Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro (original) (raw)
HCV infection induces complete UPR-autophagy. There is growing evidence showing that various host cellular responses, including autophagy, innate immunity, and apoptosis, are affected or activated by HCV in vitro (24, 25, 42–44). Here, we aimed to investigate the autophagic response upon HCV JFH1 infection and its regulatory role in virus replication in a condition that mimics natural viral infection. First, we found that the phosphatidylethanolamine-conjugated form of microtubule-associated protein 1 light chain 3 β (LC3B-II), a hallmark of autophagosome formation, was highly activated at the acute infection phase (6–9 days after infection) (Figure 1A, left panel). A UPR-activated transcriptional factor, CCAAT/enhancer binding protein (c/EBP) homologous protein (CHOP) was also greatly upregulated in these acutely HCV-infected cells (Figure 1A, left panel), suggesting that autophagosome formation in HCV infection was associated with ER stress and UPR. Further analysis of the long-term-cultured HCV-infected cells revealed that LC3B-II and CHOP levels started to diminish when the cells entered the chronic phase (15–22 days after infection) (Figure 1A, left panel). Similarly, HCV infection also led to puncta formation of GFP-LC3–labeled vacuoles in most of Huh7 cells stably expressing GFP-LC3 (Figure 1A, middle panels) and the formation of GFP-LC3-II (Figure 1A, right panel), confirming that HCV infection indeed induces the formation of autophagosomes. These results also showed a close correlation of HCV viral protein expression with CHOP induction and autophagosome formation (Figure 1A, left panel).
Induction of UPR and autophagy by HCV infection. (A) Huh7 cells were inoculated with HCV at an MOI of 10, and then harvested at different times for Western blot analysis (left panel). The asterisk indicates the nonspecific band. HCV-infected Huh7/GFP-LC3 cells (MOI of 10) were fixed at 6 days after infection and analyzed by confocal microscopy (middle panels) and Western blotting (right panel). Scale bars: 10 μm. (B) HCV-infected Huh7/mRFP-GFP-LC3 cells (MOI of 10) were maintained for 6 days, treated with (+) or without (–) CQ (50 μM) for 6 hours, and assessed for subcellular localization of the indicated proteins. Scale bars: 10 μm. (C) HCV-infected cells as described in B were processed for immuno-TEM analysis. The larger boxed images represent enlargements of the smaller boxed insets. Another set of enlarged images is shown in Supplemental Figure 2A. The black arrows indicate LD-associated core and NS5A proteins, which were respectively immmunolabeled with 12-nm and 18-nm gold particles. N, nucleus; LD, lipid droplet.
To verify whether the HCV-induced autophagosome fuses with lysosome, we made use of a tandem reporter construct, mRFP-GFP-LC3 (45). The green fluorescence of this tandem autophagosome reporter is attenuated in the acidic pH lysosomal environment by lysosomal hydrolysis, while the mRFP is not. Therefore, the green fluorescent component of the composite yellow fluorescence from this mRFP-GFP-LC3 reporter is lost upon autophagosome fusion with lysosome, whereas the red fluorescence remains detectable. In the absence of an acidification inhibitor of lysosome degradation, chloroquine (CQ), remarkable red fluorescence signals were detected in HCV-infected cells (Figure 1B, top row). And the RFP-LC3–labeled puncta structures were also colocalized with a lysosome marker, lysosome-associated membrane protein 1 (LAMP1) (Figure 1B, top row). In sharp contrast, treatment with CQ greatly restored the expression of GFP and resulted in yellow color–labeled autophagosomes in HCV-infected cells (Figure 1B, bottom row).
To confirm that HCV infection induced autophagic activation, we performed transmission electron microscopy–based (TEM-based) ultrastructural analysis. Both initial- and late-stage autophagic vacuoles (AVi and AVd, respectively) were observed in HCV-infected cells, which were judged by the detection of lipid droplets (LDs) surrounded by immunogold-labeled core or NS5A (Figure 1C and Supplemental Figure 2A), whereas no apparent AVi or AVd was detected in mock-infected cells (Supplemental Figure 2B). Moreover, the AVi and AVd detected in HCV-infected cells were, respectively, labeled with LC3B and LAMP1 antibodies (Supplemental Figure 2C, left and right panels), in agreement with the earlier studies showing that LC3B and LAMP1 are localized on the autophagosome and autolysosome, respectively, in the autophagic process (46–48). Our results confirm again that the HCV-induced autophagic process involves formation of different stages of autophagic vacuoles.
