 59-CTTGCAAGAGGAATACCACTTA AACCCAGAGAC-39 qPCR for detecting porcine RIG-I gene pRIG-I–R (q) 59-TTCTGCCACGTCCAGTCAAT ATGCCAGGTTT-39 59-CTGCTTCTGGTGATGGCTACTG-39 pIL-6-F (q) qPCR for detecting porcine IL-6 gene http://www.jimmunol.org/ pIL-6-R (q) 59-GGCATCACCTTTGGCATCTT-39 pIFN-b–F (q) 59-GCTAACAAG TGCATCCTCCAAA-39 qPCR for detecting porcine IFN-b gene pIFN-b–R (q) 59-AGCACATCATAGCTCATGGAAAGA-39 pGAPDH-F (q) 59-CAACTACATGGTCTACATGTTC-39 qPCR for detecting porcine GAPDH gene pGAPDH-R (q) 59-CTCGCTCCTGGAAGATG-39 hGAPDH-F (q) 59-AAGAAGGTGGTGAAGCAGGCATC-39 qPCR for detecting hamster GAPDH gene hGAPDH-R (q) 59-CGGCATCGAAGGTGGAAGAGTG-39 qPCR, quantitative PCR. by guest on October 7, 2021 BHK-21 cells and then infected with FMDV. DMSO-pretreated effect on FMDV proliferation, although FMDV infection induces cells followed by FMDV infection were included as control the activation of these pathways. group. The structural proteins of FMDV were examined by Surprisingly, the overexpression of IRE1a greatly suppressed Western blot to monitor the proliferation of FMDV. As shown in the expression of FMDV proteins, and depletion of IRE1a in- Fig. 4A, TG pretreatment induced UPR in both cell types, es- creased FMDV protein expression in PK-15 cells (Fig. 4D, left pecially in PK-15 cells, as evidenced by increased expression of panels). In consistence, virus yields were also signiﬁcantly sup- UPR marker protein BiP. Interestingly, the expression of viral pressed by overexpression of IRE1a but greatly promoted by proteins was greatly decreasedbyTGpretreatmentinPK-15 depletion of IRE1a (Supplemental Fig. 3, left panels). These cells compared with those in DMSO-treated cells. In BHK-21 ﬁndings suggest that IRE1a acts as an antiviral regulator for cells, the inhibitory effect on virus proliferation by TG treatment FMDV in PK-15 cells. Because IRE1a-dependent RNA decay was not as notable as those in PK-15 cells. This observation stimulates RIG-I signaling and induces the expression of IFN and reveals that FMDV proliferation is negatively regulated by UPR. antiviral responses (41, 42), IRE1a might suppress FMDV pro- To determine the molecular mechanisms by which FMDV liferation via initiation of antiviral RIG-I signaling in PK-15 cells. proliferation is regulated by UPR signaling branch, the effects of Interestingly, the proliferation of FMDV was not affected by either overexpression and knockdown of three UPR sensors (PERK, overexpression or depletion of IRE1a in BHK-21 cells (Fig. 4D, ATF6, and IRE1a) on FMDV proliferation were examined by Supplemental Fig. 3, right panels). A previous study shows that Western blot analysis (Fig. 4B–D). To ensure the overexpression RIG-I signaling pathway is defective in BHK-21 cells (43). Thus, or knockdown of these sensors is properly integrated in the met- we speculated that the lack of inhibitory effect of IRE1a on abolic networks of either PK-15 or BHK-21 cells, the recombinant FMDV proliferation might be attributed to the defect in RIG-I in constructs of PERK, ATF6, or IRE1a were cloned from either BHK-21 cells. In view of the antiviral effect of IRE1a in PK-15 porcine or hamster cells, and siRNAs were designed according cells and decrease of IRE1a by FMDV infection, we suggest that to either porcine or hamster genes. It is worth noting that over- the inhibition of IRE1a by FMDV might be an important immune expression or knockdown of these components did not affect evasion mechanism against antiviral effects of IRE1a. cell viability (Supplemental Fig. 2). As shown in Fig. 4B, 4C, the expression levels of viral proteins were not affected by either Sec62 suppresses FMDV proliferation and promotes a overexpression or knockdown of PERK/ATF6 in both cell types IRE1 phosphorylation compared