Effects of Rubicon on autophagosomal maturation and HCV RNA replication in HCV replicon cells.

<p>(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), *, <i>p</i><0.05.</p

Suppression of Rubicon expression enhanced the maturation of autophagosomes and inhibited HCV RNA replication.

<p>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.</p

HCV Induces the Expression of Rubicon and UVRAG to Temporally Regulate the Maturation of Autophagosomes and Viral Replication

<div><p>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.</p></div

Analysis of autophagosomal maturation in HCV-infected cells.

<p>(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.</p

Model for the roles of Rubicon and UVRAG in the maturation of autophagosomes in HCV-infected cells.

<p>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.</p

Over-expression of UVRAG enhanced the maturation of autophagosomes and reduced HCV RNA replication.

<p>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. *, <i>p</i> < 0.05. (C) RFP and GFP puncta in cells with the over-expression of UVRAG 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 UVRAG expression plasmid. The results represent the average of >50 cells.</p

Effects of HCV nonstructural proteins on the induction of Rubicon and autophagosomes.

<p>(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.</p

Over-expression of Rubicon inhibited the maturation of autophagosomes and enhanced HCV RNA replication.

<p>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.</p

Analysis of the effects of Rubicon and UVRAG on HCV yield.

<p>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).</p

TNF-α Induced by Hepatitis C Virus via TLR7 and TLR8 in Hepatocytes Supports Interferon Signaling via an Autocrine Mechanism

<div><p>Invasion by infectious pathogens can elicit a range of cytokine responses from host cells. These cytokines provide the initial host defense mechanism. In this report, we demonstrate that TNF-α, a pro-inflammatory cytokine, can be induced by hepatitis C virus (HCV) in its host cells in a biphasic manner. The initial induction of TNF-α by HCV was prompt and could be blocked by the antibody directed against the HCV E2 envelope protein and by chemicals that inhibit endocytosis, indicating the specificity of endocytic uptake of HCV in this induction. Further studies indicated that the induction of TNF-α was dependent on toll-like receptors 7 and 8 (TLR7/8) but not on other intracellular pattern recognition receptors. Consistently, siRNA-mediated gene silencing of the downstream effectors in the TLR7/8 signaling pathway including MyD88, IRAK1, TRAF6, TAK1 and p65 NF-κB suppressed the expression of TNF-α. The role of p65 NF-κB in the induction of TNF-α via transcriptional up-regulation was further confirmed by the chromatin immunoprecipitation assay. TNF-α induced by HCV could activate its own receptor TNFR1 on hepatocytes to suppress HCV replication. This suppressive effect of TNF-α on HCV was due to its role in supporting interferon signaling, as the suppression of its expression led to the loss of IFNAR2 and impaired interferon signaling and the induction of interferon-stimulated genes. In conclusion, our results indicate that hepatocytes can sense HCV infection via TLR7/8 to induce the expression of TNF-α, which inhibits HCV replication via an autocrine mechanism to support interferon signaling.</p></div