Next, we found that the HCV-induced LC3B-II formation was increased when infected cells were treated with lysosomal protease inhibitors (E64 and pepstatin A), CQ, and a vacuolar ATPase inhibitor (bafilomycin A1 [BAF-A1]) (Supplemental Figure 2D), which are well-known acidification inhibitors shown to block the activity of the pH-dependent lysosomal proteases and to bring about accumulation of immature autolysosome (49, 50). Further study showed that treatment with CQ and BAF-A1 led to more accumulation of LC3B-II in HCV-infected cells compared with that detected in mock-infected cells (Supplemental Figure 2E). It should be noted that interference with complete autolysosome fusion was also concomitant with reduced HCV expression (Supplemental Figure 2, D and E). In addition, the detection of free GFP fragment in HCV infection (Figure 1A, right panel) was indicative of the proceeding of autophagic vacuoles to autolysosome degradation (51). These results indicate that HCV induces the complete autophagic process through enhancing the autophagic flux.
UPR is required for HCV-induced CHOP expression and autophagosome formation. In parallel with increased CHOP expression, HCV infection also led to upregulation of the CHOP mRNA level by 16-fold compared with mock-infection (Figure 2A), suggesting that the accumulated CHOP protein level in infected cells resulted from elevated CHOP mRNA levels. Moreover, HCV infection also activated the 3 UPR modulators, as demonstrated by the cleavage of activating transcription factor 6 (ATF6) to yield p50ATF6; the splicing of XBP1, a downstream regulator of inositol requiring–1α (Ire1α); and phosphorylation of phosphorylated dsRNA-dependent protein kinase–like ER-localized eIF2α kinase (PERK) (Supplemental Figure 3A). Knockdown of CHOP by siRNA duplexes also greatly downregulated the expression of LC3B-II and HCV core expression (Supplemental Figure 3B). And siRNA-mediated transient knockdown of each of Ire1a, ATF6, and PERK specifically inhibited the HCV-triggered increase in CHOP as well as LC3B-II expression (Supplemental Figure 3C). Theses results imply that HCV infection triggers activation of UPR modulators to transactivate the CHOP expression, which in turn activates the autophagy process to enhance HCV viral protein expression.
Inhibition of HCV replication by knockdown of UPR and autophagy-related genes. (A) HCV-infected Huh7 cells (MOI of 0.1) were maintained for 3 days and analyzed for CHOP mRNA levels. (B) HCV-infected Huh7 cells (MOI of 0.01) were maintained for 24 hours and transduced with the indicated shRNA lentiviruses or treated with CQ (50 μM) or BAF-A1 (100 nM) for 24 hours. Seventy-two hours after infection, cells were assessed for intracellular viral RNA levels. (C) VEC, ATG5KD, and CHOPKD cells were used for the assessment of the replication kinetics of JFH1-Luc and HCV-N-FLuc. Data represent mean ± SEM (n = 3).
HCV-induced UPR-autophagy pathway controls viral RNA replication. Next, we examined whether HCV-induced UPR-autophagy is required for HCV RNA biogenesis. Individual knockdown of various genes involved in UPR-autophagy, such as ATG5, CHOP, and the 3 UPR regulators _Ire1_α, ATF6, and PERK, strikingly reduced the level of viral RNA in HCV-infected cells (Figure 2B) and also suppressed production of infectious virus into the culture medium (data not shown). Moreover, treatment with CQ or BAF-A1 reduced the level of intracellular viral RNA and infectious virus released into the culture medium to almost the background level (Figure 2B and data not shown).
Furthermore, we then determined whether the UPR-autophagy pathway functions in initial-stage viral RNA synthesis using CHOP- and ATG5-stable-knockdown Huh7 cells, i.e., CHOPKD and ATG5KD (Supplemental Figure 3D). A full-length (FL) bicistronic JFH1 carrying firefly luciferase, designated JFH-Luc (52) (Supplemental Figure 1, scheme 2), was examined. The replication kinetics of JFH1-Luc RNA were greatly reduced in ATG5- and CHOP-stable-knockdown Huh7 cells even at 12 hour after transfection, at which time viral assembly and release are unlikely to be attained, as compared with those observed in VEC cells, which harbored a stably transduced empty lentiviral vector (Figure 2C, left panel). Similarly, stable knockdown of ATG5 or CHOP also greatly reduced the replication kinetics of the genotype 1b HCV-N strain replicon (Figure 2C, right panel), indicating a strain-independent requirement of UPR-autophagy for HCV RNA replication. In addition, there were no apparent changes in HCVpp infection and HCV internal ribosome entry site (IRES)–mediated translation in ATG5KD and CHOPKD cells (data not shown). These results together indicate that the initial-stage RNA replication step is the primary target in the HCV life cycle most affected by the UPR-autophagy. Moreover, we found that transient knockdown of ATG5, CHOP, or LC3B by specific shRNAs in HCV-infected Huh7/RFP-LC3 cells not only disrupted RFP-LC3 puncta formation (Supplemental Figure 4A) but also greatly attenuated the HCV-induced formation of membranous web (Supplemental Figure 4B), known as the assembly site of HCV replication complex, as opposed to the obvious puncta structures and multivesicular membrane alterations seen in cells transduced with the empty lentivirus vector (Supplemental Figure 4, A and B, respectively). These results together reinforce the crucial role of the complete UPR-activated autophagy in HCV RNA replication.
