Potential conflict of interest: Nothing to report.
Autophagy is important for cellular homeostasis and can serve as innate immunity to remove intracellular pathogens. Here, we demonstrate by a battery of morphological and biochemical assays that hepatitis C virus (HCV) induces the accumulation of autophagosomes in cells without enhancing autophagic protein degradation. This induction of autophagosomes depended on the unfolded protein response (UPR), as the suppression of UPR signaling pathways suppressed HCV-induced lipidation of the microtubule-associated protein light chain 3 (LC3) protein, a necessary step for the formation of autophagosomes. The suppression of UPR or the suppression of expression of LC3 or Atg7, a protein that mediates LC3 lipidation, suppressed HCV replication, indicating a positive role of UPR and the incomplete autophagic response in HCV replication. Conclusion: Our studies delineate the molecular pathway by which HCV induces autophagic vacuoles and also demonstrate the perturbation of the autophagic response by HCV. These unexpected effects of HCV on the host cell likely play an important role in HCV pathogenesis. (HEPATOLOGY 2008.)
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Autophagy is important for removing long-lived proteins and damaged organelles in cells. During autophagy, double-membrane vesicles form to sequester part of the cytoplasm. These double-membrane vesicles, also known as autophagosomes, subsequently fuse with lysosomes to form autolysosomes for the degradation of their contents for recycling.1 Many genes that are important for autophagy have been identified. Among them is microtubule-associated protein light chain 3 (LC3), whose covalent linkage to phosphatidylethanolamine by the ubiquitin-activating enzyme E1-like protein Atg7 is necessary for the formation of autophagosomes.2
Hepatitis C virus (HCV) is a positive-stranded RNA virus with a genome size of 9.6 Kb. Infection by this virus can lead to liver cirrhosis and hepatocellular carcinoma. Based on their genetic relatedness, different HCV isolates have been grouped into six major genotypes and many more subtypes. The HCV genome codes for a polyprotein, which is proteolytically cleaved to generate the mature protein products.3
Recently, a cell culture system for efficient HCV propagation using the JFH1 strain, which belongs to HCV genotype 2a, has been developed.4–7 In this system, the HCV JFH1 RNA or its derivative was transfected into human hepatoma cells to direct the replication and release of infectious HCV particles, which could then initiate the next round of infection. In this report, we use this HCV RNA transfection/infection system to study HCV–host interactions. Our results indicate that HCV induces the accumulation of autophagosomes by activating unfolded protein response (UPR). However, HCV does not enhance autophagic protein degradation. Importantly, this induction of autophagosomes enhanced HCV replication. The persistent induction of the UPR and the perturbation of the autophagic response likely play an important role in HCV pathogenesis.
BAF, bafilomycin A1; DMEM, Dulbecco's modified Eagle's medium; DTT, dithiothreitol; ER, endoplasmic reticulum; GFP, green fluorescence protein; HCV, hepatitis C virus; LC3, microtubule-associated protein light chain 3; mRNA, messenger RNA; PERK, phosphorylated extracellular signal-regulated kinase; qRT-PCR, quantitative real-time polymerase chain reaction; siRNA, small interfering RNA; Tg, thapsigargin; UPR, unfolded protein response.
Materials and Methods
Cell Cultures, DNA Plasmids, and Small Interfering RNAs.
Huh7.5 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum and 1% nonessential amino acids (the “growth medium”). Huh7.5 cells that stably expressed GFP-LC3 were produce by transfecting Huh7.5 cells with pEGFP-LC38 followed by G418 selection. The plasmids pJFH1 and pJFH1/GND used had been described before.9 Stable Huh7 cells that contained the HCV Con1 subgenomic RNA replicon has also been described.10 The two phosphorylated extracellular signal-regulated kinase (PERK) small interfering RNAs (siRNAs)11 and LC3 siRNA12 were synthesized at the USC Microchemical Core Facility. IRE1α, ATF6, and Atg7 siRNAs were purchased from Qiagen (Germantown, MD), and the negative control siRNA was purchased from Invitrogen (Carlsbad, CA). Cells were transfected with siRNA using Lipofectamine 2000 (Invitrogen, Carlsbad, CA) per the manufacturer's instructions. The siRNA sequences are shown in Supplementary Table 1.