with those in vector- or NC siRNA-transfected cells. Sec62 is responsible for importing nascent proteins across the ER FMDV yields in the culture medium were also not affected, as membrane in complex with other Sec family proteins, such as evidenced by TCID50 assay (Supplemental Fig. 3). These results Sec61 and Sce63. Sec62 is also an ER-resident autophagy receptor demonstrate that the activation of PERK and ATF6 has no obvious that delivers selected ER constituents to the autophagy-lysosomal The Journal of Immunology 435 Downloaded from http://www.jimmunol.org/

FIGURE 4. IRE1a signaling suppresses FMDV proliferation. (A) Initiation of UPR by TG treatment does not support FMDV proliferation, especially in PK-15 cells. PK-15 cells and BHK-21 cells were pretreated with DMSO or TG (100 nM), followed by FMDV infection. DMSO or TG was maintained in the culture medium during FMDV infection. Cells were harvested at the indicated time points, and the expression levels of BiP and viral proteins were b

analyzed with Western blot. -Actin was used as a loading control. The band intensities of FMDV proteins were determined by densitometry and nor- by guest on October 7, 2021 malized to the intensities of b-actin bands. The value at 4 h.p.i. in DMSO+FMDV–treated group was set as 1. The fold change of FMDV proteins at indicated time points was shown in the table. (B–D) IRE1a is involved in the suppression of FMDV proliferation in PK-15 cells. PK-15 cells and BHK-21 cells were transfected with cognate Flag-PERK, Flag-ATF6, Flag-IRE1a, or vector for 24 h, or transfected with cognate PERK/ATF6/IRE1a siRNA or NC siRNA for 36 h, followed by FMDV infection. Cell lysates were harvested at indicated time points and subjected to Western blot analysis. The fold change of FMDV proteins was shown in the table as described above in (A). pathway and contributes to reestablishment of ER homeostasis (Supplemental Fig. 4A). However, in BHK-21 cells, neither over- (26, 28). It has been reported that Sec62 interacts with BiP (44), expression nor depletion of Sec62 had an effect on FMDV prolif- and we also found that FMDV infection increases the expression eration (Fig. 5B, 5C, right panels, Supplemental Fig. 4B). These of Sec62 (29). In this study, we found that Sec62 protein was results suggest that Sec62 plays an antiviral role in PK-15 cells. slightly upregulated at the early stages of infection (2–6 h.p.i.) but We next asked whether Sec62 regulates UPR branches during greatly decreased at 8–10 h.p.i. in both cell types (Fig. 5A). TG FMDV infection. Sec62 was overexpressed in PK-15 and BHK-21 treatment also upregulated Sec62 expression by 1.4–1.8-fold cells, followed with FMDV infection, and Western blot analysis (Fig. 5A), suggesting that UPR is involved in Sec62 expression. was performed to examine the three UPR branches signaling. As To assess the role of Sec62 on FMDV proliferation, porcine- shown in Fig. 5D, the levels of BiP, phospho-eIF2a, and ATF6-N derived Sec62 with Myc tag and hamster-derived Sec62 with were not changed by overexpression of Sec62 compared with Flag tag were constructed. PK-15 and BHK-21 cells were trans- those in vector-transfected cells. However, the level of IRE1a was fected with porcine-derived Myc-Sec62 or hamster-derived Flag- stabilized by overexpression of Sec62 in both cell types compared Sec62, respectively, and infected with FMDV. The successful with the decreased levels of IRE1a along with infection time expression of Sec62 was confirmed by Western blotting using anti- course in vector-transfected cells. In addition, phosphorylation of Myc or anti-Flag Ab (Fig. 5B). Meanwhile, Sec62 was knocked IRE1a was enhanced in Sec62-overexpressed cells. These results down by transfecting siRNA targeting