Type-I-IFN-Stimulated Gene TRIM5γ Inhibits HBV Replication by Promoting HBx Degradation

Abstract

To understand the molecular mechanisms that mediate the anti-hepatitis B virus (HBV) effect of interferon (IFN) therapy, we conduct high-throughput bimolecular fluorescence complementation screening to identify potential physical interactions between the HBx protein and 145 IFN-stimulated genes (ISGs). Seven HBx-interacting ISGs have consistent and significant inhibitory effects on HBV replication, among which TRIM5γ suppresses HBV replication by promoting K48-linked ubiquitination and degradation of the HBx protein on the K95 ubiquitin site. The B-Box domain of TRIM5γ under overexpression conditions is sufficient to trigger HBx degradation and is responsible both for interacting with HBx and recruiting TRIM31, which is an ubiquitin ligase that triggers HBx ubiquitination. High expression levels of TRIM5γ in IFN-α-treated HBV patients might indicate a better therapeutic effect. Thus, our studies identify a crucial role for TRIM5γ and TRIM31 in promoting HBx degradation, which may facilitate the development of therapeutic agents for the treatment of patients with IFN-resistant HBV infection.

Keywords: HBV; HBx; TRIM31; TRIM5γ; type I IFN.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare that they have no conflict of interest.

Published by Elsevier Inc.

Figures

Figure 1.. Screening of ISGs That Interact with HBx by BiFC Assay

(A) Schematic illustration of the BiFC assay. (B) Expression plasmids for YFPc-GBP2 and YFPc-TRIM62 were transfected individually with YFPn-HBx plasmid into 293T cells. YFP fluorescence intensity was measured 36 h post-transfection. Scale bars represent 100 μm. (C) A total of 145 YFPc-ISG plasmids were transfected individually with the YFPn-HBx plasmid into 293T cells, and YFP fluorescence was measured after 36 h by flow cytometry. YFP fluorescence mean intensity (MFI) of >2 was deemed to signify interaction with HBx (labeled in orange) and

Figure 2.. Higher TRIM5γ Induction Indicates a Better Therapeutic Outcome of HBV Patients Undergoing IFN-α Treatment

(A) HepG2 cells were treated with IFN-α (10 ng/mL) or untreated; qPCR was performed to analyze the mRNA expression level of 7 ISGs, as indicated. (B) Blood samples of 31 HBV-infected patients before and 17 weeks after IFN-α treatment were collected for ALT, AST, HbeAg, and HBV DNA detection. (C and D) PBMCs from HBV patients before or after IFN treatment were isolated and subjected to RNA extraction; qPCR was performed to evaluate the TRIM5γ (C) or TRIM22 (D) mRNA level. (E) Seventeen weeks after IFN treatment, the outcome of the two HBV-infected patients groups (TRIM5γ high or low, TRIM22 high or low) were analyzed by comparing the AST, ALT, HbeAg, and HBV DNA levels. Mean ± SD values from three independent experiments are shown. *p 0.05.

Figure 3.. TRIM5γ Inhibits HBV Infection and Replication by Targeting HBx

(A) HepG2 cells were co-transfected with constructs expressing TRIM5γ proteins and either pHBV1.2 WT or DX, as indicated. After 72 h, cells and supernatant were harvested and subjected to qPCR or ELISA. (B) Scheme of HBV infection. (C) HepG2-NTCP cells were infected with lentiviral-ctrl or lentiviral-TRIM5γ for 16 h, and later, cells were washed with PBS and inoculated for 24 h with HBV. After infection, cells were washed three times with PBS and were maintained for another 9 days; the medium was changed every 2 days. The supernatant and cells were collected for detection of HBV DNA, pgRNA, and HBsAg by qPCR or ELISA. (D) HepG2-NTCP WT or TRIM5α/γ KO cells were infected with HBV for 24 h with or without IFN-α treatment (10 ng/mL) and analyzed as in (C). Mean ± SD values from three independent experiments are shown. *p

Figure 4.. TRIM5γ Specifically Promotes HBx Degradation

(A) 293T cells were co-transfected with constructs expressing FLAG-HBV proteins and either GFP-TRIM5γ or EV as indicated. After 36 h, cells were harvested and whole-cell lysates were subjected to immunoblotting using anti-FLAG, anti-GFP, or anti-Tubulin antibody. (B) 293T cells were co-transfected with FLAG-HBx and either GFP-TRIM5γ, -USP2, -TRIM38, -UBE3A, or -EV expression plasmids as indicated and were analyzed as in (A). (C) HepG2 cells were co-transfected with plasmids expressing FLAG-HBx and either HA-TRIM5γ or TRIM5α or hemagglutinin (HA) empty vector as indicated, and cells were collected 28 h after transfection. Whole-cell lysates were immunoblotted with antibodies as indicated. (D) FLAG-HBx and either HA-TRIM5γ or HA empty vector was transfected into HepG2 cells as indicated; 48 h later, cells were fixed in acetone-methanol and subjected to immunofluorescence staining analysis using FLAG and HA antibodies. Scale bars represent 20 μm. (E) TRIM5α and TRIM5γ were both knocked out in HepG2 cells by CRISPR/Cas9 technology; WT or TRIM5α/γ KO HepG2 cells were transfected with FLAG-HBx; and 24 h later, cells were treated with IFN-α as indicated. Post-treatment, whole-cell lysates were immunoblotted with the indicated antibodies. (F) HepG2 cells were co-transfected with HA-TRIM5γ and FlAG-HBx as indicated; 24 h later, cells were treated with 10 mM MG132 or Z-VAD for 8 h and collected for immunoblotting with antibodies as indicated. (G) 293T cells were co-transfected with HA-Ub and FLAG-HBx, with or without GFP-TRIM5γ; 24 h later, cells were treated with MG-132 as indicated for 8 h and subjected to coIP analysis. Cell lysates and precipitated samples were subjected to immunoblot analysis using the indicated antibodies. (H) 293T cells were co-transfected with K48 or K63-Ub and Flag-HBx, with or without GFP-TRIM5γ; 24 h later, cells were treated with MG132 (10 μM) for 8 h; and cells were collected and analyzed as in (G). Data are representative of at least three independent experiments.

