Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2 and either pRK5-HA-Ub or pRK5-HA-Ub-K6 and with either pUC19-HBV-C-AT_JPN, carrying a 1.3-mer overlength HBV, or pUC19-HBV-C-AT_JPN(ΔHBx), carrying a 1.3-mer overlength HBV lacking HBx gene expression. At 48 h posttransfection, cells were harvested and lysed under denaturing conditions. Cell lysates were immunoprecipitated with anti-FLAG M2 affinity beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-FLAG MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-FLAG MAb (fourth panel), anti-HBx PAb (fifth panel), anti-HBc MAb (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pEF1A-HBx-Myc-His 6 , pCAG-HUWE1, and either pRK5-HA-Ub or pRK5-HA-Ub-K6. At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (C) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pEF1A-HBx-Myc-His 6 , and the HUWE1 siRNA-resistant plasmid pCAG-HUWE1_R. At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-FLAG MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. HUWE1_R, siRNA-resistant HUWE1. (D) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pEF1A-HBx-Myc-His 6 and either pCAG-HUWE1(WT) or the catalytically inactive mutant pCAG-HUWE1(C4341A). At 48 h posttransfection, cells were harvested. Cell lysates were subjected to immunoprecipitation with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-HUWE1 PAb (fourth panel), anti-Nrf2 MAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. Data in panels A to D are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Gene Expression, Immunoprecipitation, Western Blot, Control, Plasmid Preparation, Mutagenesis

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-c-Myc MAb (top panel) or anti-HA PAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-c-Myc MAb (fourth panel), and anti-GAPDH MAb (fifth panel). GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and either pUC19-HBV-C-AT_JPN or pUC19-HBV-C-AT_JPN (ΔHBx). At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-HBx PAb (top panel), anti-HBc MAb (second panel), or anti-HA PAb (third panel). Input samples were immunoblotted with anti-HA PAb (fourth panel), anti-HBx PAb (fifth panel), anti-HBc Mab (sixth panel), and anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. (C) HepG2 cells were transfected with pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HUWE1 PAb or rabbit IgG (control), followed by immunoblotting with anti-c-Myc MAb (top panel) or anti-HUWE1 PAb (second panel). Input samples were immunoblotted with anti-c-Myc MAb (third panel), anti-HUWE1 PAb (fourth panel), and anti-GAPDH MAb (fifth panel). GAPDH served as a loading control. Data are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were transfected with pCAG-HA-HUWE1 alone, pEF1A-HBx-Myc-His6 alone, or both plasmids. At 48 h posttransfection, cells were fixed and subjected to indirect immunofluorescence staining with anti-HUWE1 rabbit PAb followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (green), and anti-c-Myc mouse MAb followed by Alexa Fluor 594-conjugated goat anti-mouse IgG (red). Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 μm. (B) HepG2 cells were co-transfected with pCAG-HA-HUWE1 and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were fixed and subjected to an in situ proximity ligation assay (PLA) using both anti-HA rabbit PAb and anti-c-Myc mouse MAb. Nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 10 μm. Data in panels A and B are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunofluorescence, Staining, In Situ, Proximity Ligation Assay

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-HA-HUWE1, pCAG-FLAG-Nrf2, and pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-HA PAb, followed by immunoblotting with anti-Nrf2 MAb (top panel), anti-c-Myc MAb (second panel), and anti-HUWE1 PAb (third panel). Input samples were immunoblotted with the indicated antibodies, and GAPDH served as a loading control. (B) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2 and pCAG-HA-HUWE1 and either pEGFP-C3 (upper panel) or pEGFP-C3-HBx (bottom panel). At 48 h posttransfection, the cells were fixed and incubated with anti-FLAG mouse MAb and anti-HA rabbit PAb, followed by an in situ PLA. Scale bar, 10 μm. (C) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, pCAG-HUWE1, and pEF1A-HBx-Myc-His 6. At 48 h posttransfection, cells were harvested. Cell lysates were immunoprecipitated with anti-FLAG beads, followed by immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with anti-HA PAb (third panel), anti-Nrf2 MAb (fourth panel), anti-HUWE1 PAb (fifth panel), anti-c-Myc MAb (sixth panel), or anti-GAPDH MAb (seventh panel). GAPDH served as a loading control. Data in panels A to C are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Immunoprecipitation, Western Blot, Control, Incubation, In Situ

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A to D) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pCAG-FLAG-Nrf2 and either pEF1A-Myc-His 6 (panels A and B) or pEF1A-HBx-Myc-His 6 (panels C and D). At 48 h posttransfection, cells were treated with 100 µg/mL cycloheximide (CHX) for 0, 1, 2, 4, or 6 h prior to harvesting. Cell lysates were then subjected to immunoblotting with the indicated antibodies. GAPDH served as a loading control. (B, D) Specific signals were quantified by densitometry, and the percentage of remaining FLAG-Nrf2 at each time point was compared with that at time zero. Closed triangles (▴) indicate control siRNA, and closed circles (●) indicate HUWE1 siRNA. Data are mean ± standard errors of the mean (SEM) of three independent experiments. * P < 0.05 compared with the control siRNA by Student’s t test. (E) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were further transfected with pCAG-FLAG-Nrf2 and either pEF1A-Myc-His 6 or pEF1A-HBx-Myc-His 6 . At 48 h posttransfection, cells were harvested and subjected to subcellular fractionation. Cytoplasmic and nuclear factions were analyzed by immunoblotting with the indicated antibodies. The relative levels of nuclear Nrf2 were quantified by densitometry and are indicated below each lane. IĸBα and histone H3 served as cytoplasmic and nuclear markers, respectively. The western blots are representative of three independent experiments.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Western Blot, Fractionation

