Interferon alpha (IFNα)-induced TRIM22 interrupts HCV replication by ubiquitinating NS5A

Abstract

TRIM22, a tripartite-motif (TRIM) protein, is upregulated upon interferon alpha (IFNα) administration to hepatitis C virus (HCV)-infected patients. However, the physiological role of TRIM22 upregulation remains unclear. Here, we describe a potential antiviral function of TRIM22's targeting of the HCV NS5A protein. NS5A is important for HCV replication and for resistance to IFNα therapy. During the first 24 h following the initiation of IFNα treatment, upregulation of TRIM22 in the peripheral blood mononuclear cells (PBMCs) of HCV patients correlated with a decrease in viral titer. This phenomenon was confirmed in the hepatocyte-derived cell line Huh-7, which is highly permissive for HCV infection. TRIM22 over-expression inhibited HCV replication, and Small interfering RNA (siRNA)-mediated knockdown of TRIM22 diminished IFNα-induced anti-HCV function. Furthermore, we determined that TRIM22 ubiquitinates NS5A in a concentration-dependent manner. In summary, our results suggest that TRIM22 upregulation is associated with HCV decline during IFNα treatment and plays an important role in controlling HCV replication in vitro.

Figure 1

TRIM22 is induced in PBMCs by Type I IFN treatment of HCV patients and is associated with a decrease in HCV levels. (a) The TRIM22 mRNA level was significantly increased in the PBMCs of HCV patients approximately 12 h after initiation of IFNα treatment, as measured by real-time PCR. Comparison of the HCV mRNA levels with the baseline HCV mRNA level at the remaining time points revealed significant differences. ***P<0.001 as determined by Student's t-test. (b) HCV viral titers (shown as the logarithm) in patients' blood decreased markedly, as measured by COBAS (as described in the section on ‘Materials and methods'). Comparison of the HCV titer with the baseline virus titration at other time points revealed significant differences. ***P<0.001, as determined by Student's t-test. (c) TRIM22 is induced in patient PBMC samples, as determined by western blotting (a representative patient result is shown). HCV, hepatitis C virus; IFN, interferon; PBMC, peripheral blood mononuclear cell; TRIM, tripartite motif.

Figure 2

TRIM22 is upregulated in Huh-7 cells treated with IFNα. (a, b) TRIM22 mRNA is upregulated in Huh-7 cells treated with IFNα (50 IU/ml). Time-series (time points are shown in the figure) samples were collected and prepared for RT-PCR or western blotting. The primers are described in the section on ‘Materials and methods'. (c) TRIM22 is induced by JFH-1 (MOI=0.1) 24 h after JFH-1 infection but at a lower level than induced by IFNα (50 IU/ml). HCV, hepatitis C virus; IFN, interferon; MOI, multiplicity of infection.

Figure 3

TRIM22 has anti-viral activity against HCV. (a) Western blot analysis of TRIM22 expression with anti-TRIM22 antibody after transfection of cells with FLAG-TRIM22 (0.5 µg/well, 24-well plate). Endogenous TRIM22 is shown as a positive control in the lane labeled ‘IFNα' in Huh-7 cells that were treated with IFNα (50 IU/ml) for 24 h and collected for western blotting. (b, c) A marked decrease in the virus replicon levels was observed in TRIM22-over-expressing Huh-7-con1-replicon cells 48–96 h after infection. Huh-7-con1-replicon cells were transfected with GFP, TRIM22 and the anti-viral ISG and IRF1 plasmids. Samples were collected at 48 h, 72 h and 96 h after transfection. Real-time RT-PCR and western blotting were performed as described in the section on ‘Materials and methods'. (d) The knockdown efficiency of the siRNAs was determined by western blotting. Huh-7 cells were transfected with siRNA or TRIM22 (siRNA: 50 nmol per well/24-well plate, TRIM22: 100 ng per well/24-well plate), and cells were collected for western blotting 24 h after transfection. (e, f) Knockdown of TRIM22 diminished the anti-viral effects of IFNα. Huh-7 cells were transfected with siRNA (50 nmol per well/24-well plate). After 24 h, the transfected cells were infected with JFH1 (MOI=0.1). At 24 h after infection, the cells were treated with IFNα (50 IU/ml) for an additional 48 h. Samples were collected for real-time PCR assays and western blotting. *P<0.05 as determined by Student's t-test. ***P<0.001 as determined by Student's t-test. HCV, hepatitis C virus; IFN, interferon; IRF, IFN-regulatory factor; ISG, interferon-stimulated gene; PBMC, peripheral blood mononuclear cell; TRIM, tripartite motif.