to porcine Sec62 mRNA reveal that Sec62 is involved in the maintenance of the steady or hamster Sec62 mRNA, and cells were then infected with FMDV. level and activation of IRE1a during FMDV infection. The knockdown efficiency was confirmed by Western blotting using anti-Sec62 Ab (Fig. 5C). As shown in Fig. 5B, 5C, the over- Sec62 promotes innate immune responses against FMDV expression of Sec62 greatly suppressed FMDV protein synthesis Recent studies show that the degradation of host mRNAs via (Fig. 5B, left panel), whereas FMDV protein expression was in- regulated IRE1-dependent decay (RIDD) activates the antiviral creased by Sec62 knockdown in PK-15 cells (Fig. 5C, left panel). RNA helicase RIG-I (42, 45). This newly delineated pathway is Consistent with these findings, virus yields were also decreased by termed IRE1a-RIDD–RIG-I (42). Based on the findings that overexpression of Sec62 and were increased by depletion of Sec62 IRE1a activates RIG-I signaling by cleaving both viral and host 436 Sec62 PROTEIN IS INVOLVED IN ANTI-FMDV IMMUNE RESPONSE Downloaded from

FIGURE 5. Sec62 suppresses FMDV proliferation by stabilization and promotion of IRE1a activation. (A) PK-15 cells and BHK-21 cells were mock infected or infected with FMDV. TG-treated cells were included as control group. Sec62 and viral proteins were analyzed with Western blot. b-Actin served as a loading control. The band intensities of Sec62 were determined by densitometry and normalized to the intensities of b-actin bands. The value at 0 h.p.i. http://www.jimmunol.org/ in the mock-infected group was set as 1, and the fold change of Sec62 at indicated time points was shown in the table. (B and C) Sec62 is involved in the suppression of FMDV proliferation in PK-15 cells. PK-15 cells or BHK-21 cells were transfected with cognate Myc-Sec62– or Flag-Sec62–expressing plasmid or vector for 24 h (B) or transfected with NC siRNA or cognate Sec62 siRNA for 36 h (C), followed by FMDV infection. Cell lysates were harvested at indicated time points and subjected to Western blot analysis. The fold change of FMDV proteins was shown in the table. (D) Sec62 recovers the levels of IRE1a and promotes IRE1a phosphorylation in both PK-15 cells and BHK-21 cells. Cells were transfected with the vector or cognate Sec62- expressing plasmid for 24 h, followed by FMDV infection. Cells were collected at indicated time points. Myc, BiP, phospho-eIF2a, eIF2a, ATF6, and phospho-IRE1a, viral proteins, and b-actin were detected by Western blot analysis. The band intensities of corresponding proteins were determined by densitometry and normalized to that of eIF2a or b-actin. The value at 0 h.p.i. in the mock-infected group was set as 1. The fold change of proteins at indicated time points was shown in the table. by guest on October 7, 2021 mRNA (41, 42) and that IRE1a plays an antiviral role in RIG-I– (Fig. 6B, left panels). In contrast, silencing of Sec62 greatly reduced functional PK-15 cells but not in RIG-I–defective BHK-21 cells, the FMDV-stimulated expression of RIG-I, IL-6, and IFN-b we speculated that IRE1a might trigger RIG-I–IFN-b signaling (Fig. 6B, right panels). To validate these results, the secretion of during FMDV infection. We next investigated whether Sec62 IFN-b and IL-6 protein in the PK-15 cell culture medium was regulates RIG-I–IFN-b signaling by regulating IRE1a. Hence, we measured by ELISA. The overexpression of Sec62 promoted the examined the activation of NF-kB, IRF3, and IFN-b promoter by secretion of these cytokines (left panels), and silencing of this luciferase reporter systems in Sec62-overexpressing cells. PK-15 protein decreased their secretion (right panels) (Fig. 6C, 6D). Al- and BHK-21 cells were cotransfected with Sec62 and