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A to C) HepG2 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 24 h post-siRNA transfection, cells were co-transfected with pUC19-HBV-D-IND60 and the HUWE1 siRNA-resistant plasmid pCAG-HUWE1_R. At 48 h post-plasmid transfection, cells were harvested. Total RNA was extracted, and HBV RNA (A) and pgRNA (B) were quantified by RT-qPCR and normalized to GAPDH mRNA. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. HUWE1_R, siRNA-resistant HUWE1. (C) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (D-F) Hep38.7-Tet cells were transfected with 40 nM HUWE1 siRNA or control siRNA and subsequently cultured for 7 days without doxycycline to induce HBV replication. Total RNA was extracted, and HBV RNA (D) and pgRNA (E) were quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (F) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (G) HepG2-NTCP-Myc-His 6 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with recombinant HBV expressing the NanoLuc (NL) reporter gene (HBV/NL) at 100 genome equivalents (GEq)/cell. At 4 days postinfection (dpi), cells were lysed to measure NL activity as an indicator of HBV replication. Results represent the mean ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (H-J) HepG2-NTCP-Myc-His 6 cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with HBV at 1,000 GEq/cell, and harvested at 5 dpi. Total RNA was extracted, and HBV RNA (H) and pgRNA (I) were quantified by qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (J) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (K-L) PXB cells were transfected with 40 nM HUWE1 siRNA or control siRNA. At 48 h posttransfection, cells were infected with HBV at 1,000 GEq/cell and harvested at 5 dpi. Total RNA was extracted, and HUWE1 mRNA (K) was quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (L) Cell lysates were subjected to immunoblotting using anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH as the loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Western Blot, Cell Culture, Infection, Recombinant, Expressing, Activity Assay

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: (A) HepG2 cells were co-transfected with pCAG-FLAG-Nrf2, pRK5-HA-Ub-K6, and pEF1A-HBx-Myc-His 6 . At 24 h posttransfection, cells were treated with 10 µM BI8626 or equivalent volume of dimethyl sulfoxide (DMSO; vehicle control) for 48 h prior to harvesting. Cell lysates were immunoprecipitated with anti-FLAG beads and subjected to immunoblotting with anti-HA PAb (top panel) or anti-Nrf2 MAb (second panel). Input samples were immunoblotted with the indicated antibodies. GAPDH served as a loading control. (B) Cell viability of HepG2 cells treated with 10 µM BI8626 for 48 h, assessed by CellTiter-Glo v2.0 ATP assay. Results represent the mean values ± SEM from three biological replicates. (C to E) HepG2-NTCP-Myc-His 6 cells were infected with HBV at 1,000 GEq/cell. At 24 h postinfection, cells were treated with 50 µM BI8626 and maintained until harvesting. At 10 dpi, total RNA was extracted, and HBV RNA (C) and pgRNA (D) were quantified by RT-qPCR. Data represent the means ± SEM from three biological replicates. Values for control cells were arbitrarily set to 1.0. * P < 0.05 by Student’s t test. (E) Cell lysates were analyzed by immunoblotting with anti-HUWE1 PAb (top panel), anti-HBc MAb (middle panel), and anti-GAPDH MAb (bottom panel), with GAPDH serving as a loading control. The relative levels of HBc were quantified by densitometry and are indicated below each lane. (F) Cell viability of HepG2-NTCP-Myc-His 6 cells treated with 50 µM BI8626 for 48 h, assessed by CellTiter-Glo 2.0. Data represent the means ± SEM from three biological replicates.

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Transfection, Control, Immunoprecipitation, Western Blot, ATP Assay, Infection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: E3 ubiquitin ligase HUWE1 mediates K6-linked polyubiquitylation and stabilization of Nrf2 in an HBx-dependent manner, thereby inhibiting hepatitis B virus replication

doi: 10.64898/2026.04.20.719611

Figure Lengend Snippet: Under normal conditions (upper panel), HUWE1 does not associate with Nrf2. Keap1 bridges Nrf2 to the Cullin3 E3 ligase complex, leading to K48-linked polyubiquitylation and proteasomal degradation of Nrf2. Upon HBV infection (lower panel), HBx recruits HUWE1 to form a ternary HUWE1/HBx/Nrf2 complex in the cytoplasm. HUWE1 then adds K6-linked polyubiquitylation chains to Nrf2, switching the ubiquitin code from K48 to K6 linkage. K6-linked polyubiquitylation stabilizes Nrf2, promotes its nuclear translocation, and leads transcriptional activation of antioxidant response element (ARE)-driven genes. In the nucleus, Nrf2 suppresses HBV core, PreS1, and PreS2/S promoter activities, thereby suppressing HBV replication. Meanwhile, Nrf2 also suppresses HBV replication through its target gene Prdx1, which facilitates HBV RNA degradation, as we reported previously .

Article Snippet: The HUWE1 inhibitor BI8626 (TargetMol Chemicals, Wellesley Hills, MA) were dissolved in dimethyl sulfoxide (DMSO).

Techniques: Infection, Ubiquitin Proteomics, Translocation Assay, Activation Assay