The authors have declared that no competing interests exist.
Conceived and designed the experiments: LW JHJO. Performed the experiments: LW YT. Analyzed the data: LW YT JHJO. Wrote the paper: LW JHJO.
Hepatitis C virus (HCV) induces autophagy to enhance its replication. However, how HCV regulates the autophagic pathway remains largely unclear. In this report, we demonstrated that HCV infection could induce the expression of Rubicon and UVRAG, which inhibited and stimulated the maturation of autophagosomes, respectively. The induction of Rubicon by HCV was prompt whereas the induction of UVRAG was delayed, resulting in the accumulation of autophagosomes in the early time points of viral infection. The role of Rubicon in inhibiting the maturation of autophagosomes in HCV-infected cells was confirmed by siRNA knockdown and the over-expression of Rubicon, which enhanced and suppressed the maturation of autophagosomes, respectively. Rubicon played a positive role in HCV replication, as the suppression of its expression reduced HCV replication and its over-expression enhanced HCV replication. In contrast, the over-expression of UVRAG facilitated the maturation of autophagosomes and suppressed HCV replication. The HCV subgenomic RNA replicon, which expressed only the nonstructural proteins, could also induce the expression of Rubicon and the accumulation of autophagosomes. Further analysis indicated that the HCV NS4B protein was sufficient to induce Rubicon and autophagosomes. Our results thus indicated that HCV, by differentially inducing the expression of Rubicon and UVRAG, temporally regulated the autophagic flux to enhance its replication.
HCV induces autophagy to benefit its replication. In this report, we demonstrated that HCV infection could induce the expression of both Rubicon and UVRAG, which inhibited and stimulated the maturation of autophagosomes, respectively. The induction of Rubicon by HCV was prompt whereas the induction of UVRAG was delayed, resulting in the accumulation of autophagosomes in the early time points of viral infection. Rubicon positively regulated HCV replication, apparently by increasing the pool of autophagosomes, which could serve as the sites for HCV RNA replication. On the contrary, UVRAG, which reduced the pool of autophagosomes, inhibited HCV replication if it was over-expressed prior to HCV infection. The analysis of HCV genes indicated that the HCV NS4B protein was sufficient to induce the expression of Rubicon and the accumulation of autophagosomes. Our results thus revealed a novel mechanism used by a virus to temporally regulate the autophagic flux for its replication.
Hepatitis C virus (HCV) is an important human pathogen that can cause severe liver diseases including cirrhosis and hepatocellular carcinoma. It belongs to the flavivirus family and has a 9.6 Kb positive-stranded RNA genome. This genome encodes a polyprotein with a length of slightly more than 3,000 amino acids. The translation of the HCV polyprotein is mediated by an internal ribosomal entry site (IRES) that comprises most of the 5’-untranslated region and the first few codons of the polyprotein coding sequence. After its synthesis, the HCV polyprotein is processed into structural and nonstructural proteins by cellular and viral proteases [
HCV is a hepatotropic virus. It can induce autophagy in its host cells to enhance its replication [
Many protein factors that are important for autophagy have been identified. Class III phosphatidylinositol-3-kinase (PI3KC3) is one of these factors. It catalyzes the formation of phosphatidylinositol-3-phosphate (PI3P) and is important for the initiation of autophagy [
HCV can induce the accumulation of autophagosomes and use autophagosomal membranes as the site for its RNA replication [
To understand how HCV induces autophagy, we infected Huh7.5 cells with a variant of the HCV JFH1 isolate. This variant replicated more efficiently than its parental virus [
(A) Western-blot analysis of HCV-infected cells at various time-points post-infection (p.i.) (m.o.i. = 1). Numbers under Rubicon and UVRAG panels indicate their protein levels at different time points relative to their levels in mock-infected cells (i.e., 0 hour p.i.). (B) Fluorescence imaging of HCV infected cells. Stable Huh7.5 cells that expressed mRFP-GFP-LC3 were infected with HCV and fixed at the time points indicated for the analysis of RFP and GFP puncta. Nuclei were stained with DAPI. The boxed areas were enlarged and shown to the right. (C) Upper panel, levels of RFP and GFP puncta after HCV infection relative to those in mock-infected cells, which were arbitrarily defined as 1. Lower panel, percentages of RFP puncta that were positive for GFP (i.e., Yellow/Red ratio). The results represent the average of >50 cells that were analyzed. (D) Fluorescence imaging of RFP, GFP and LAMP1 in HCV-infected cells expressing mRFP-GFP-LC3. The lysosomal marker LAMP1 was stained in blue color. DAPI was not used to stain the nuclei. The colocalization of RFP and GFP will generate the yellow color, and the colocalization of RFP with LAMP without GFP will generate the purple color. The boxed areas were enlarged and shown to the right.