Interference with UPR-autophagy activates the innate immune response. HCV escapes the innate immune defense and establishes chronic infection by NS3/4A-dependent proteolysis of MAVS, also called IFN-β promoter stimulator 1 (IPS1)/virus-induced signaling adaptor (VISA)/CARD adaptor–inducing IFN-β (Cardif), the downstream effector of pathogen recognition receptors, RIG-I, and MDA5 (53, 54). As the loss of UPR-autophagy activation by gene silencing led to inhibition of HCV RNA replication and infectious virus production (Figure 2B and data not shown), which was reminiscent of the antiviral state activated by the innate immune response, we wondered whether signaling of type I IFN is activated in these UPR-autophagy gene–knockdown cells. To test this hypothesis, we compared RLR signaling among VEC, ATG5KD, and CHOPKD cells. The activation of RLR signaling was determined by measurement of the IFNB promoter activity induced by ectopic expression of an N-terminal fragment of RIG-I (RIGI-N), which was shown to activate the IFNB promoter through the phosphorylation, dimerization, and translocation of IFN regulatory factor 3 (IRF3) into the nucleus (55). In VEC cells, HCV infection inhibited RIGI-N–mediated IFN-β activation, as opposed to uninfected VEC cells, in which RIGI-N–activated IFNB promoter increased 40-fold (Figure 3A), presumably due to the inactivation of MAVS signaling by NS3/4A protease activity in infected cells. In contrast, RIGI-N maintained a greater ability to activate the IFNB promoter in infected ATG5KD and CHOPKD cells compared with infected VEC cells (Figure 3A). HCV core expression was also greatly reduced in ATG5KD or CHOPKD cells compared with that observed in infected VEC cells (Figure 3E). And ectopically expressed RIGI-N in HCV-infected ATG5KD and CHOPKD cells also coincidently reduced the expression of core protein compared with that observed with HCV-infected VEC cells (Figure 3E). These results reveal a critical role of UPR-autophagy in modulation of innate immune response and HCV expression.
Activation of RIG-I–mediated IFN-β induction by knockdown of signaling molecules in UPR-autophagy. (A) VEC, ATG5KD, and CHOPKD cells were infected with HCV at an MOI of 10. Three days after infection, infected cells along with uninfected VEC cells were cotransfected with pIFN-β/Fluc reporter and pCMV22-Rluc plasmids together with an empty vector (–) or a RIG-I N expression construct (+). Twenty-four hours after DNA transfection, cells were harvested for the dual luciferase assay. The Renilla luciferase unit was used to normalize the transfection efficiency. The results are expressed as fold increase in IFNB promoter activity in the presence of RIGI-N relative to the basal level of the empty vector. (B) HCV infection and reporter transfection were performed as described in A, except that the ISRE promoter luciferase reporter was used instead of pIFN-β/Fluc and RIG-I N expression constructs. Twenty-four hours after transfection, cells were treated with or without 100 U/ml IFN-α for 16 hours before the dual luciferase assay. The results represent the fold induction of ISRE promoter activity in the presence of IFN-α relative to the basal level of cells without IFN-α treatment. (C) The indicated cells were analyzed for RIGI-N–mediated IFNB promoter activation as described in B. (D) The indicated cells were assessed for IFN-α–stimulated ISRE promoter activity as described in B. (E and F) Cell samples obtained from A and C and from B and D were analyzed by Western blotting, and the results are shown in E and F, respectively. Data represent mean ± SEM (n = 3) (A–D).
To further assess the role of UPR-autophagy in IFN-mediated downstream signaling, we determined the promoter activity of IFN-stimulated gene-responsive element (ISRE) upon IFN stimulation. Infection of HCV did not greatly affect the IFN-α–stimulated activation of the ISRE promoter, as shown by the comparable ISRE-Fluc activities in VEC cells infected or not infected with HCV (Figure 3B). This phenomenon was consistent with a previous report that HCV infection does not block IFN-stimulated activation of the ISRE promoter (42). Interestingly, HCV-infected ATG5KD or CHOPKD cells still possessed a greater capacity for IFN-α–stimulated activation of the ISRE promoter, compared with infected VEC cells (Figure 3B). The IFN-α–triggered ISRE promoter activation also led to inhibition of HCV core protein expression in VEC, ATG5KD, and CHOPKD cells (Figure 3F). Similarly, in the absence of HCV infection, ATG5KD and CHOPKD cells still showed a higher potential for RIGI-N–mediated activation of the IFNB promoter as well as IFN-α–stimulated activation of the ISRE promoter than VEC cells (Figure 3, C and D, respectively), indicating that the incremental increase in innate immune signaling triggered by the loss of UPR-autophagy does not necessarily result from a decrease in NS3/4A expression due to the reduced HCV RNA replication, but may also proceed in an HCV-independent manner.
