Subversion of cellular autophagy machinery by hepatitis B virus for viral envelopment

Abstract

Autophagy is a conserved eukaryotic mechanism that mediates the removal of long-lived cytoplasmic macromolecules and damaged organelles via a lysosomal degradative pathway. Recently, a multitude of studies have reported that viral infections may have complex interconnections with the autophagic process. The findings reported here demonstrate that hepatitis B virus (HBV) can enhance the autophagic process in hepatoma cells without promoting protein degradation by the lysosome. Mutation analysis showed that HBV small surface protein (SHBs) was required for HBV to induce autophagy. The overexpression of SHBs was sufficient to induce autophagy. Furthermore, SHBs could trigger unfolded protein responses (UPR), and the blockage of UPR signaling pathways abrogated the SHB-induced lipidation of LC3-I. Meanwhile, the role of the autophagosome in HBV replication was examined. The inhibition of autophagosome formation by the autophagy inhibitor 3-methyladenine (3-MA) or small interfering RNA duplexes targeting the genes critical for autophagosome formation (Beclin1 and ATG5 genes) markedly inhibited HBV production, and the induction of autophagy by rapamycin or starvation greatly contributed to HBV production. Furthermore, evidence was provided to suggest that the autophagy machinery was required for HBV envelopment but not for the efficiency of HBV release. Finally, SHBs partially colocalized and interacted with autophagy protein LC3. Taken together, these results suggest that the host's autophagy machinery is activated during HBV infection to enhance HBV replication.

Fig. 1.

HBV induces autophagosome formation in hepatoma cells without promoting protein degradation by the lysosome. (A) Huh7 cells were treated with rapamycin (50 nM) or transfected with pUC19 or pHBV1.3. After transfection for 46 h, pHBV1.3-transfected cells were left untreated or were treated with 3-MA (10 mM) for 2 h. After 48 h of transfection or treatment, the levels of LC3-II expression were determined by Western blot analysis. β-Actin expression was examined as a protein loading control. (B) The LC3-II/β-actin ratios (% of rapamycin) were determined by Western blot analysis, and the bands were quantified by densitometric analysis using NIH ImageJ software. Results represent the mean data from three independent experiments. *, P < 0.05. (C) Huh7 cells were transfected with GFP-LC3, and after 12 h they were treated or transfected as described for panel A. The nuclei were stained by DAPI (blue stain), and the distribution of GFP-tagged LC3 protein was visualized with a confocal fluorescence microscope. Representative confocal images are shown. (D) Quantification of the frequency of Huh7 cells displaying a punctate distribution of GFP-tagged LC3 protein was performed as described in Materials and Methods. Results represent the mean data from three independent experiments. *, P < 0.05. (E) Huh7 cells were treated or transfected as described for panel A. After 48 h of transfection or treatment, cells were subjected to transmission electron microscopy. Arrows indicate representative autophagosomes. (F) Quantification of numbers of autophagosomes was performed as described in Materials and Methods. Results represent the mean data from two independent experiments. *, P < 0.05. (G) Huh7 cells were transfected with pUC19 or pHBV1.3 or treated with DMSO or rapamycin. After 48 h of transfection or treatment, the levels of p62 protein expression were determined by Western blot analysis. β-Actin expression was examined as a protein loading control. (H) The p62/β-actin ratios (% of rapamycin) were determined as described for panel B. Results represent the mean data from three independent experiments. *, P < 0.05.

Fig. 2.

SHBs is required for HBV to induce autophagy. (A) Huh7 cells were transfected with pHBV1.3, pHBV1.3-Pol⁻, pHBV1.3-HBx⁻, pHBV1.3-ENV⁻, pHBV1.3-SHBs⁻, pcDNA3.1-Flag, or pcDNA3.1-Flag-SHBs together with pEGFP-C2 plasmid. After 48 h of transfection, the levels of LC3-II expression were determined by Western blot analysis. β-Actin and EGFP expression levels were examined to control for loading and transfection efficiency. (B) The LC3-II/β-actin ratios (% of pHBV1.3) were determined as described in the legend to Fig. 1B. Results represent the mean data from three independent experiments. *, P < 0.05. (C) Huh7 cells were transfected with GFP-LC3 plasmid, and after 12 h they were transfected with the plasmids described for panel A (excluding pEGFP-C2). The nuclei were stained by DAPI, and the distribution of GFP-tagged LC3 protein was visualized with a confocal fluorescence microscope. Representative confocal images are shown. (D) Quantification of the frequency of Huh7 cells displaying a punctate distribution of GFP-tagged LC3 protein was performed as described in Materials and Methods. Results represent the mean data from three independent experiments. *, P < 0.05. (E) Huh7 cells were transfected with pcDNA3.1-Flag, pcDNA3.1-Flag-SHBs, or pcDNA3.1-Flag-SHBs (Cys48Ala) or treated with BSA or HBsAg (200 ng/ml). After 48 h of transfection or treatment, the levels of LC3-II and p62 expression were determined by Western blot analysis. β-Actin expression was examined as a protein loading control. (F) The ratios of LC3-II/β-actin or p62/β-actin (% of pcDNA3.1-Flag) were determined as described in the legend to Fig. 1B. Results represent the mean data from two independent experiments. *, P < 0.05.