To investigate why the autophagic protein degradation appeared to be inefficient in the first 24 hours of HCV infection, we infected stable Huh7.5 cells that expressed the mRFP-GFP-LC3 tripartite fusion protein with HCV. This fusion protein is localized diffusely in the cytosol, but upon the induction of autophagy, it is localized to autophagic vacuoles due to the lipidation of LC3. As the red fluorescence signal produced by mRFP of this fusion protein is not sensitive to acid whereas the green fluorescence signal produced by GFP is [
Rubicon and UVRAG have antagonistic activities in the regulation of maturation of autophagosomes [
To test whether Rubicon indeed negatively regulated the maturation of autophagosomes in the first 24 hours after HCV infection, we performed the siRNA knockdown experiment to suppress the expression of Rubicon in Huh7.5 cells, which were then infected with HCV for either 24 hours or 48 hours. As shown in
Huh7.5 cells were transfected with the negative control siRNA (NC) or the Rubicon (Rb) siRNA for 48 hours and then infected with 1 m.o.i. of HCV. (A) Western-blot analysis of cell lysates at different time points after HCV infection. Actin served as the loading control. (B) Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *, p < 0.05. siNC, negative control siRNA; siRb, Rubicon siRNA. (C) Fluorescence imaging of RFP and GFP puncta in cells transfected with the control siRNA (top two panels) or the Rubicon siRNA (bottom two panels). Cells were fixed at 24 and 48 hours after HCV infection for the analysis. Boxed areas in merged images are enlarged and shown to the right. (D) Percentages of RFP puncta that were also positive for GFP in Huh7.5 cells treated with either the control siRNA or the Rubicon siRNA. The results represent the average of >50 cells.
To further confirm the role of Rubicon in the maturation of autophagosomes, we transfected stable Huh7.5 cells that expressed mRFP-GFP-LC3 with the control or the Rubicon siRNA followed by HCV infection. As shown in
If Rubicon indeed negatively regulated the maturation of autophagosomes in HCV-infected cells, then its over-expression using an expression vector should further inhibit the maturation of autophagosomes, even at 48 hours post-infection. To test this possibility, we transfected Huh7.5 cells with an expression plasmid of Flag-tagged Rubicon. The transfection efficiency was determined by immunostaining, which revealed that most cells were positive for the Flag-tagged Rubicon (
Huh7.5 cells were transfected with the control vector or the Flag-tagged Rubicon expression plasmid for 24 hours followed by infection with HCV. (A) Western-blot analysis of cell lysates at different time points after infection. Mock-infected cells were lysed at 48 hours post-transfection. (B) Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *, p < 0.05. (C) RFP and GFP puncta in cells with the over-expression of Rubicon at different time points after HCV infection. Merged images are shown to the right. (D) Percentages of RFP puncta that were also positive for GFP in Huh7.5 cells transfected with either the control vector or the Rubicon expression plasmid. The results represent the average of >50 cells.