Interference with UPR-autophagy enhances HCV PAMP-triggered innate immune activation. The homopolymeric uridine and cytidine (poly-U/UC; hereafter abbreviated as PU/UC) sequence located within the 3′-UTR of the genomes of various HCV genotypes has been identified as an HCV PAMP motif, mainly for its ability to activate RIG-I–triggered innate immunity (56, 57). It was imperative to examine whether stable silencing of ATG5 or CHOP also increases HCV PAMP-mediated activation of type I IFN, since ATG5 and CHOP function in the inhibition of RLR signaling (Figure 3). First, the HCV JFH1 3′-UTR and PU/UC PAMP RNAs were synthesized in vitro and checked for their authenticity (Supplemental Figure 5), and then used to test this hypothesis. As expected, both HCV 3′-UTR and PU/UC PAMPs efficiently increased IFNB promoter activity (~10-fold) and stimulated IFNB mRNA expression (~80-fold) in uninfected VEC cells (Figure 4, A and B, respectively). Stable knockdown of ATG5 or CHOP further upregulated HCV PAMP–triggered IFNB promoter activity and IFNB mRNA level in uninfected VEC cells (Figure 4, A and B, respectively). HCV infection inhibited both HCV PAMP–triggered IFNB promoter activation and IFNB mRNA expression in VEC cells (Figure 4, A and B, respectively). In contrast, 3′-UTR and PU/UC PAMP each still greatly stimulated IFNB promoter activation as well as IFNB mRNA production in HCV-infected ATG5KD and CHOPKD cells (Figure 4, A and B, respectively). These results confirm again that silencing of ATG5 or CHOP enhances HCV PAMP–mediated IFNB promoter activation even in the context of HCV infection.
Activation of HCV PAMP–mediated IFN responses by knockdown of UPR-autophagy signaling molecules. (A) The indicated cells were infected with or without HCV (MOI of 10). Three days after infection, cells were transfected with pIFN-β/Fluc promoter reporter and pCMV22-Rluc plasmids. Twenty-four hours after transfection, cells were transfected with HCV 5′-UTR (Control), 3′-UTR, or PU/UC PAMP RNAs, followed by dual luciferase assay 24 hours after PAMP transfection. The fold increase is calculated by normalization to the basal level of control RNA transfection. (B) Virus infection and PAMP RNA transfection were performed as described in A but without DNA transfection. Cells were harvested for determination of the IFNB mRNA level, and fold increase was determined by normalization to the basal level of control RNA transfection. (C) Cell samples from B were assessed for the indicated proteins by Western blotting. (D) The indicated cells were infected (MOI of 10), and 3 days after infection, infected cells along with uninfected VEC cells were transfected with the 3′-UTR PAMP RNA motif for 24 hours. Culture supernatants were harvested, filtered, and used as conditioned medium. For inhibition experiments, Huh7 cells were transfected with JFH1-Luc RNA for 4 hours and incubated for an additional 48 hours with the indicated percentages of conditioned medium collected from different treatments as indicated in the total culture medium, which are expressed as percent 3′-UTR PAMP–treated supernatant (sup.) or percent PU/UC PAMP–treated supernatant. Replication of JFH1-Luc was monitored by the firefly luciferase activity. (E) Generation of PU/UC-treated conditioned medium and JFH1-Luc inhibition assay were performed as described in D. Data represent mean ± SEM (n = 3) (A, B, D, and E).
UPR-autophagy–modulated activation of HCV PAMP–triggered innate immunity correlates with its paracrine antiviral activity. Since stable knockdown of ATG5 or CHOP upregulated IFNB promoter activation and IFN-α–stimulated ISRE promoter activation in infected cells (Figure 3, A and B, and Figure 4, A and B), we then determined whether interference with UPR-autophagy can trigger ISG expression. HCV 3′-UTR and PU/UC each were able to induce the expression of IFN-induced protein with tetratricopeptide repeats 1 (IFIT1), also named ISG56 (58, 59), in HCV-infected ATG5KD and CHOPKD cells, but not in HCV-infected VEC cells (Figure 4C). Next, we examined whether stable knockdown of ATG5 or CHOP would exert a paracrine antiviral action on neighboring cells through secreted IFN-β. The supernatants collected from VEC cells transfected with 3′-UTR or PU/UC RNA specifically inhibited expression of JFH1-Luc reporter virus in a dose-dependent manner (Figure 4, D and E, respectively). As expected, HCV infection greatly inhibited the antiviral activities in the supernatants of 3′-UTR– and PU/UC RNA–transfected VEC cells on JFH-Luc replication, as shown in HCV-infected VEC cells (VEC HCV IF in Figure 4, D and E, respectively). Remarkably, the supernatants from 3′-UTR and PU/UC RNA–transfected, HCV-infected ATG5KD and CHOPKD cells possessed mediated a more potent inhibitory effect on HCV replication than the supernatants obtained from HCV-infected VEC cells (Figure 4, D and E). These results together highlight the notion that interference with UPR-autophagy not only amplifies HCV PAMP–mediated IFN-β activation but also elevates the downstream innate immune response to inhibit HCV replication in a paracrine fashion.
