Chronic hepatitis B virus infection is a leading cause of cirrhosis and liver cancer1,2. Hepatitis B virus encodes the regulatory HBx protein whose primary role is to promote transcription of the viral genome, which persists as an extrachromosomal DNA circle in infected cells3,4,5. HBx accomplishes this task by an unusual mechanism, enhancing transcription only from extrachromosomal DNA templates6. Here we show that HBx achieves this by hijacking the cellular DDB1-containing E3 ubiquitin ligase to target the ‘structural maintenance of chromosomes’ (Smc) complex Smc5/6 for degradation. Blocking this event inhibits the stimulatory effect of HBx both on extrachromosomal reporter genes and on hepatitis B virus transcription. Conversely, silencing the Smc5/6 complex enhances extrachromosomal reporter gene transcription in the absence of HBx, restores replication of an HBx-deficient hepatitis B virus, and rescues wild-type hepatitis B virus in a DDB1-knockdown background. The Smc5/6 complex associates with extrachromosomal reporters and the hepatitis B virus genome, suggesting a direct mechanism of transcriptional inhibition. These results uncover a novel role for the Smc5/6 complex as a restriction factor selectively blocking extrachromosomal DNA transcription. By destroying this complex, HBx relieves the inhibition to allow productive hepatitis B virus gene expression.
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We are most grateful to M. Rivoire for providing liver samples, L. Lefrancois and M. Michelet for help in preparing hepatocytes, U. Protzer for the HBV(ΔX)-producing cell line, C. J. Gloeckner for the StrepII/Flag TAP-tag construct, C. E. P. Goldring for the tetracyclin-inducible HepG2 cell line, S. Elledge for the lentiviral pINDUCER vectors, A. R. Lehmann for anti-Smc5 and anti-Smc6 antibodies, M. A. Petit for anti-HBsAg and anti-HBcAg antibodies, D. Garcin for the Cardif shRNA construct, Q. Seguín-Estévez, Y. Grimaldi and P. Ferrari for help with the ChIP assay, P. Arboit and the Geneva Proteomics Core Facility for mass spectrometry analysis, the Centre d’imagerie Quantitative Lyon-Est for help in confocal microscopy, A. Joshi for statistical analysis support, and J. Curran for reading the manuscript. This study was supported by grants from CLARA (Lyon) and the French National Agency for Research against AIDS and viral hepatitis (ANRS) (to O.H.) and from the Swiss National Science Foundation (31003A-127384 and 310030-149626) (to M.S.) and by the Canton of Geneva (to M.S).
This study was partly funded by Gilead Sciences, Inc., and R.K.B., C.M.L., C.N. and S.P.F. are employees of Gilead Sciences, Inc. The other authors declare no competing financial interests.
Extended data figures and tables
a, Stable HepG2 cell lines were generated expressing GFP or the indicated GFP-tagged viral proteins carrying an N-terminal tandem Flag/StrepII (FS) tag from a doxycycline-inducible promoter (ref. 29 and see Methods). After doxycycline induction, whole-cell extracts were prepared and the FS-tagged and associated proteins were purified by TAP. Eluted proteins were separated by gel electrophoresis and stained with Coomassie blue. CUL4 was identified by mass spectrometry. No protein candidate was identified showing a profile predicted for an HBx substrate: that is, co-purifying specifically with HBx and WHx but not with SV5-V, or co-purifying only with the DDB1-binding-defective HBx(R96E) mutant because of its otherwise unstable interaction with HBx and WHx when recruited to the E3 ligase. In particular, the Smc5/6 complex was not detected in these purifications. b, Purification of SV5-V and associated proteins. FS-tagged SV5-V was purified from a stably expressing HepG2 cell line by TAP as above. Proteins were eluted under native conditions and processed for analysis by peptide fragmentation sequencing (nano-LC–ESI MS/MS). The number of unique peptides for each protein and the percentage of total protein sequence covered by these peptides are indicated. Listed are proteins identified with 100% certainty and represented by at least two