pGL4– together, these results suggest that Sec62 positively regulates the NF-kB–luc, Sec62 and pGL4-IRF3-luc, or Sec62 and pGL4–IFN- innate immune responses against FMDV infection in PK-15 cells. b–luc, respectively, as well as pRL-TK plasmid as an internal a control, followed with FMDV infection. Vector- and reporter Positive regulation of IRE1 -RIDD–RIG-I pathway by Sec62 plasmid-transfected group was included in the parallel experiment during FMDV infection as control. Cells were harvested at 5 h.p.i. and analyzed for firefly Based on the fact that Sec62 induces the expression of antiviral and Renilla luciferase activity. As shown in Fig. 6A, FMDV in- cytokines, we next examined the regulation of RIG-I and its fection slightly activated transcription factor NF-kB and IRF3, downstream signaling by Sec62. The levels of RIG-I, p65, and IFN-b promoter in both cell types compared with mock in- phospho-p65, and phospho-IRF3 were analyzed by Western blot in fection group. As expected, overexpression of Sec62 significantly Sec62 overexpression or knockdown cells. As shown in Fig. 7A, activated both NF-kB and IRF3 as well as IFN-b promoter only in the level of RIG-I was greatly increased by overexpression of mock- and FMDV-infected PK-15 cells (Fig. 6A, left panels) but Sec62 compared with that in vector-transfected cells in FMDV- not in BHK-21 cells (Fig. 6A, right panels). These observations infected PK-15 cells. In addition, the degradation of p65 trig- confirm that Sec62 indeed stimulates innate antiviral immune gered by FMDV infection in vector-transfected cells was prevented signaling in PK-15 cells, independent of virus infection. in Sec62-overexpressing cells. Accordingly, phosphorylation of p65 Because Sec62 stimulates the transcription factors of IFN-b only and IRF3 was increased by overexpression of Sec62 (Fig. 7A, left in PK-15 cells, we next analyzed the effect of Sec62 on triggering panel). Consistent with the above results, silencing of Sec62 de- endogenous transcription of RIG-I, IFN-b, and IL-6 in PK-15 cells creased the levels of phospho-p65 and phospho-IRF3 compared by quantitative real-time RT-PCR (Table I). As shown in Fig. 6B, with those in NC siRNA-transfected cells (Fig. 7B, left panel). It overexpression of Sec62 significantly upregulated the transcription was worth to note that in both NC siRNA and Sec62 siRNA- of RIG-I, IFN-b, and IL-6 in both mock- and FMDV-infected cells transfected PK-15 cells, along with infection course, FMDV not The Journal of Immunology 437 Downloaded from http://www.jimmunol.org/

FIGURE 6. Sec62 positively regulates the expression of type I IFN and cytokines. (A) Sec62 positively regulates NF-kB, IRF3, and IFN-b promoter by guest on October 7, 2021 activity in PK-15 cells. PK-15 cells and BHK-21 cells were cotransfected with myc-Sec62 or vector (1 mg) combined with pGL4–NF-kB–luc (0.1 mg), pGL4-IRF3-luc (0.1 mg), or pGL4–IFN-b–luc (0.1 mg), with pRL-TK plasmid (0.02 mg) as an internal control to normalize the transfection efficiency. At 24 h after transfection, cells were infected with FMDV or mock infected for 5 h. The dual-specific luciferase assay kit was used to analyze the luciferase activities of firefly and Renilla. The data represent the means and SD from three independent experiments (**p , 0.01). (B) Sec62 enhances the transcription of RIG-I, IL-6, and IFN-b in PK-15 cells. PK-15 cells were transfected with either Myc-Sec62 plasmid/vector for 24 h or transfected with NC siRNA/Sec62 siRNA for 36 h, followed with FMDV infection or mock infected for 5 h. Total RNA was extracted. RIG-I, IFN-b, and IL-6 mRNA were detected with quantitative real-time RT-PCR. Data are representative of three independent experiments (*p , 0.05, **p , 0.01). (C and D) Sec62 stimulates