UPR-autophagy suppressing antiviral innate immunity can occur independently of HCV infection. In addition, we examined the effects of silencing of UPR-autophagy on upregulation of the HCV PAMP–mediated innate immune response in the context of no viral infection. Knockdown of LC3B, ATG5, or CHOP by siRNA duplexes was attained by transient transfection of siRNA duplexes (Figure 5A, top left panel). HCV 3′-UTR PAMP–mediated IFNB mRNA induction and IFN-triggered ISG56 expression were greatly amplified in Huh7 cells in which LC3B, ATG5, and CHOP were individually knocked down (Figure 5A, top right and bottom panels, respectively). Also, IFNB mRNA and ISG56 protein expression were further increased in HeLa cells transiently knocked down for ATG5 or CHOP, albeit with a smaller effect than those observed with Huh7 cells (Supplemental Figure 6). Additionally, as shown in Figure 5B, left and right panels, respectively, HCV PAMP RNA was able to trigger further induction of IFNB promoter and ISG56 expression in ATG5-knockout, i.e., Atg5–/–, mouse embryonic fibroblasts (MEFs) (60).
Enhancement of HCV PAMP–mediated IFN response by disruption of UPR-autophagy activation. (A) Huh7 cells were first transfected with 400 pmol each of control, LC3B, ATG5, or CHOP siRNA duplexes for 72 hours and then harvested for analysis of LC3B, ATG5, CHOP, and β-actin expression (top left panel). The asterisks indicate nonspecific background signals. An uncropped image of A is shown in Supplemental Figure 9, left panel. A portion of siRNA-transfected cells was then transfected with the control HCV 5′-UTR RNA or HCV 3′-UTR PAMP RNA 36 hours after siRNA transfection. Twenty-four hours after PAMP RNA transfection, the cells were harvested for analyses of the IFNB mRNA level (top right) and ISG56 expression (bottom). The fold increase in IFNB mRNA level was determined by normalization to the basal level of control RNA transfection. (B) WT and Atg5–/– MEFs were transfected with pIFN-β/Fluc promoter reporter and pCMV22-Rluc plasmids. Twenty-four hours later, cells were transfected with control HCV 5′-UTR RNA (Control), HCV 3′-UTR PAMP RNA, or DEV PAMP RNA. An additional 24 hours after PAMP RNA transfection, cells were harvested and dual luciferase activity determined (left panel). The fold increase was calculated by normalization to the basal level of control RNA transfection. In parallel, the cells were also analyzed for ISG56, ATG5-ATG12, and β-actin expression by Western blotting (right panel). Data represent mean ± SEM (n = 3) (A, middle panel, and B).
We also examined whether knockdown of UPR modulators upregulates the HCV PAMP RNA–mediated innate immune response, as these UPR modulators controlled the HCV-induced expression of CHOP (Supplemental Figure 3). Stable silencing of each of Ire1α, ATF6, and PERK greatly increased the fold increases in HCV 3′-UTR– and PU/UC PAMPs–mediated IFNB promoter activation (Supplemental Figure 7A).
UPR-autophagy is also required for DEV PAMP–mediated suppression of innate immunity. Given that several RNA viruses such as DEV and coxsackievirus may activate the UPR and autophagic pathway in their life cycle to benefit their infection (61–63), we then determined whether UPR-autophagy is also required for suppression of other RNA viral PAMP–mediated innate antiviral immunity. The PAMPs derived from DEV, rabies virus (RV), and Ebola virus were examined, since poly-U sequences are also present in the genomes of these 3 RNA viruses, which are known to trigger RIG-I–mediated innate immune signaling (56, 57, 64). As observed in HCV, stable knockdown of ATG5 or CHOP resulted in a greater capacity to activate the IFNB promoter in response to PAMP RNA motifs derived from these 3 viruses (Supplemental Figure 5 and Supplemental Figure 7B). In particular, the DEV PAMP RNA, just like the HCV PAMP, also enhanced IFNB promoter activation and ISG56 expression in Atg5–/– MEFs (Figure 5B). Collectively, these results indicate that HCV and, perhaps, DEV and its flaviviral relatives require the UPR-autophagy pathway to repress innate antiviral immunity.