Fig. 3.

SHBs triggers ER stress. (A) Huh7 cells were transfected with pcDNA3.1-Flag, pcDNA3.1-Flag-SHBs, pHBV1.3-SHBs⁻, or pHBV1.3 for 48 h or were treated with DTT (2 mM) for 2 h. The phosphorylation levels of PERK and eIF2α were determined by Western blot analysis. β-Actin expression was examined as a protein loading control. (B) The ratios of p-PERK/β-actin or p-eIF2α/β-actin (% of DTT) were determined as described for Fig. 1B. Results represent the mean data from two independent experiments. *, P < 0.05. (C) Huh7 cells were transfected or treated as described for panel A. The cleavage of ATF6 was determined by Western blot analysis. β-Actin expression was examined as a protein loading control. (D) The ratios of ATF6/β-actin or GRP94/β-actin (% of DTT) were determined as described for Fig. 1B. Results represent the mean data from two independent experiments. *, P < 0.05. (E) Huh7 cells were transfected or treated as described for panel A. Total cellular RNA was analyzed for XBP1 mRNA by RT-PCR as described in Materials and Methods. XBP1 (U) and XBP1 (S) represent DNA fragments derived from unspliced and spliced XBP1 RNA, respectively. The GAPDH mRNA was analyzed to serve as an internal control. (F) The XBP1 (S)/GAPDH ratios (% of DTT) were determined as described for Fig. 1B. Results represent the mean data from two independent experiments. *, P < 0.05.

Fig. 4.

Blockage of UPR signaling pathways by RNA interference abrogated the SHBs-induced lipidation of LC3-I. (A) Huh7 cells were transfected with 40 nM siRNA duplexes targeting EGFP or PERK (siPERK). At 24 h posttransfection, the cells were transfected with 20 nM the same siRNA duplexes together with pcDNA3.1-Flag, pcDNA3.1-Flag-SHBs, pHBV1.3, or pHBV1.3-SHBs⁻ for 48 h as indicated. The levels of LC3-II expression were determined by Western blot analysis. The effectiveness of siRNA duplexes was detected by Western blot analysis using the antibodies against PERK or eIF2α. β-Actin expression was examined as a protein loading control. (B) Huh7 cells were transfected as described for panel A, except that siRNA duplexes targeting ATF6 (siATF6) were used in place of the siRNA duplexes targeting PERK. The levels of LC3-II expression were determined by Western blot analysis. The effectiveness of siRNA duplexes was detected by Western blot analysis using antibodies against ATF6 or GRP94. β-Actin expression was examined as a protein loading control. (C) Huh7 cells were transfected as described for panel A, except that siRNA duplexes targeting IRE1α (siIRE1α) were used in place of the siRNA duplexes targeting PERK. The levels of LC3-II expression were determined by Western blot analysis. The effectiveness of siRNA duplexes was detected by the Western blot analysis of the expression of IRE1α or by RT-PCR analysis of XBP1 splicing. β-Actin expression was examined as a protein loading control. (D) The ratios of LC3-II/β-actin (% of pcDNA3.1-Flag) in panels A, B, and C were determined as described for Fig. 1B. Results represent the mean data from two independent experiments. *, P < 0.05.

Fig. 5.