peptides identified at a 95% confidence level. CUL4A and CUL4B are closely related paralogs. Note that no peptides were detected, even at low confidence level, for STAT1 that is well known to be recruited by SV5-V to the DDB1 E3 ligase for ubiquitin-mediated degradation. By contrast MDA5, a cytoplasmic sensor of viral RNA to which SV5-V also binds but, in contrast to STAT1, without causing its degradation44, was present in the SV5-V purifications. This suggests that, in contrast to MDA5, STAT1 associates with SV5-V very transiently, presumably because of its rapid degradation or dissociation from the E3 ligase complex. We considered that the same may be true for the HBx target, thus precluding its identification by regular affinity purification. c, As a proof of principle for the use of a fusion strategy to identify the HBx target, we covalently linked SV5-V to wild-type DDB1 or to a CUL4-binding-defective DDB1 mutant that could not incorporate in the E3 ligase complex. See text for details. d, HepG2 cells were mock transfected (−) or transfected with plasmid DNA expressing the indicated FS-tagged SV5-V–DDB1 fusions. Whole-cell extracts were prepared and the fusion proteins purified by a single round of affinity purification. The amounts of fusion proteins recovered (IP) and the presence of STAT1 in the eluates (co-IP) were assessed by western blotting. The fusion proteins were revealed using anti-Flag antibodies and were expressed at too low levels for detection in the crude extracts (input). Note that high amounts of STAT1 are recovered only with the mutant SV5-V–DDB1 fusion. e, Architecture of the Smc5/6 complex. The core of the Smc5/6 complex is formed by a heterodimer of Smc5 and Smc6. The two proteins form a V-shaped structure and associate with four non-SMC proteins, designated Nse1–Nse4 (refs 15, 17). Note that depletion of any of the Smc5/6 complex subunit, other than Nse2, results in destabilization and degradation of the entire complex21.
Extended Data Figure 2 HBx induces rapid depletion of the Smc5/6 complex by an MG132 proteasome inhibitor-sensitive pathway to stimulate extrachromosomal reporter gene activity.
a, Same experiment as in Fig. 2a except that luciferase activity and Smc6 protein levels were monitored at the indicated time points after transduction of GFP or GFP–HBx. Luciferase activities are expressed in arbitrary units, with the fold increase in HBx-expressing cells relative to GFP control cells indicated on top. GFP–HBx was detected using anti-GFP antibodies. One out of two independent experiments. b, HepG2 cells were transduced with lentiviral vectors encoding GFP or GFP–HBx. Then, 16 h later, DMSO (control) or 10 μM MG132 was added to the culture to inhibit proteasome activity. Cells were harvested 8 h later and the level of the indicated proteins and of global ubiquitination was analysed by western blotting. GFP–HBx was detected using anti-GFP antibodies. One out of two independent experiments. c, The effect of the indicated viral proteins on Smc5, Smc6, and luciferase mRNA levels was determined by real-time RT–PCR. The values are relative to those measured in GFP-transduced cells, which were set to 1. d, Similar experiment as in Fig. 2b except that two concentrations of MLN4924 were used. e, Similar experiment as in Fig. 2c. f, Similar experiment as in Fig. 2d but with HepG2 cells containing the HBV Enhancer-I-driven firefly luciferase reporter gene (HBV-FLuc) integrated into the chromosome and an episomal Renilla luciferase construct driven by the CMV promoter (CMV-RLuc). Note that the shRNAs against Smc5 and Smc6 used in this experiment differ from those in Fig. 2d (see Methods). g, Same with the Renilla luciferase CMV promoter construct chromosomally integrated and a firefly luciferase reporter gene driven by the EF1α promoter episomal. Data in c, f and g represent the mean ± s.e.m. of three independent experiments.
Extended Data Figure 3 Smc5/6 degradation by HBx does not alter the cell cycle or promote chromosomal integration of the reporter but prevents the binding of Smc5/6 to the episomal DNA template.