Enhanced UPR-autophagy by chemical inducers inhibits IFN-β activation. To ascertain the role of UPR-autophagy in modulation of the innate immune response, we then investigated whether induction of the UPR-autophagy signaling may directly downregulate the HCV PAMP RNA–mediated activation of innate immunity. Because nutrient starvation and rapamycin have been shown to induce the autophagic pathway through interference with the insulin receptor and mammalian target of rapamycin (mTOR) signaling cascades (17, 18), we treated Huh7/RFP-LC3 cells with Earle’s balanced salt solution (EBSS), HBSS, or fresh medium containing the mTOR inhibitor rapamycin, and cells were then quantified for the formation of autophagic vacuoles. A large portion of EBSS-, HBSS-, and rapamycin-treated RFP-LC3 reporter cells displayed RFP-LC3–labeled puncta structures (Figure 6A). Consistent with this finding, a higher percentage of autophagic cells containing AVi and AVd structures were detected in treated cells compared with untreated cells (Figure 6B), indicating that nutrient starvation and rapamycin can activate the complete autophagic process in a manner similar to that observed in HCV infection. In these autophagy-activated cells, the HCV- and DEV-PAMP RNA–mediated IFNB promoter activation was greatly reduced (Figure 6, C and D, respectively).
Downregulation of HCV- and DEV-PAMP RNA–triggered IFN-β activation by autophagy inducers. (A) Huh7/RFP-LC3 cells were treated with EBSS, HBSS, or with fresh medium containing 4 mM rapamycin for 6 hours. Cells were fixed and analyzed for the formation of RFP-LC3B–labeled autophagic vacuoles by confocal microscopy (scale bars: 10 μm). A set of confocal images is shown (left panel). The degree of cells forming autophagic vacuoles was also quantified (right panel). (B) Huh7/RFP-LC3 cells were starved or treated with rapamycin as described in A, and cells were fixed and subjected to TEM analysis. The ratio of autophagy (autophagic cells/total cells) was determined by counting the number of cells containing autophagic vacuoles among the total 30 randomly selected cells. (C) Huh7/RFP-LC3 cells were transfected with the pIFN-β/Fluc promoter reporter and cultured for 24 hours. Cells were then transfected with control HCV 5′-UTR RNA or 3′-UTR PAMP RNA, and maintained for an additional 12 hours. The transfected cells were then treated with chemicals as described in A prior to determination of IFNB promoter activation. The fold increase in IFNB promoter of the PAMP RNA–transfected cells was determined by normalization to the basal level of the control RNA–transfected cells. (D) The effects of EBSS, HBSS, and rapamycin on DEV PAMP RNA–induced IFNB promoter reporter activation were determined as described in C. Data represent mean ± SEM (n = 3) (A, C, and D).
Moreover, we examined whether activation of UPR may lead to inhibition of HCV PAMP RNA–mediated IFN-β activation through the activated autophagic process. DTT and tunicamycin are two well-known inducers of UPR (20–22, 65) and have been reported to trigger the activation of autophagy through the UPR signaling (21, 25). Treatment with either DTT or tunicamycin led to more accumulation of RFP-LC3–labeled puncta structures (Figure 7A), upregulated expression of both CHOP and LC3B-II (Figure 7B), and a higher percentage of cells containing autophagic vacuoles (Figure 7C) in the Huh7/RFP-LC3 cells, indicating their ability to trigger the UPR-autophagy pathway in Huh7 cells. Meanwhile, DTT and tunicamycin reduced HCV- and DEV-PAMP RNA–mediated IFNB promoter activation (Figure 7, D and E, respectively). Likewise, the repressing effects of activated UPR-autophagy by these autophagy and UPR inducers on HCV- and DEV-PAMP RNA–mediated IFNB promoter induction were also observed in HeLa cells (Supplemental Figure 8). Taken together, these results clearly demonstrate that activated UPR-autophagy suppresses the Flaviviridae PAMP–mediated innate immune response.
Suppression of the HCV- and DEV-PAMP RNA–induced IFNB promoter activation by UPR inducers. (A–C) Huh7/RFP-LC3 cells were left untreated or treated with 2 mM DTT or 4 μg/ml tunicamycin for 6 hours, and then subjected to quantification of RFP-LC3–labeled puncta structure (scale bars: 10 μm) (A), Western blot analysis of the expressions of indicated proteins (B), or TEM analysis of autophagic vacuoles (C). The asterisk in B indicates the nonspecific background signal. An uncropped image of B is shown in Supplemental Figure 9, right panel. The ratio of autophagy in C was determined as described in Figure 6B. (D) Huh7/RFP-LC3 cells were transfected with the pIFN-β/Fluc promoter, followed by transfection with HCV PAMP RNA. The transfected cells were then treated with DTT or tunicamycin as described in A prior to assessment of IFNB promoter activation. The fold increase in IFNB promoter of the PAMP RNA–transfected cells was determined by normalization to the basal level of the control RNA–transfected cells. (E) The effects of DTT and tunicamycin on DEV PAMP RNA–mediated IFNB promoter reporter induction were determined as described in D. Data represent mean ± SEM (n = 3) (A, D, and E).