the secretion of IFN-b (C) and IL-6 (D) in PK-15 cells. PK-15 cells were transfected with either Myc-Sec62 plasmid/vector for 24 h or transfected with NC siRNA/Sec62 siRNA for 36 h, followed with FMDV infection or mock infected for 5 h. The culture medium was collected to quantify the secretion of IFN- b and IL-6 by ELISA. Data are representative of three independent experiments (*p , 0.05, **p , 0.01). only reduced the levels of phospho-IRE1a but also decreased the cells, the levels of MAVS and TRAF3 were increased, and the levels level of RIG-1; moreover, FMDV infection resulted in cleavage of of phospho-TBK1, phospho-IRF3, and phospho-IRF7 were also p65 into smaller bands (Fig. 7B, left panel). In contrast to the enhanced in both mock-infected and FMDV-infected cells com- observations in FMDV-infected PK-15 cells, in FMDV-infected pared with those in vector-transfected cells (Fig. 8A). In contrast, BHK-21 cells, no obvious change of RIG-I, p65, phospho-p65, the levels of MAVS, TRAF3, phospho-TBK1, phospho-IRF3, and and phospho-IRF3 was observed either by overexpression or si- phospho-IRF7werereducedbydepletionofSec62inFMDV- lencing of Sec62 compared with those in control group (Fig. 7A, 7B, infected PK-15 cells (Fig. 8B). These results demonstrate that right panels). It was interesting to note that knockdown of Sec62 FMDV infection activates IRF3 and IRF7, and Sec62 augments further reduced the levels of phospho-IRE1a in both PK-15 and this activation. Examination of NF-кB–signaling molecules BHK-21 cells compared with those in NC siRNA-transfected cells showed that FMDV infection stimulated the phosphorylation (Fig. 7B), further confirming the positive role of Sec62 on the of IKKa/b and IкBa, promoted the degradation of IкBa, and phosphorylation of IRE1a. In all, these results suggest that Sec62 stimulated the phosphorylation of p65, although p65 degradation positively regulates NF-кB/IRF3 signaling in FMDV-infected PK-15 was observed (Fig. 8). In Sec62-overexpressing cells, more cells via maintenance of the protein levels of IRE1a and RIG-I. TRAF6, phospho-IKKa/b, phospho-IкBa, p65, and phospho- To further dissect the above observation, the signaling molecules p65 were detected in both mock-infected and FMDV-infected involved in IRF3/IRF7 and NF-кB pathway were examined in cells compared with those in vector-transfected cells (Fig. 8A), FMDV-infected PK-15 cells by either overexpression or knockdown demonstrating that Sec62 also augments the FMDV-stimulated of Sec62. As shown in Fig. 8, FMDV infection stimulated the NF-кB signaling. When Sec62 was knocked down, the levels of phosphorylation of TBK1, IRF3, and IRF7. In Sec62-overexpressing TRAF6, phospho-IKKa/b, phospho-IкBa, and phospho-p65 were 438 Sec62 PROTEIN IS INVOLVED IN ANTI-FMDV IMMUNE RESPONSE

FIGURE 7. Sec62 recovers the levels of RIG-I and p65 as well as promotes phosphorylation of p65 and IRF3 in PK-15 cells. PK-15 cells and BHK-21 cells were transfected with vector or cognate Myc-Sec62/ Flag-Sec62 plasmid for 24 h (A) or transfected with control siRNA (NC) or cognate Sec62 siRNA for 36 h (B), followed by FMDV infection. Cells were harvested at 0, 2, 4, 6, 8, and 10 h.p.i. Myc-Sec62, Flag-Sec62, phospho-IRE1a, RIG-I, p65, phospho-p65, phospho-

IRF3, and b-actin expression levels were analyzed Downloaded from using Western blot. The band intensities of the indicated proteins were normalized to that of b-actin. The value at 0 h.p.i. in vector+FMDV group or in NC siRNA+FMDV group was set as 1. The fold change of proteins at indicated time points was shown in the table. http://www.jimmunol.org/ by guest on October 7, 2021