Autophagy machinery is required for HBV production. (A) Huh7 cells were transfected with pHBV1.3 and after 36 h were left untreated or were treated with 3-MA for 12 h. The amounts of nucleocapsid-associated DNA and viral RNA were determined by Southern blot and Northern blot analyses, respectively, using a ³²P-radiolabeled HBV DNA probe. After the detection of HBV RNA, the blots were stripped and rehybridized with a ³²P-radiolabeled GAPDH DNA probe to control for gel loading. The positions of HBV relaxed circular (RC), single-stranded (SS) DNAs and the positions of viral pregenomic RNA (3.5 kb), pre-S1/S RNA (2.4 kb), and pre-S2/S RNA (2.1 kb) are indicated. (B) The percentage of the inhibitory effect of 3-MA (% of control) on the abundance of HBV DNA and RNA shown in panel A was calculated. The bands were quantified by densitometric analysis using NIH ImageJ software. Results represent the mean data from two independent experiments. *, P < 0.05. (C) Huh7 cells were transfected and treated as described for panel A. Enveloped HBV virions in culture supernatant and nucleocapsids in cell lysates were precipitated with antibody against HBsAg or antibody against HBcAg, respectively, and assayed by EPA as described in Materials and Methods. SHBs in the precipitate from culture supernatant was determined by Western blot analysis. dsDNA, double-stranded DNA. (D) The percentage of inhibitory effect of 3-MA (% of control) on the abundance of extracellular virions and intracellular nucleocapsids shown in panel C was calculated as described for panel B. Results represent the mean data from two independent experiments. *, P < 0.05. (E) Huh7 cells were transfected with pHBV1.3. After 12 h of transfection they were treated with rapamycin for 36 h, or after 46 h of transfection they were transferred to a starvation medium (Earle's balanced salts) for 2 h. Enveloped HBV virions in culture supernatant and nucleocapsids in cell lysates were assayed by EPA as described in Materials and Methods. SHBs in the precipitate from culture supernatant was determined by Western blot analysis. (F) The percentage of the enhancive effect of autophagy inducers (% of control) on the abundance of extracellular virions and intracellular nucleocapsids shown in panel E was calculated as described for panel B. Results represent the mean data from two independent experiments. *, P < 0.05. (G) Huh7 cells were transfected with 40 nM siRNA duplexes targeting EGFP (siEGFP), Beclin1 (siBeclin1), or ATG5 (siATG5). At 24 h posttransfection, the cells were transfected with 20 nM the same siRNA duplexes together with pHBV1.3 for 48 h. Enveloped HBV virions in culture supernatant and nucleocapsids in cell lysates were assayed by EPA as described in Materials and Methods. The effectiveness of siRNA duplexes targeting Beclin1 or ATG5 was detected by Western blot analysis of the expression of Beclin1, ATG5, or LC3-II. (H) The percentage of the inhibitory effect of siRNA duplexes (% of control) on the abundance of extracellular virions and intracellular nucleocapsids shown in panel G was calculated as described for panel B. Results represent the mean data from two independent experiments. *, P < 0.05. (I) Huh7 cells were transfected as described for panel G, except that the siRNA duplexes targeting PERK (siPERK), ATF6 (siATF6), or IRE1α (siIRE1α) were used in place of the siRNA duplexes targeting Beclin1 or ATG5. Enveloped HBV virions in culture supernatant and nucleocapsids in cell lysates were assayed by EPA as described in Materials and Methods. (J) The percentage of the inhibitory effect of siRNA duplexes (% of control) on the abundance of extracellular virions and intracellular nucleocapsids shown in panel I was calculated as described for panel B. Results represent the mean data from two independent experiments. *, P < 0.05.

Fig. 6.

Autophagy machinery is required for HBV envelopment. (A) Huh7 cells were transfected and treated as described for Fig. 5A. The abundance of extracellular or intracellular enveloped virions and intracellular nucleocapsids was analyzed by EPA as described in Materials and Methods. dsDNA, double-stranded DNA; IP, immunoprecipitation. (B) The percentage of the inhibitory effect of 3-MA (% of control) on the abundance of extracellular or intracellular enveloped virions and intracellular nucleocapsids was calculated as described for Fig. 5B. Results represent the mean data from two independent experiments. (C) Huh7 cells were transfected as described for Fig. 5G. The abundance of extracellular or intracellular enveloped virions and intracellular nucleocapsids was analyzed by EPA as described in Materials and Methods. siEGFP, siBeclin1, and siATG5 indicate siRNA complexes targeting EGFP, Beclin1, and ATG5, respectively. (D) The percentage of the inhibitory effect of siRNA duplexes targeting Beclin1 or ATG5 (% of control) on the abundance of extracellular or intracellular enveloped virions and intracellular nucleocapsids was calculated as described for Fig. 5B. Results represent the mean data from two independent experiments. (E) pHBV1.3-ENV⁻ was transfected into Huh7 cells. The cell lysates were precipitated with antibodies against HBsAg or HBcAg, respectively, and then subjected to EPA as described in Materials and Methods.

Fig. 7.

SHBs partially colocalized and coimmunoprecipitated with autophagy protein LC3. (A) Huh7 cells were transfected with pHBV1.3 for 48 h. Cells were fixed, blocked, and incubated with anti-SHBs or anti-HBx antibodies together with anti-LC3 antibody, followed by being stained with Alexa Fluor 488-conjugated anti-mouse secondary antibody and Cy3-conjugated anti-rabbit secondary antibody. The nuclei were stained with DAPI, and the colocalization of SHBs or HBx (green stain) with LC3 (red stain) was observed with a confocal fluorescence microscope. Representative confocal images are shown. (B) Huh7 cells were transfected with pcDNA3.1-Flag-SHBs or pCDNA3.1-Flag-HBx together with pCMV-Myc-LC3 for 48 h. Coimmunoprecipitation analysis was performed with anti-Flag antibody as described in Materials and Methods. The presence of exogenous LC3 in the immunoprecipitate (IP) was detected by Western blot analysis (WB) with anti-Myc antibody. (C) Huh7 cells were transfected with pHBV1.3 for 48 h. Coimmunoprecipitation analysis was performed with anti-SHBs antibody as described in Materials and Methods. The presence of endogenous LC3 in the immunoprecipitate was detected by Western blot analysis with anti-LC3 antibody.