Deficiency in UPR/autophagy-related genes disrupts chemical inducer–mediated downregulated antiviral innate immunity. Next, we explored whether EBSS- and HBSS-mediated suppression of IFNB promoter activity truly relies on the activated autophagic process. First, a large number of endogenous LC3B-labeled puncta structures were detected in EBSS- and HBSS-treated VEC cells, but not in the ATG5KD cells treated in parallel (Figure 8, A and B, top left and bottom panels), indicating that nutrient starvation–triggered activation of autophagy is lost in ATG5KD cells. In contrast to the VEC cells, which exhibited reduced HCV PAMP–triggered IFNB promoter activation upon HEBS or EBSS treatment (Figure 8A, top right panel), the HBSS- and EBSS-treated ATG5KD cells still retained a high capacity for IFNB activation in a fashion similar to that of the untreated cells (Figure 8B, top right panel).
The effect of UPR-autophagy gene silencing on chemical inducer–mediated suppression of IFN-β activation. (A and B) VEC (A) and ATG5KD (B) Huh7 cells were transfected with the IFNB promoter reporter and cultured for 24 hours. The cells were then transfected with HCV control HCV 5′-UTR RNA or 3′-UTR PAMP RNA and maintained for an additional 12 hours. Transfected cells were left untreated, or treated with EBSS or HBSS for 6 hours, and then IFNB promoter reporter activation determined (upper right panels). Another set of cells without DNA and RNA transfections was handled in parallel prior to quantification for endogenous LC3B-labeled puncta structures by immunostaining (upper left and bottom panels, scale bars: 10 μm). (C and D) VEC (C) and CHOPKD (D) Huh7 cells were transfected with the IFNB promoter reporter and HCV 3′-UTR PAMP RNA and then treated with 2 mM DTT or 4 μg/ml tunicamycin for 6 hours before assessment of IFNB promoter reporter activation (upper right panels). Another set of cells without DNA and RNA transfections was handled in parallel and then quantified for LC3B-labeled puncta structures (upper left and bottom panels; scale bars: 10 μm). Data represent mean ± SEM (n = 3).
Moreover, DTT and tunicamycin failed to induce expression of CHOP (data not shown) and formation of endogenous LC3B puncta structures in CHOPKD cells, as compared with those observed in VEC cells (Figure 8, C and D, top left and bottom panels), supporting again the vital role of CHOP in the UPR-induced autophagic process. Intriguingly, the DTT- and tunicamycin-mediated reduction in HCV PAMP RNA–triggered IFNB activation observed in VEC cells was strikingly alleviated in CHOPKD cells (Figure 8, C and D, top right panels). These results together indicate that the downregulation of antiviral innate immunity triggered by chemical inducers requires UPR-autophagy.
Similarly, despite enhancing formation of endogenous LC3B puncta structures and reducing HCV PAMP RNA–mediated IFNB promoter activation in control MEFs (Figure 9A), HBSS and rapamycin lost their inhibitory effect on innate antiviral immunity in Atg5–/– MEFs, which did not show the activated autophagic LC3B punta vacuoles (Figure 9B). These observations are in line with the earlier report that Atg5–/– MEFs are more susceptible to RLR signaling (37). The results indicate that activated autophagy represses viral PAMP–induced innate immunity in a broad spectrum of cells derived from different species.
Effect of Atg5 gene knockout in MEFs on chemical inducer–mediated autophagic activation and suppression of IFN-β activation. WT (A) and Atg5–/– (B) MEFs were examined for the effects of HBSS and rapamycin on HCV 3′-UTR PAMP RNA–mediated IFNB promoter (upper right panels). Another parallel set of cells without DNA and RNA transfections was quantified for LC3B-labeled puncta structures (upper left and bottom panels, scale bars: 10 μm). Data represent mean ± SEM (n = 3).
CQ ablates rapamycin- and DTT-mediated suppression of antiviral innate immunity. Since HCV-induced complete autolysosome formation was critical for HCV RNA replication (Figure 2B) as well as viral protein expression (Supplemental Figure 2, D and E), we then examined whether the complete autolysosome formation is analogously necessary for the repression of antiviral innate immunity by UPR-autophagy. CQ was shown to block the maturation of autolysosomes and lead to accumulated autophagic vacuoles in cells (49, 66). Cotreatment of rapamycin- and DTT-treated Huh7/mRFP-GFP-LC3 cells with CQ led to the restoration of yellow-colored autophagic vesicles and accumulation of LC3B-II expression, as opposed to rapamycin- and DTT-treated dual reporter cells without CQ administration (Figure 10, A and B, respectively). Similarly, administration of rapamycin- and DTT-treated cells with CQ showed an even higher percentage of autophagic cells harboring large, partial, or even nondegraded AVd (unAVd), which contained a majority of undigested organelles, as opposed to the large population of cells containing only AVi and AVd structures in rapamycin and DTT treatment (Figure 10C), in agreement with a previous study showing that CQ impairs the digestion of autolysosome content (66). Meanwhile, CQ hindered rapamycin- and DTT-mediated repression of HCV PAMP RNA–mediated IFN-β activation (Figure 11, A and B). Rapamycin- and DTT-mediated repression of DEV PAMP RNA–triggered IFN-β activation was also palliated by CQ treatment (Figure 11, C and D). Moreover, pretreatment of CQ and BAF-A1 augmented the HCV PAMP RNA–mediated induction of IFNB mRNA and ISG56 expression (data not shown).