To confirm the role of Rubicon in autophagosomal maturation, we also expressed Flag-tagged Rubicon in stable mRFP-GFP-LC3 cells followed by infection with HCV. As shown in
In contrast to the first 24 hours of infection, the maturation of autophagosomes and the autophagic protein degradation were efficient at 48 hours post-infection. As there was a significant increase of the UVRAG level at 48 hours (
Huh7.5 cells were transfected with the control vector or the Flag-tagged UVRAG expression plasmid for 24 hours followed by infection with HCV. (A) Western-blot analysis of cell lysates at different time points after infection. Mock-infected cells were lysed at 48 hours post-transfection. (B) Real-time RT-PCR analysis of HCV RNA at 24 and 48 hours post-infection. *,
The results described above indicated that Rubicon and UVRAG had opposite effects on HCV core protein and RNA levels in cells. To determine whether Rubicon and UVRAG also affected the yield of progeny virus, we harvested the incubation media of HCV-infected cells at 24 and 48 hours post-infection and used them to infect naive cells for the determination of viral titers and for Western-blot analysis of the HCV core protein. As shown in
Cells transfected with the control siRNA (siNC), the Rubicon siRNA (siRb), the Rubicon expression plasmid (pRubicon) or the UVRAG expression plasmid (pUVRAG) were infected with HCV (m.o.i. = 1). The incubation media were harvested at 24 and 48 hours post-infection and used to infect naïve Huh7.5 cells. Cells were either fixed and stained for the HCV core protein for the determination of viral titers (A) or lysed for Western-blot analysis of the HCV core protein (B) two days after infection. The results shown in (A) represent the average of three independent experiments, and the numbers under the core protein panels in (B) indicate the relative core protein levels, with the core protein level of control siRNA transfected cells arbitrarily defined as 1. Actin served as the loading control in (B).
To further determine how Rubicon affected HCV replication, we analyzed the HCV subgenomic RNA replicon, which expressed only the HCV nonstructural proteins NS3-NS5B and could induce autophagosomes [
(A) Increase of Rubicon, UVRAG, p62 and LC3-II in HCV subgenomic RNA replicon cells. Actin served as the loading control. Numbers under Rubicon and UVRAG indicate the protein levels of Rubicon and UVRAG in replicon cells relative to their levels in control Huh7 cells. (B) Effects of nutrient starvation on Rubicon, p62 and LC3 in Huh7 cells. Huh7 cells were nutrient-starved for 2 or 6 hours as indicated and lysed for Western-blot analysis. The replicon cells were used as the control for comparison. (C) Colocalization analysis of GFP-LC3 puncta and lysosomes. Stable Huh7 cells that expressed GFP-LC3 were nutrient-starved for 2 hours and stained with Lysotracker-red for lysosomes. The HCV replicon cells were also stained with Lysotracker-red for comparison. (D) Colocalization efficiency of GFP puncta with Lysotracker-red shown in (C). The results represent the average of >30 cells. (E) Effect of Rubicon knockdown on parental Huh7 cells and HCV replicon cells. Huh7 cells and HCV replicon cells were treated with the control siRNA or the Rubicon siRNA for two days. Cells were then lysed for Western-blot analysis. (F) Relative HCV RNA levels as measured by real-time RT-PCR. HCV replicon cells treated with either the control siRNA or the Rubicon siRNA for two days were lysed for quantification of HCV RNA by real-time RT-PCR. (G) Effect of UVRAG overexpression on HCV replicon cells. HCV replicon cells were transfected with the control vector or flag-UVRAG plasmid for two days. Cells were then lysed for Western-blot analysis. (H) Relative HCV RNA levels as measured by real-time RT-PCR. HCV replicon cells transfected with either the control vector or the flag-UVRAG plasmid for two days were lysed for quantification of HCV RNA by real-time RT-PCR. In (F) and (H), *,
To test whether Rubicon also inhibited the maturation of autophagosomes in replicon cells, we suppressed the expression of Rubicon with its siRNA. As shown in
Rubicon may enhance HCV replication via enhancing viral protein translation or viral RNA replication. To distinguish between these two possibilities, we transfected Huh7 cells with a control siRNA or the Rubicon siRNA. These cells were then transfected with a DNA plasmid that expressed a bicistronic HCV RNA, which encoded the renilla luciferase at its 5’-end and the firefly luciferase at its 3’-end. In this bicistronic RNA, the translation of renilla luciferase was cap-dependent whereas the translation of the firefly luciferase was mediated by the HCV IRES. As shown in
As the HCV subgenomic RNA replicon, which expressed HCV NS3, NS4A, NS4B, NS5A and NS5B, was sufficient to induce Rubicon, we tested whether any of these HCV gene products could induce Rubicon. We transfected Huh7 cells with the plasmids that expressed GST, NS3/4A, NS4B, NS5A and NS5B, which were all HA-tagged. The GST protein served as the negative control. As shown in
(A) Western-blot analysis of Huh7 cells transfected with the expression plasmids of HA-tagged GST and various HCV nonstructural proteins. Cells were lysed 48 hours after transfection for analysis. The localizations of molecular weight markers are indicated. The asterisk denotes a nonspecific protein band. (B) Analysis of GFP-LC3 puncta in stable Huh7 cells that expressed GFP-LC3. Cells were transfected with various expression plasmids for 48 hours and immunostained with the anti-HA antibody (red color). GFP-LC3 puncta were apparent in cells that expressed HCV NS4B. (C) The average number of GFP-LC3 puncta per cell shown in (B). The results represent the mean of >30 cells.