In Vitro Transcription of HCV RNA and Electroporation of Huh7.5 Cells.
Plasmids pJFH-1 and pJFH-1/GND were linearized with the restriction enzyme Xba1 for RNA synthesis using the MEGA-script kit (Ambion, Foster City, CA). Huh7.5 cells were then electroporated with the HCV RNA. Unless specifically indicated, all the analyses were conducted on cells between 2 and 5 days after electroporation.
Confocal Microscopy and LysoTracker-Red Staining.
Cells were fixed with 4% formaldehyde and incubated with the rabbit anti-HCV core antibody and then with the rhodamine-conjugated goat anti-rabbit antibody for confocal microscopy. For LysoTracker red staining, cells were treated with 50 nM LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA) at 37°C for 3 hours. Depending on the experiments, cells might be nutrient starved in Hank's balanced salt solution or treated with 2.5 mM dithiothreitol (DTT) in the growth media for the induction of endoplasmic reticulum (ER) stress at 37°C for an additional 20 minutes in the presence of LysoTracker-red. The colocalization coefficient, which measures the fraction of green fluorescent protein (GFP) pixels that are also positive for LysoTracker-red, was performed on randomly selected GFP-positive cell images (n > 20) using the Zeiss LSM 510 imaging software. A colocalization coefficient of 1 means complete colocalization, whereas 0 means no colocalization.
Long-Lived Protein Degradation Assay.
Cells were labeled with L-[4,5-3H] leucine (50 μCi/mL) for 24 hours in growth media, rinsed with DMEM, and further incubated in growth media for 24 hours. Cells were then rinsed with DMEM or, for nutrient starvation, with Hank's balanced salt solution, and treated with or without 200 nM bafilomycin A1 in DMEM or Hank's balanced salt solution at 37°C for 1 or 4 hours. Cell lysates and media were collected at the end of treatment, precipitated with trichloroacetic acid and analyzed by scintillation counting. The protein degradation rate was determined by the following equation: (trichloroacetic acid–soluble counts in media) / (total counts in media and cell lysates) × 100%. Cells treated with thapsigargin (Tg) were analyzed using the same procedures, with the exception that Tg was added into the growth media 12 hours after the removal of the 3H-leucine.
Total cellular RNA was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using the TaqMan Gold RT-PCR Kit (Applied Biosystems, Foster City, CA) following the manufacturer's instructions. HCV JFH1 primers 5′-TCTGCGGAACCGGTGAGTA-3′ (sense) and 5′-TCAGGCAGTACCACAAGGC-3′ (antisense) and the probe 5′-CACTCTATGCCCGGCCATTTGG-3′ were used for the qRT-PCR. Control glyceraldehyde 3-phosphate dehydrogenase primers and its probe were purchased from Applied Biosystems (Foster City, CA).
Analysis of XBP1 Messenger RNA Splicing by IRE1.
Total cellular RNA was reverse transcribed and amplified using the sense primer (5′-CCTTGTAGTTGAGAACCAGG-3′) and antisense primer (5′-GGGGCTTGGTATATATGTGG-3′). The PCR products were further digested with the restriction enzyme Pst1. For internal control, the complementary DNA of the β-actin messenger RNA (mRNA) was also amplified using the sense primer (5′-ATCTGGCACCACACCTTCTACAATGAG-3′) and the antisense primer (5′-CGTCATACTCCTGCTTGCTGATCC-3′).
Induction of Autophagosomes by HCV.
We transfected the HCV JFH1 RNA or its replication defective GND mutant RNA into Huh7.5 cells to study HCV–host interactions. The GND mutant RNA served as a negative control, because it cannot replicate and is rapidly degraded in transfected cells. By performing electron microscopy, we found that HCV JFH1 induced the accumulation of membrane vesicles with the morphology of autophagosomes (Supplementary Fig. 1A), which were rarely observed in GND RNA transfected cells (Supplementary Fig. 1B). To investigate whether HCV could indeed induce the accumulation of autophagosomes, we established stable Huh7.5 cells that expressed the GFP-LC3 fusion protein. Cells transfected by the GND RNA displayed no detectable HCV core protein signal and in general a weak and diffused cytoplasmic signal of the GFP-LC3 fusion protein (Fig. 1A). In contrast, most of the cells transfected by the JFH1 RNA stained strongly for the HCV core protein with most of them (approximately 80% at day 5 posttransfection) also displaying bright and punctuate GFP-LC3 signal (Fig. 1B). At a higher magnification, many of the core protein signals were often found to be circular, which is consistent with previous reports that the core protein is associated with lipid droplets. Importantly, the GFP-LC3 signals were also often found to be circular, consistent with their localization on autophagosomal membranes (Fig. 1C). Circular GFP-LC3 signals were also observed when JFH1 cells were stained for the HCV NS5A protein (Supplementary Fig. 2).