decreased in FMDV-infected cells (Fig. 8B), further conﬁrming suggesting that UPR plays an antiviral role on FMDV infection. the positive regulation of NF-кB signaling by Sec62. Taken to- Overexpression or knockdown of three UPR sensors shows that gether, these results demonstrate that Sec62 performs the antiviral PERK and ATF6 have no effect on FMDV proliferation, but function during FMDV infection by positively regulating both IRE1a suppresses FMDV proliferation in PK-15 cells but not in IRF3/IRF7 signaling and NF-kB signaling (Fig. 9). BHK-21 cells. FMDV infection decreased the level of Sec62, which is a component of ER translocation machinery at the late Discussion stage of infection. Furthermore, downregulated expression of UPR is one of the important cellular responses to cope with invasive IRE1a and its phosphorylation was recovered by the over- viruses, and it is also regarded as one of the antiviral responses that expression of Sec62 in both FMDV-infected PK-15 cells and helps cells eliminate pathogens via shutting off protein synthesis or BHK-21 cells. Hence, FMDV replication was inhibited by Sec62 by inducing IFN signaling (46–48). For instance, transmissible in PK-15 cells but not in BHK-21 cells. The fact that PK-15 cells gastroenteritis virus infection activates all three UPR pathways, harbor RIG-I signaling but BHK-21 cells are defect in RIG-I and the activation of PERK-eIF2a branch negatively regulates suggests the RIG-I signaling as an antiviral effector of IRE1a- viral replication by suppressing protein translation and promoting and Sec62-mediated inhibition of FMDV replication. IRE1a en- type I IFN production (49). It has been found that UPR is selec- donuclease cleaves mRNA and activates RIG-I through the RIDD, tively activated and hijacked by various viruses to facilitate their resulting in the activation of NF-кB under ER stress (42). Hence, own replication. For example, hepatitis C virus suppresses the we hypothesize that IRE1a plays an antiviral function by cleaving IRE1-XBP1 pathway to stimulate the synthesis of viral proteins mRNA and activates the RIG-I as well as its downstream effector (50), and hepatitis B virus activates ATF6 and IRE1-XBP1 path- molecules. Sec62 plays an antiviral role by stabilizing the levels of ways to promote viral genome replication (51). IRE1a. In this study, we investigated the role of UPR on FMDV in- A previous study showed that RIG-I suppressed FMDV repli- fectioninPK-15andBHK-21cells.FMDV-triggeredUPR,which cation, and FMDV infection in turn decreased the level of RIG-I is characterized by the phosphorylation of PERK and eIF2a, (52–54). In this study, we indeed observed that FMDV infection increased expression of ATF4, CHOP, BiP, and nuclear transloca- decreased the level of RIG-I in PK-15 cells and overexpression of tion of ATF6. However, FMDV inhibited IRE1a-XBP1 signaling by Sec62 reversed FMDV-induced inhibition of RIG-I. Thus, in ad- reducing the level of IRE1a. Induction of UPR by pharmacological dition to the stabilization of the level of IRE1a and activation treatment inhibited virus proliferation, especially in PK-15 cells, of IRE1a signaling, Sec62 also involves in regulation of RIG-I The Journal of Immunology 439 Downloaded from

FIGURE 8. Sec62 positively regulates IRF3 and NF-kB upstream sig- http://www.jimmunol.org/ naling in PK-15 cells. PK-15 cells were transfected with Myc-Sec62 plasmid or vector for 24 h (A) or transfected with NC siRNA or Sec62 siRNA for 36 h (B), followed with FMDV infection or mock infection. Cells were collected at 5 h.p.i. Sec62, FMDV proteins, MAVS, TRAF3, phospho-TBK1, phospho-IRF3, IRF3, phospho-IRF7, TRAF6, phospho- a b k a k a IKK / ,I B , phospho-I B , p65, and phospho-p65 were detected using FIGURE 9. Schematic picture of the role of Sec62 in FMDV infection b Western blot analysis. -Actin was used as a loading