a, HepG2 cells were transfected with the HBV Enh1 luciferase reporter plasmid, split, and then either mock-transduced or transduced in triplicates with lentiviral vectors encoding GFP or GFP–HBx. Luciferase activity and protein expression (left panel) were monitored as in Fig. 2 at 5 days after transfection. In parallel, cells were analysed by FACS for GFP positivity (middle inset) and for DNA content (right panels) after propidium iodide staining. b, HBx does not promote stable integration of the reporter gene into chromosomes. HepG2 cells were transduced with a lentiviral vector to chromosomally integrate a Renilla luciferase reporter gene and subsequently transfected with a firefly reporter construct. Cells were then split equally and transduced with lentiviral vectors encoding GFP or GFP–HBx. Activity of the episomal firefly (left) and integrated Renilla (right) luciferase genes was monitored at the indicated time points after transduction of GFP or GFP–HBx. The values are expressed in arbitrary units relative to those measured at day 1 in the GFP control, which was set to 1.0. Indicated above the columns in the left panel is the remaining luciferase activity (expressed in per cent) relative to that measured 3 days after transfection. Note that in the absence of HBx (black bars in the left panel) the episomal firefly luciferase signal decreases rapidly with time, presumably because of loss of the reporter plasmid. In the presence of HBx (red bars in the left panel), the signal slightly increases to reach sixfold higher levels relative to the GFP control at 3 days after transfection, and then drops with kinetics close to that observed in the GFP control. In contrast, expression of the integrated Renilla reporter remains constant (right panel). This argues against HBx stimulating episomal reporter activity by promoting its integration into chromosomes. c, Left panels, the same ChIP experiment using anti-HA antibodies as in Fig. 2e but with HepG2 cells containing the HBV Enhancer-I-driven firefly luciferase reporter (HBV Enh1-FLuc) integrated into the chromosome and an episomal EF1α Renilla luciferase construct (EF1α-RLuc) as a control. Data represent the mean ± s.e.m. of three independent experiments. In one experiment, the unbound material from the indicated samples expressing no HA-tagged Nse4 was further purified using anti-Nse4 antibodies (lower right). Data are expressed as the percentage of input DNA recovered by ChIP. Note that in this experiment, the episomal reporter was delivered using an integrase-defective lentiviral vector32.
a, PHHs were infected with normalized stocks of wild-type (WT) or HBx-deficient (ΔX) HBV as in Fig. 3. HBeAg secretion (top) was assessed 10 days later by ELISA. In parallel, the amounts of cytoplasmic DNA replicative intermediates produced during reverse transcription of the viral pregenomic RNA (middle) and of the nuclear episomal HBV template extracted using a modified Hirt procedure (bottom) were analysed by Southern blot as reported34. b, PHHs were either mock transduced (none) or transduced with a lentiviral construct expressing an shRNA specific for Smc6 and the next day infected with HBx-deficient HBV particles (HBV(ΔX) as in Fig. 3c. HBeAg secretion was assessed 12 days later by ELISA (top). In parallel, the amount of nuclear HBV genome was quantified by real-time FRET–PCR34 both directly or after treatment with Plasmid-safe DNase (Epicentre), an exonuclease that degrades single-stranded and double-stranded linear DNA but not the episomal HBV DNA (bottom left). Shown as a control for Plasmid-safe DNase treatment are the results for the chromosomal β-globin gene (bottom right).
a, Purified PHHs were either left uninfected (0) or infected with wild-type HBV particles at the indicated viral-genome equivalents per cell (see Methods). HBeAg and surface antigen (HBsAg) secretion into the culture supernatants was assessed 10 days later by ELISA. Concentrations are expressed in, respectively, national clinical units (NCU) per millilitre (HBeAg) and nanograms per millilitre (HBsAg) and were determined according to the manufacturers’ guidelines (Autobio Diagnostics). The noise signals measured with the supernatants from the uninfected cells were 0.50 NCU ml−1 and 0.88 ng ml−1. b, PHHs infected with normalized stocks of wild-type (WT) or HBx-deficient (ΔX) HBV at the indicated viral genome equivalents per cell were examined for HBsAg expression at 9 days after infection by indirect immunofluorescence confocal microscopy. Cell nuclei were stained with Hoechst dye (blue). c, Quantification of confocal images. Data are mean ± s.d. of at least three fields. Note that infection in Fig. 3 was with 200 viral genome equivalents per cell or more.