These observations indicated that HCV could induce the accumulation of autophagosomes. This possibility was further confirmed by western blot analysis of LC3, which is converted from the cytosolic form (LC3-I) to the lipidated, autophagosome-associated form (LC3-II) during autophagy. Although little LC3-II could be detected in mock transfected cells or cells transfected with the GND mutant RNA, a significant amount of LC3-II was detected in cells transfected with the JFH1 RNA (Fig. 2A). The ability of HCV to induce the lipidation of LC3 is not limited to the HCV JFH1 isolate; a similar result was observed in Huh7 cells harboring a high replication level of the subgenomic HCV RNA replicon derived from the genotype 1b Con-1 virus (Fig. 2B).10
Lack of Enhancement of Autophagy by HCV.
The results shown in Figs. 1 and 2 indicated that HCV could induce the accumulation of autophagosomes in cells. To investigate whether HCV could also enhance autophagic protein degradation, we analyzed the degradation rate of long-lived proteins in cells. Huh7.5 cells were metabolically labeled with 3H-leucine for 24 hours followed by the chase of another 24 hours to allow the degradation of labeled, short-lived proteins. Cells were then rinsed and incubated in fresh media in the presence and absence of bafilomycin A1 (BAF), a vacuolar adenosine triphosphatase inhibitor that suppresses the fusion between autophagosomes and lysosomes.13 The protein degradation rate was determined by measuring the amount of trichloroacetic acid–soluble radiolabel released into the medium per hour. The overall protein degradation rates and those sensitive to BAF and hence mediated by autophagy are shown in Fig. 3A and 3B, respectively. As shown in the figures, nutrient starvation, which induces autophagy and served as the positive control, increased both overall and BAF-sensitive protein degradation rates. Interestingly, HCV JFH1 did not increase, but rather slightly reduced, overall and BAF-sensitive protein degradation rates. These results suggested that, although HCV JFH1 was able to enhance the accumulation of autophagosomes, it did not enhance autophagic protein degradation. To further confirm this observation, we analyzed the p62/SQSTM1 protein level in cells. The p62/SQSTM1 protein binds to LC3 and is degraded by autophagy.14 There was a continuous increase, albeit small, of the p62/SQSTM1 protein level in cells during the first 24 hours after the transfection of the HCV RNA (Fig. 3C). In contrast, nutrient starvation reduced the p62 level by approximately 65%. These results again indicated that HCV JFH1 did not enhance autophagic protein degradation.
To further investigate why the accumulation of autophagosomes induced by HCV did not lead to a higher protein degradation rate, we analyzed the level of autolysosomes in cells by staining stable GFP-LC3 cells with LysoTracker-red, which stains for acidic organelles such as lysosomes. GFP-LC3 cells that were starved for nutrients were used as a positive control. Bright GFP puncta (in other words, autophagic vacuoles) were detected in a large fraction of cells that were nutrient-starved (Fig. 4). Nearly half (colocalization coefficient 0.43) of these GFP puncta were also positive for LysoTracker-red. In contrast, when HCV cells were also analyzed, the fraction of GFP-LC3 puncta that was also positive for LysoTracker-red was significantly lower, with a colocalization coefficient of only 0.14 (P < 0.005). These results, together with the low autophagic protein degradation rate of HCV cells, indicated that the fusion between autophagosomes and lysosomes was inefficient in HCV cells.
Induction of ER Stress by HCV.