It has previously been shown that HCV infection can induce autophagy. In this report, we demonstrated that the induction of the autophagic response by HCV was rapid and could be detected as early as six hours post-infection (
In the normal autophagic pathway, UVRAG, in complex with Beclin-1, p150 and Vps34, facilitates the fusion between autophagosomes and lysosomes to form autolysosomes. The induction of Rubicon by HCV in the early stage of infection inhibits the UVRAG activity and the fusion between autophagosomes and lysosomes. This leads to the accumulation of autophagosomes, which enhance HCV RNA replication. The induction of UVRAG in the late stage of HCV infection overcomes the inhibitory effect of Rubicon and results in the maturation of autophagosomes. In the model illustrated, the effect of HCV on the initiation of autophagy is not addressed.
Our studies also demonstrated that Rubicon enhanced HCV replication whereas UVRAG inhibited HCV replication (
We had also studied the mechanism of Rubicon induction by HCV. Our results indicated that HCV NS4B was sufficient to induce its expression and the accumulation of autophagosomes (
Many viruses can perturb the autophagic pathway to enhance their replications and, similar to HCV, some viruses can inhibit the fusion between autophagosomes and lysosomes [
Huh7 and its derivative Huh7.5 (gift of Dr. Charles Rice, Rockefeller University) are human hepatoma cell lines [
The plasmid DNA was mixed with the BioT transfection reagent (Bioland) in serum-free DMEM to a final concentration of 2μg/mL per the manufacturer’s protocol. This transfection mixtures was incubated at room temperature for 20 min prior to inoculation into cells. Two days after transfection, cells were harvested for further studies.
The primary antibodies used in this study included the rabbit anti-Rubicon antibody (Abcam), rabbit anti-UVRAG antibody (Sigma-Aldrich), rabbit anti-p62 antibody (Cell Signaling), mouse anti-HCV NS5A monoclonal antibody (Millipore), rabbit anti-LC3 antibody (MBL), and rabbit anti-core antibody [
For the siRNA knockdown experiment, siRNAs (100 μM) against Rubicon (SASI_Hs02_00346051) and UVRAG (SASI_Hs01_00113688) (Sigma-Aldrich) were transfected into cells using Lipofectamine RNAiMAX (Invitrogen) in Opti-MEM (Invitrogen). Briefly, 4 × 104 cells seeded in a 35-mm dish were transfected with 2 μl of siRNAs (100 μM each) for 6 h and then the transfection mixture was replaced by fresh DMEM. Replicon cells were harvested 48 hours post-transfection for protein and RNA analyses, and Huh7.5 cells were infected with HCV using a multiplicity of infection (m.o.i.) of 1. Infected cells were then harvested at various time points for further analysis.
Huh7.5 cells were seeded onto the 8-well chamber slide (2x104 cells/well) and inoculated with serially diluted HCV the next day. Forty-eight hours after infection, cells were washed with phosphate-buffered saline (PBS) and fixed with 3.7% paraformaldehyde for 15 minutes. Cells were then stained with the rabbit anti-core primary antibody for 2 hours and then with the Alexa-488-conjugated goat anti-rabbit secondary antibody for 2 more hours. After washing, cells on the slide were mounted with VectorShield with DAPI. The HCV core-positive cells were counted under the microscope for titration
Cells were washed with PBS and lysed with M-PER Mammalian Protein Extraction Reagent (Thermo). After centrifugation to remove cell debris, cell lysates were subjected for SDS-PAGE electrophoresis. After the semi-wet transfer, the membrane was blocked with 5% skim milk for 1 hour and incubated with the primary antibody overnight. After three washes with PBS containing 1% Tween 20 (PBST), the membrane was incubated with the HRP-conjugated secondary antibody for 1 hour. After further washes with PBST, chemiluminescent substrates (Pierce) were applied on the membrane, and the image was captured using the LAS-4000 imaging system (FujiFilm).