Extended Data Figure 6 Silencing of Smc5/6 restores HBx-negative HBV transcription and rescues wild-type HBV on a DDB1 knockdown background.
a, Biological replicate of Fig. 3a using PHHs from a different donor. b, Control for lentiviral shRNA-mediated depletion in PHHs. PHHs were transduced with lentiviral constructs expressing the indicated shRNAs and infected with HBV as in Fig. 3c. HBsAg secretion and the amounts of the indicated proteins were assessed 9 days later by ELISA and western blot analysis. HBsAg concentrations are relative to those measured in mock-transduced cells infected with wild-type HBV, which were taken as 100 (not shown). c, Independent northern blot analysis of HBV RNA production as in Fig. 3c using PHHs from a different donor. All lanes are from the same gel and exposure. Shown in the upper panel is the corresponding ELISA for HBeAg secretion. d, Smc5/6 silencing restores expression of HBx-negative HBV in HepaRG cells. Differentiated HepaRG cells were either left uninfected or infected with normalized stocks of wild-type (WT) or HBx-deficient (ΔX) HBV particles. Twenty-four hours later, cells were either mock transfected (none) or transfected with the indicated siRNA. HBeAg secretion was measured 10 days after infection by ELISA. The values are in arbitrary units relative to the wild-type HBV control, which was given a value of 100. Shown in the upper and lower panels are two independent experiments. CyPB, cyclophilin B. e, HBV mRNA expression. Total RNA was extracted from the samples analysed for HBeAg secretion in Fig. 3e. HBV mRNA levels were measured by real-time RT–PCR. The values normalized to β-actin are given in arbitrary units relative to those measured in mock-transfected cells, which were set to 100. Data represent the mean ± s.e.m. of n = 4 independent experiments performed with two different PHH donors. f, Control of siRNA efficacy. DDB1 (grey bars) and Smc6 (black bars) mRNA levels in Fig. 3e were measured by real-time RT–PCR and normalized to β-actin as in e. The values are given in arbitrary units relative to those measured in mock-transfected cells, which were set to 100. Data represent the mean ± s.e.m. of n = 4 independent experiments performed with two different PHH donors. g, Control western blot analysis of siRNA-mediated protein depletion. PHHs were transfected with the indicated siRNAs 3 days after plating. Protein levels were measured 10 days later. h, Data of one of the four experiments used in Fig. 3e that includes an siRNA targeting Smc5. Mean ± s.e.m. of triplicate measurements. i, Same experiment as in h but measuring HBV mRNA levels. Mean ± s.e.m. of triplicate measurements.
a, Same as in Fig. 3b but 4 × 4 contiguous images were acquired and stitched together to produce a single image for examination of 200–300 PHHs. Shown are Smc6 (green), HBcAg (red), and DAPI (blue). b, Representative images of uninfected PHHs stained with mouse isotype-control-matched IgG2a primary antibodies or Alexa Fluor 488- and 594-conjugated secondary antibodies alone. Samples were imaged and processed as in Fig. 3b.
a, Same as in Fig. 4a but with different animal samples. b, Specificity of detection of human Smc6 in humanized mouse liver tissues. Fresh-frozen uninfected (animal 62) and infected (animal 12) humanized mouse liver tissues were stained with DAPI nuclear stain (blue) and with antibodies against human albumin (red) and human Smc6 (green). In the middle and right panels, a yellow dotted line delineates the interface between the human (Hu) and mouse (Ms) hepatocyte populations.
Extended Data Figure 9 Human albumin and human cytokeratin-18 as markers for human hepatocytes in humanized mouse liver tissues.
Fresh-frozen uninfected (animal 54) or infected (animal 12) humanized mouse liver tissues were stained with DAPI nuclear stain (blue) and with antibodies against human albumin (red) and human cytokeratin-18 (CK-18, green). A yellow dotted line delineates the interface between the human (Hu) and mouse (Ms) hepatocyte populations in the third column from left. Shown on the right are control staining of liver tissue from animal 12 with secondary antibody only or IgG1 isotype control.
This file contains the raw data for Figures 1-4 and Extended Data Figures 1,2, 3, 4, and 6. (PDF 7763 kb)
This file contains the full mass spectrometric data of the HBx-DDB1 interactions of Fig. 1b. (XLS 31 kb)
This file contains the siRNA and ChIP primers used. (XLSX 12 kb)
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Decorsière, A., Mueller, H., van Breugel, P. et al. Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor. Nature 531, 386–389 (2016). https://doi.org/10.1038/nature17170
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