Several HCV gene products, as well as the subgenomic RNA replicon, have been shown to induce ER stress, although sometimes with conflicting results.15–18 Because ER stress has been shown to induce autophagy,19, 20 we decided to investigate whether HCV could induce the accumulation of autophagosomes via the induction of ER stress. We first examined whether HCV could induce ER stress during productive replication. ER stress, which is caused by the accumulation of misfolded proteins in the ER, can activate the UPR via three different sensors: PERK, ATF6, and IRE1.21 HCV induced the phosphorylation of PERK and its downstream effector eIF2α, which in turn led to the increased expression of ATF4, CHOP/GADD153, and the protein chaperon GRP78 (Fig. 5A). We then examined the effect of HCV on IRE1, which, on its activation, induces the splicing of the xbp1 mRNA to stimulate the expression of UPR target genes.21 Splicing of xbp1 mRNA was assayed by RT-PCR: the unspliced RNA generates a 442-bp product that can be digested by the restriction enzyme Pst1, whereas the spliced RNA generates a 416-bp Pst1-resistant product.22 The spliced xbp1 RNA was easily detectable in cells transfected with the JFH1 RNA but not in cells transfected with the GND RNA or in untransfected cells (Fig. 5B). As a positive control, we treated naïve Huh7.5 cells with DTT, a chemical that causes protein misfolding and ER stress.22 As expected, this treatment induced the splicing of xbp1 mRNA in Huh7.5 cells (Fig. 5B).
We also investigated the effect of HCV on the ATF6 signaling pathway. ATF6 is cleaved from the 90-kDa inactive form to a 50-kDa active fragment on ER stress induction. A low level of activated ATF6 was detected in cells that were transfected with the HCV JFH1 RNA but not in cells that were transfected with the replication defective GND RNA (Fig. 5C). Huh7.5 cells treated with DTT served as the positive control, because they showed efficient ATF6 cleavage (Fig. 5C).
Induction of Autophagosomes by HCV via ER Stress.
The results shown in Fig. 5 indicated that HCV could induce ER stress to activate all three arms of the UPR. Next, we examined whether ER stress could induce autophagy in Huh7.5 cells. Treatment of cells with the ER stress inducer Tg or DTT induced the formation of LC3-II (Fig. 6A), the colocalization of GFP-LC3 puncta with LysoTracker-red (Fig. 6B), and an increase of overall and BAF-sensitive protein degradation rate (Fig. 6C). These observations indicated that ER stress could induce autophagy in Huh7.5 cells. To test whether HCV JFH1 indeed induced the accumulation of autophagosomes via the induction of ER stress, we treated HCV JFH1 cells with siRNAs directed against PERK, IRE1, and ATF6. Two different siRNAs directed against PERK reduced significantly the PERK protein level in HCV JFH1 cells (Fig. 7A). In contrast, a control siRNA had no effect on the total PERK protein level. The suppression of PERK function by the siRNAs was confirmed by the observation that the phosphorylation of eIF2α, a substrate of PERK, was reduced by PERK siRNAs (Fig. 7A). Neither the control siRNA nor the PERK-specific siRNAs affected the total eIF2α protein level in HCV JFH1 cells. We then examined whether the suppression of PERK expression by siRNA would affect the lipidation of LC3 by HCV. Although the LC3-II level was not affected by the control siRNA, it was significantly reduced by the PERK siRNAs (Fig. 7A). These results indicated a critical role of PERK in the autophagic response induced by HCV.
In the IRE1 knockdown experiment, the siRNA directed against IRE1 but not the control siRNA significantly reduced the IRE1 protein level (Fig. 7B). The IRE1 siRNA but not the control siRNA also reduced the LC3-II protein level. Similarly, in the ATF6 knockdown experiment, the siRNA directed against ATF6 significantly reduced uncleaved and cleaved ATF6 protein levels and also the LC3-II protein level (Fig. 7C).
As a control, we also analyzed the effects of PERK, IRE1, and ATF6 siRNAs on LC3 in naïve Huh7.5 cells. None of these siRNAs had any effect on LC3 (Supplementary Fig. 3). These results indicated that PERK, IRE1, and ATF6 pathways were also important for the lipidation of LC3 induced by HCV. The role of ER stress in LC3 lipidation was further confirmed by the treatment of cells with the chemical chaperon 4-phenylbutyric acid, which reduced the LC3-II level in HCV cells (Supplementary Fig. 4).
Positive Effect of ER Stress and Autophagic Pathway in HCV RNA Replication.