control. The band via regulation of IRE1a–RIG-I signaling. FMDV infection induces b intensities of the indicated proteins were normalized to that of -actin. The UPR, resulting in activation of PERK and ATF6 pathway. However, value of vector group or NC siRNA group was set as 1. The fold change of IRE1a activity is suppressed by unknown mechanisms. Sec62 positively by guest on October 7, 2021 proteins at indicated time points was shown in the table. regulates IRE1a and promotes IRE1a–RIG-I antiviral signaling. In turn, FMDV decreases the protein level of Sec62 and IRE1a at late infection signaling by recovering the level of RIG-I. Moreover, over- times. expression of Sec62 promoted the levels of MAVS and TRAF3, as well as prevented the degradation of p65 during FMDV infection, of antiviral IFN and cytokines (Fig. 9). Therefore, Sec62 inhibits which also contribute to antiviral IFN signaling. Our studies thus FMDV proliferation via regulation of IRE1a–RIG-I pathway. unveil the novel functions of Sec62 in UPR and innate immune Because Sec62 is an important antiviral regulator of FMDV in- signaling pathways. fection, FMDV-mediated inhibition of Sec62, IRE1a, and RIG-I Sec62 is an integral membrane protein located in the rough ER might be a critical immune evasion mechanism of FMDV to en- that functions as a central component of the protein translocation sure their proliferation in host cells. We thus proposed in this apparatus of the ER membrane. Together with Sec61 and Sec63, paper that FMDV-mediated inhibition of Sec62, IRE1a, and RIG-I Sec62 contributes to translocation of posttranslational protein might be a good target to develop therapeutic strategies to combat into the ER lumen or backward transport of ER proteins that FMDV infection or broad-spectrum antiviral therapies. are subjected to the ubiquitin–proteasome–dependent pathway (26, 28), thereby attenuating ER stress by delivering proteins to Acknowledgments their right place. Previous studies showed that modulation of We greatly appreciate Dr. Shaobo Xiao at Huazhong Agricultural University Sec61-mediated cotranslational translocation selectively im- for kindly providing the luciferase reporter plasmids pGL4–NF-kB–luc, paired glycoprotein proteostasis of influenza virus, HIV, and pGL4-IRF3-luc, pGL4–IFN-b–luc, and pRL-TK plasmid and Prof. Jae dengue virus and further led to the inhibition of viral growth and Hyang Lim at Ewha Women’s University at South Korea for helpful sci- infectivity (55). Despite its role in positive regulation of pro- entific and editorial advice. teostasis and virus proliferation by Sec family protein, the un- derlying molecular mechanisms by which Sec62 suppresses Disclosures FMDV proliferation as well as promotes the levels of IRE1a, The authors have no financial conflicts of interest. RIG-I, MAVS, TRAF3, and p65 are not understood yet and need to be further investigated. References Taken together, we report in this study that Sec62 stabilizes the 1. Domingo, E., E. Baranowski, C. Escarmı´s, and F. Sobrino. 2002. Foot-and- level of IRE1a and stimulates the phosphorylation of IRE1a, mouth disease virus. Comp. Immunol. Microbiol. Infect. Dis. 25: 297–308. thereby maintaining the steady levels of RIG-I and positively 2. Mason, P. W., M. J. Grubman, and B. Baxt. 2003. Molecular basis of pathogenesis of FMDV. Virus Res. 91: 9–32. regulating its downstream signaling, resulting in restoring the 3. Han, S. C., H. C. Guo, and S. Q. Sun. 2015. Three-dimensional structure of foot- levels of MAVS, TRAF3, and p65 and promoting the production and-mouth disease virus and its biological functions. Arch. Virol. 160: 1–16. 440 Sec62 PROTEIN IS INVOLVED IN ANTI-FMDV IMMUNE RESPONSE

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