Figure 5.. BBox Domain of TRIM5γ Is Indispensable for TRIM5γ-Induced HBx Degradation

(A) Diagrams of mutant TRIM5γ constructs. (B) 293T cells were co-transfected with FLAG-HBx and TRIM5γ-derived constructs or EV as indicated, and whole-cell lysates were immunoblotted with anti-FLAG, HA, or GAPDH antibodies. (C) 293T cells were transfected as indicated; 28 h later, coIP was carried out, and cell lysates and precipitated samples were analyzed by immunoblotting using an anti-FLAG or anti-HA antibody. (D) 293T cells were transfected with glutathione S-transferase (GST)-BBox, flag-HBx, or EV as indicated and were subjected to coIP analysis as in (C). (E) 293T cells were co-transfected with FLAG-HBx and GST-BBox or HA-TRIM5γ plasmids and analyzed as in (B). (F) HepG2 cells were transfected with FLAG-HBx, HA-Ub, GST-Bbox, or GST-EV plasmids as indicated; 24 h later, cells were treated with MG132 for 8 h, collected, and subjected to coIP analysis. Data are representative of at least three independent experiments.

Figure 6.. TRIM5γ Interacts with the C Terminus of HBx and Promotes HBx K95 Site Ubiquitination

(A) Diagram of mutant HBx constructs. the numbers indicate the amino acids in the HBx constructs. (B–E) 293T cells were co-transfected with the expression vectors for Flag-TRIM5γ and GST-HBx WT or mutants (B, R96E, Del; C, X1, X2, X3; D, X2a, X2b, X3a, X3b; E, X3bD, DD, X2bD) or EV as indicated. After 28 h, cells were harvested, coIP with FLAG-HBx, and cell lysates and precipitated samples were analyzed by immunoblotting with the indicated antibodies. (F) HBx WT or truncations were co-transfected with TRIM5γ or EV into HepG2 cells; 28h later, cell lysates were subjected to immunoblot analysis using the indicated antibodies. (G) Plasmids of expression of HBx WT, K90R, or K95R were co-transfected with TRIM5γ or EV into HepG2 cells; 28h later, cell lysates were subjected to immunoblot analysis using the indicated antibodies. (H) HepG2 cells were co-transfected with the expression vectors for HA-TRIM5γ, HA-Ub, and FLAG-HBx WT or mutants as indicated; 28h later, cells were harvested and coIP with FLAG-HBx, and cell lysates and precipitated samples were analyzed by immunoblotting with the indicated antibodies. Data are representative of at least three independent experiments.

Figure 7.. TRIM5γ Recruits TRIM31 to Degradate HBx

(A) HepG2 cells were co-transfected with FLAG-HBx and HA-TRIM31 or HA empty vector plasmids; 36 h later, cells were left untreated or were treated with MG132 (10 μM) or Z-VAD (10 μM) as indicated. Cells were harvested after treatment, and whole-cell lysates were immunoblotted with the indicated antibodies. (B) HepG2 cells were transfected with FLAG-HBx, HA-Ub, HA-TRIM5γ, or TRIM31 expression plasmids as indicated; 24 h later, cells were treated with MG132 for 6 h and were collected and subjected to coIP analysis. (C) HepG2 cells were transfected with FLAG-HBx or co-transfected with TRIM31 plasmids (WT or mutant); 36 h later, cells were harvested and analyzed as in (A). (D) HepG2 WT or TRIM31 KO cell lysates were immunoblotted with TRIM31 or Tubulin antibody. (E) HepG2 WT orTRIM31KO cells were transfected withFLAG-HBxwithor without the TRIM5γ expression vector; 28h later, cells were collected and analyzed asin(A). (F) WT HepG2 cells or TRIM31 KO cells were transfected as indicated; 28 h later, cells were collected and analyzed as in (A). (G and H) Expression vectors for HA-TRIM5γ (G) or GST-BBox (H) were co-transfected into 293T cells with or without FLAG-TRIM31; 36 h later, cells were subjected to coIP using FLAG-TRIM31. Immunoblot analyses were carried out using anti-FLAG, anti-HA, or anti-GST antibody. (I) 293T cells were transfected with FLAG-HBx, HA-TRIM5γ, or HA-TRIM31 expression plasmids as indicated; 24 h later, cells were treated with MG132 (10 μM) for 8 h and subjected to coIP analysis. Immunoblotting was carried oout using an anti-FLAG or anti-HA antibodies. Data are representative of at least three independent experiments. (J) HepG2 WT or TRIM31 KO cells were transfected with pHBV1.3 plasmids or together with TRIM5γ or TRIM31 expression plasimds as indicated; 72 h later, the supernatant was tested for HBeAg and HBsAg content using ELISA. Mean ± SD values from three independent experiments are shown. **p