Total RNA was isolated from Huh7.5 cells using TRIZOL (Invitrogen) following the manufacturer’s protocol. RNA thus isolated was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen) and oligo(d)T primers in the presence of RNasin (Promega). Gene-specific primers were used to amplify cDNA. qPCR was performed using the Taqman PCR core reagent system (Roche) and analyzed by the Fast Real-Time PCR system (ABI). Semi-quantitative PCR was performed using GoTaq Green Master Mix (Promega) and the products were analyzed by DNA gel electrophoresis.
For Lysotracker staining, cells were incubated in growth media containing 50 nM LysoTracker Red DND-99 (Invitrogen, Carlsbad, CA) at 37°C for 1.5 hours. After the incubation, cells were rinsed with phosphate-buffered saline (PBS) and then fixed with 3.7% formaldehyde. Cells were permeabilized with PBS containing 0.1% saponin, 1% bovine serum albumin (BSA) and 0.05% sodium azide for 5 minutes, and incubated with antibodies for immunofluorescence microscopy. Cover-slips were mounted in VectorShield (Vector) containing DAPI, which stained the DNA. Images were acquired with the Keyence All-in-one fluorescence microscope. 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 cells (>50) using the Image J imaging software.
(A) Huh7.5 cells were infected by the HCV JFH-1 variant (m.o.i. = 1) and stained for the HCV core protein (green color) at 48 hours post-infection. Mock-infected cells were used as the control. Nuclei were stained with DAPI (blue color). (B) Stable Huh7.5 cells that expressed the mRFP-GFP-LC3 were infected with HCV (m.o.i. = 1). The HCV core protein was stained with the anti-core antibody (blue color). The inset (boxed) was enlarged and shown to the right.
(TIF)
Huh7 cells were infected by HCV (m.o.i. = 1) and lysed at different time points after infection for Western-blot analysis. Numbers under Rubicon and UVRAG panels indicated the expression levels of these proteins relative to the mock-infected control (i.e., 0 hours p.i.)
(TIF)
Huh7.5 cells infected by HCV were lysed at different time points for the isolation of total cellular RNA. The levels of Rubicon and UVRAG RNAs were then analyzed by the semi-quantitative RT-PCR. The actin RNA was also analyzed to serve as an internal control. Numbers under the Rubicon and UVRAG panels indicated the fold increase of the RNA level relative to the 0 hour.
(TIF)
Huh7.5 cells were transfected with the control vector or the expression plasmid of Flag-tagged Rubicon or Flag-tagged UVRAG. The transfection efficiency was then analyzed by immunofluorescence staining using the anti-Flag antibody.
(TIF)
(A) Huh7.5 cells were transfected with either the control siRNA (NC) or the UVRAG siRNA (UV) for 48 hours followed by HCV infection for 24 hrs. Cells were then lysed for Western-blot analysis. (B) HCV subgenomic replicon cells were transfected with either the control siRNA or the UVRAG siRNA for 48 hours. Cell lysates were then subjected to Western-blot analysis.
(TIF)
Huh7 cells were transfected with either the control siRNA (siNC) or the Rubicon siRNA (siRb) for two days followed by the transfection of the reporter plasmid pHL-RL. pHL-RL expressed a bicistronic RNA (see illustration on the top of the Fig), which encoded the renilla luciferase at the 5’-end and the firefly luciferase at the 3’-end. The translation of the renilla luciferase was cap-dependent whereas that of the firefly luciferase was under the control of the HCV IRES. The relative HCV IRES activity was determined by dividing the firefly luciferase activity of siRb-transfected cells with that of siNC-transfected cells after the normalization of the firefly luciferase activity against the renilla luciferase activity. n.s., statistically not significant.
(TIF)
We thank members of JHJO’s lab for their constructive comments and suggestions during the studies. We are also grateful to Dr. Jae Jung for providing us with Rubicon and UVRAG expression plasmids and Dr. David Ann for the plasmid that expressed the tandem reporter mRFP-GFP-LC3.