Effect of CQ on rapamycin- and DTT-triggered autophagic process. (A–C) Huh7/mRFP-GFP-LC3 cells were left untreated or treated with 4 mM rapamycin or 4 mM rapamycin in the presence (+) or absence (–) of 100 μM CQ. Six hours after treatment, cells were analyzed by Western blot analysis (A and B, left panels) and quantified for RFP-LC3–labeled (RFP-positive) and RFP-GFP-LC3–labeled (RFP-GFP-positive, yellow-colored) puncta structures by confocal microscopy (A and B, middle and right panels; scale bars: 10 μm). Another set of reporter cells was treated with drugs according to the procedure described above and fixed and analyzed by TEM for formation of autophagic vacuoles (C). The black arrowheads indicate the AVi or AVd, and the white arrows unAVd. Data represent mean ± SEM (n = 3) (A and B).
Interference with the UPR-autophagy inducer–triggered repression of IFN-β activation by CQ. (A and B) Huh7/mRFP-GFP-LC3 cells were transfected with the pIFN-β/Fluc promoter reporter and cultured for 24 hours. Cells were then transfected with control HCV 5′-UTR RNA or 3′-UTR PAMP RNA and then maintained for an additional 12 hours. Transfected cells were left untreated or treated with rapamycin (A) or DTT (B) in the presence or absence of CQ as described in the legend to Figure 10, A and B. The fold increase in the IFNB promoter activity of viral PAMP RNA–transfected cells was determined by normalization to the basal level of the control RNA–transfected cells. (C and D) The effect of CQ on rapamycin-induced (C) or DTT-induced (C) suppression of DEV PAMP–mediated IFNB promoter activation was assessed as described in A and B. Data represent mean ± SEM (n = 3).
Knockdown of LAMP2 and Rab7 relieves EBSS- and tunicamycin-induced repression of antiviral innate immunity. Various cellular proteins such as lysosome-associated membrane protein 2 (LAMP2) and the small GTP-binding protein Rab7 are essential for lysosome biogenesis and participate in the maturation step of autolysosomes (45–47). Disruption of LAMP2 expression and interference with the biological function of Rab7 were shown to result in accumulated autophagosomes and/or partially undigested autolysosomes due to blocking of the final maturation step of late autophagic vacuoles (45–47). We first found that individual silencing of LAMP2 and Rab7 resulted in the rescued GFP signals in EBSS- or tunicamycin-treated Huh7/mRFP-GFP-LC3 cells (Figure 12A), indicating that individual knockdown of LAMP2 and Rab7 specifically inhibits the maturation of autolysosomes. Meanwhile, EBSS- and tunicamycin-activated autophagy lost its ability to repress HCV PAMP RNA–mediated IFNB promoter activation in LAMP2- and Rab7-knockdown cells (Figure 12B). In parallel, knockdown of LAMP2 and Rab7 also reduced the intracellular viral RNA level and NS5A expression in HCV-infected cells (Figure 12, C and D, respectively). Collectively, these results demonstrate that the complete autophagic process is indispensable not only for suppression of the innate immune response, but also for productive HCV replication.
Abrogation of the UPR-autophagy–mediated suppression of IFN-β activation by knockdown of LAMP2 and Rab7. (A) Huh7/mRFP-GFP-LC3 cells were transfected with 400 pmol each of control, LAMP2, or Rab7 siRNA duplexes for 48 hours, and cells were quantified for RFP-LC3– (RFP-positive) and RFP-GFP-LC3–labeled (RFP-GFP-positive) puncta structures by confocal microscopy (bottom panel). The yellow-colored image represents the RFP-GFP-LC3–labeled puncta structure. A set of representative confocal images is also shown (top panel; scale bars: 10 μm). (B) Huh7/mRFP-GFP-LC3 cells were transfected with 400 pmol each of the indicated siRNA duplexes. Forty-eight hours later, cells were transfected with the pIFN-β/Fluc promoter reporter and cultured for 24 hours. The cells were then transfected with control HCV 5′-UTR RNA or 3′-UTR PAMP RNA and maintained for an additional 12 hours. The cells were left untreated or treated with EBSS or tunicamycin for 6 hours. Cells were then analyzed for IFNB promoter reporter activation (left panel). A set of cells 48 hours after siRNA transfection was analyzed by the Western blotting (right panel). (C and D) Huh7/mRFP-GFP-LC3 cells were infected with HCV at an MOI of 10 and cultured for 6 days. The cells were then transfected with the indicated siRNA duplexes as described in A. Seventy-two hours after transfection, cells were harvested and analyzed for the intracellular HCV RNA level (C) and analyzed for the indicated proteins by Western blotting (D). Data represent mean ± SEM (n = 3) (A–C).