To investigate the possible effect of ER stress and the autophagic pathway on HCV RNA replication, cells treated with control siRNA or siRNA directed against PERK, IRE1, or ATF6 were analyzed for HCV RNA levels by qRT-PCR. The siRNAs directed against PERK, IRE1, and ATF6 reduced the HCV RNA level by approximately 80%, 98%, and 80%, respectively, as compared with HCV RNA levels in cells treated with the control siRNA (Fig. 8A). These results indicated that ER stress and likely also autophagosomes played a positive role in HCV RNA replication. To confirm the role of the autophagosomes in HCV RNA replication, we conducted additional knockdown experiments using siRNAs directed against LC3 and Atg7. The LC3 siRNA reduced both LC3-I and LC3-II protein levels, whereas the Atg7 siRNA reduced the Atg7 protein level and the LC3-II level (Fig. 8B). The LC3 siRNA and the Atg7 siRNA also reduced the HCV RNA level by approximately 70% and 55% in JFH1 cells (Fig. 8A), confirming a positive role of autophagosomes or the pathway leading to its accumulation in HCV RNA replication.
Several viruses have been shown to induce autophagy.23 Our results demonstrated that HCV also induces the accumulation of autophagosomes in its host cells (Figs. 1 and 2). The ability of HCV to induce autophagosomes is not limited to the genotype 2a HCV JFH1 strain, as this induction was also observed in Huh7 cells harboring the genotype 1b Con1 virus subgenomic RNA replicon (Fig. 2B). While this manuscript was in preparation, it was also reported that the HCV genotype 1a virus could induce the accumulation of autophagic vacuoles.24 Thus, the ability of HCV to induce autophagosomes is shared by HCV strains across different genotypes. Although HCV JFH1 was able to induce autophagosomes, however, it was not able to enhance autophagic protein degradation (Fig. 3). This lack of enhancement of autophagic degradation by HCV is apparently attributable to the inefficient fusion between autophagosomes and lysosomes (Fig. 4).
How viruses induce autophagy has remained largely unclear. Our results demonstrated that HCV induced the accumulation of autophagosomes via the induction of ER stress (Figs. 5 and 6). This induction of autop-hagosomes involved all three arms of the UPR (Fig. 7), as the suppression of any of these three pathways suppressed the lipidation of LC3, an essential step for the formation of autophagosomes. This observation is interesting, because it indicated that the target genes activated by these different signaling pathways must work in concert with one another to induce autophagy. ER stress can enhance autophagy in Huh7.5 cells (Fig. 6). However, the autophagy activated by HCV is incomplete, because autophagic degradation is not increased (Fig. 3). It is likely that HCV factors or other signaling pathways activated by HCV perturb the maturation of autophagic vacuoles (Fig. 4).
The suppression of any of the three signaling pathways of UPR decreased HCV RNA replication (Fig. 8). This is at least partially through the suppression of the autophagic response, because suppressing the expression of Atg7 and LC3, two proteins necessary for autophagosome formation, also led to a reduced level of HCV RNA replication (Fig. 8). Thus, our results indicated that ER stress and the autophagic pathway play a positive role in HCV replication. It does not appear likely that HCV uses autophagosomes as sites for virion morphogenesis, because the HCV core protein and autophagosomes do not colocalize (Fig. 1B). The replication complexes of several positive RNA viruses localize to autophagosomes,25–28 but it remains to be determined whether the HCV RNA replication complex also resides on autophagosomes or on membranes derived from autophagosomes.
In conclusion, our studies demonstrated that HCV induced ER stress and an incomplete autophagic response. The persistent induction of ER stress and the incomplete activation of autophagy likely play an important role in HCV pathogenesis. Our results also suggest that the many viruses that are capable of inducing ER stress have the potential to induce an autophagic response. The outcome of this response, however, would likely vary for individual viruses, because autophagy is a cellular mechanism to clear viral infection, whereas, conversely, viruses such as HCV appear to have used this cellular response to enhance their own replication.
The authors thank Dr. Amy Lee for helpful discussions on ER stress during the course of our studies and Dr. David Ann for helpful suggestions on autophagy studies and for critical reading of this manuscript. We also thank Dr. Charles Rice for providing us with the Huh7.5 cells, Michelle McVeigh at the USC Research Center for Liver Diseases for help with confocal microscopy, and Sandra Huling of the SFVAMC/NCIRE Microscopy Core for electron microscopy.