Figure 4

TRIM22 interacts with HCV NS5A. (a) Both FLAG-TRIM22 (1.5 µg/well, six-well plate) and HA-NS5A (1.5 µg/well, six-well plate) were expressed in 293T cells. For lane 1, cells were transfected with only HA-NS5A. For lane 2, cells were transfected with only FLAG-TRIM22. For lane 3, cells were transfected with both HA-NS5A and FLAG-TRIM22. Co-IP experiments were performed with anti-FLAG and anti-HA antibodies 24 h after transfection of cells with the plasmids. The results of the co-IP experiments revealed that TRIM22 interacts with HCV NS5A. (b) The endogenous interaction of TRIM22 and NS5A was evaluated. Twenty-four hours after adding IFNα (50 IU/ml) to the HCV Huh-7-Con-1 replicon, the cells were harvested and subjected to endogenous IP with anti-TRIM22 and anti-NS5A antibodies. (c) Schematic diagram of the four truncated forms of TRIM22: TRIM22A (lacks coiled-coil and SPRY domains), B (lacks SPRY domain), C (lacks RING and B-Box domains) and D (lacks RING domain). (d) The SPRY and coiled-coil domains are important for the interaction of TRIM22 with NS5A because only TRIM22 C and TRIM22 D interact with NS5A. All TRIM22 truncations (1.5 µg/well, six-well plate) and HA-NS5A (1.5 µg/well, six-well plate) were expressed in 293T cells. Co-IP experiments were performed using anti-FLAG and anti-HA antibodies 24 h after transfection of cells with the plasmids. (e) Schematic diagram of the three truncated forms of NS5A: NS5A-D1 (contains only domain 1), NS5A-D12 (contains domains 1 and 2) and NS5A-D23 (contains domains 2 and 3). (f) Domain 1 of NS5A is important for the interaction of TRIM22 with NS5A because only NS5A-D23 does not interact with NS5A. All HA-NS5A truncations (1.5 µg/well, six-well plate) and FLAG-TRIM22 (1.5 µg/well, six-well plate) were expressed in 293T cells. Co-IP experiments were performed using anti-FLAG and anti-HA antibodies 24 h after transfection of cell with the plasmids. (j) IFNα (50 IU/ml) treatment was initiated 24 h after Huh-7 cells were infected with the JFH-1 virus (MOI of 0.1). TRIM22 was colocalized with NS5A in the cytoplasm. Immunofluorescence was performed IFNα using anti-TRIM22 and anti-NS5A 12 h after IFNα treatment. LD staining was conducted with bodipy dye (Life Technologies D2228). The region of co-localization is indicated in white in the figure labeled ‘Co-local'. TRIM22 and NS5A were colocalized near the lipid droplet, which is reported to be important for HCV assembly. co-IP, co-immunoprecipitation; HCV, hepatitis C virus; IFN, interferon; MOI, multiplicity of infection; TRIM, tripartite motif.

Figure 5

TRIM22 is an ubiquitin ligase of NS5A. (a) TRIM22 specifically degrades NS5A. 293T cells are transfected. HCV proteins are transfected together with TRIM22 and ubiquitin. Western blotting was performed 48 h after plasmid transfection. (b) Exogenous NS5A: Huh-7 cells transfected with HA-NS5A plasmid (100 ng/well in a 24-well plate) were transfected with FLAG-TRIM22 (upper left: 0, 200 and 1000 ng/well in a 24-well plate) and HIS-Ubiquitin (40 ng/well in a 24-well plate) plasmids or treated with IFNα (upper right: IFNα 0, 50 and 100 IU/ml triggered 24 h after HA-NS5A plasmid transfection). After 48 h, samples were collected for western blotting. TRIM22 degraded NS5A (100 ng/well in a 24-well plate) in a dose-dependent manner. Endogenous NS5A: Huh-7-Con-1 replicon cells were transfected with FLAG-TRIM22 (lower left: 0, 200 and 1000 ng/well in a 24-well plate) and HIS-Ubiquitin (40 ng/well in a 24-well plate) or treated with IFNα (lower right: 50 IU/ml). Samples were detected by western blotting 48 h after plasmid transfection or IFNα treatment. TRIM22 degraded endogenous NS5A (100 ng/well in a 24-well plate) in a dose-dependent manner. (c) Upper panel: FLAG-TRIM22 (1.5 µg/well, six-well plate), HA-NS5A (1.5 µg/well, six-well plate) and HIS-Ubiquitin (0.3 µg/well, six-well plate) were expressed in 293T cells. Immunoprecipitation of NS5A with anti-HA antibody and western blotting of ubiquitin with an anti-HIS antibody were performed 24 h after plasmid transfection. TRIM22 efficiently ligated ubiquitin to NS5A. Lower panel: endogenous ubiquitination of NS5A was detected. Huh-7-Con-1 replicon cells were treated with IFNα (50 IU/ml). Twenty-four hours after IFNα treatment, cells were harvested and immunoprecipitated with an anti-NS5A antibody. Then, western blotting was performed with an anti-ubiquitin antibody. (d) MG132 (20 nM) successfully blocked the endogenous ubiquitination of NS5A and NS5A degradation. HCV, hepatitis C virus; IFN, interferon; TRIM, tripartite motif.