Seneca Valley virus 3C protease targets the Nrf2/HO-1 pathway to antagonize its antiviral activity

Abstract

Seneca Valley virus (SVV) infection gives rise to severe vesicular diseases in pigs, presenting a substantial threat to the global swine industry. The redox imbalance resulting from oxidative stress is an essential pathogenic mechanism during viral infections. Nevertheless, the regulatory mechanisms of oxidative stress by viral and host factors during SVV infection remain elusive. In this study, we discovered that SVV elicited cellular oxidative stress through the induction of reactive oxygen species production and the suppression of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Our findings indicated that the overexpression of Nrf2/HO-1 exerted a remarkable anti-SVV effect. Conversely, the inhibition of Nrf2/HO-1 expression facilitated the proliferation of SVV. HO-1 metabolic products carbon monoxide and biliverdin inhibit SVV replication. HO-1 promotes type I interferon response and interferon-stimulated gene expressions, which contribute to its antiviral mechanism. Furthermore, our findings reveal that the SVV 3C proteinase targets the Nrf2/HO-1 axis for degradation via caspase pathway, thereby promoting viral replication. Collectively, these results clarify the convoluted molecular mechanisms by which SVV weakens the host's antioxidant defense system and suggest potential targets for therapeutic interventions regarding SVV infections.

Importance: Nrf2 is a crucial redox regulator responsible for initiating the expression of downstream antioxidant genes, including HO-1 and superoxide dismutase. HO-1, an enzyme induced by stress, performs protective roles through the conversion of heme into carbon monoxide, biliverdin, and iron. Nevertheless, the function of Nrf2/HO-1 during Seneca Valley virus (SVV) infection is yet to be clearly defined. In this study, we showed that SVV infection led to a reduction in the expression of Nrf2/HO-1, and the overexpression of Nrf2/HO-1 induced a potent anti-SVV effect. SVV 3C proteinase promoted the caspase-dependent degradation of Nrf2/HO-1. As a result, it attenuated the cell's ability to resist oxidative stress and counteracted the antiviral function of Nrf2/HO-1. Our research further uncovered a novel mechanism through which SVV eludes the host's antiviral effects by disrupting cellular redox balance, offering important targets for preventing and controlling SVV infection.

Keywords: 3C protease; Seneca Valley virus (SVV); heme oxygenase-1 (HO-1); nuclear factor erythroid 2-related factor 2 (Nrf2).

Fig 1

SVV infection inhibits Nrf2-HO-1 pathway. (A and B) BHK-21 cells were infected with SVV (multiplicity of infection [MOI] = 1 and 5). The cell lysates were collected at 0, 4, 8, 12, and 24 h post-infection (hpi) and analyzed by immunoblotting with antibodies against Nrf2, Keap1, HO-1, NQO-1, VP1, and β-actin as an internal control. (C) Quantification analysis of Nrf2, Keap1, HO-1, and NQO-1 protein expression levels from (A) using ImageJ. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.0010). (D) The transcriptional expression levels of Nrf2, Keap1, HO-1, and NQO-1 were analyzed using quantitative RT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (MOI = 1) (***, P < 0.001). (E) BHK-21 cells were inoculated with SVV (MOI = 0.1, 0.5, and 5.0) and incubated for 12 h. Reactive oxygen species production was evaluated through the utilization of the fluorescent indicator DCFH-DA. A fluorescence microscope was used to take the images. (F) The fluorescence intensity ratio was analyzed by ImageJ. (G and H) BHK-21 cells were infected with SVV (MOI = 0.1, 1.0, and 5.0). The MDA and GSH levels were measured using an MDA assay kit and a GSH quantification kit, respectively. NS, not significant.

Fig 2

SVV infection retains Nrf2 in the cytoplasm. (A and C) BHK-21 cells infected with SVV (MOI = 0.5) or mock-infected with phosphate-buffered saline (PBS) with MG132 treatment (10 µM) (A) were monitored with confocal microscopy to examine subcellular localization of Nrf2 and HO-1 at 3, 6, and 12 hpi, respectively. Cells were stained with antibodies for detecting endogenous Nrf2 and HO-1 proteins (red), VP1 monoclonal antibody (green), and DAPI (blue), then examined by confocal microscopy. (B) BHK-21 cells mock-infected with PBS or infected with SVV (MOI = 0.5) with MG132 treatment (10 µM). Cells were stained with Nrf2 antibody (red), VP1 monoclonal antibody (green), and DAPI (blue), then examined by confocal microscopy. (D) BHK-21 cells mock-treated with dimethyl sulfoxide or CoPP or infected SVV (MOI = 0.5) with CoPP treatment (50 µM). Cells were stained with HO-1 antibody (red), VP1 monoclonal antibody (green), and DAPI (blue), then examined by confocal microscopy. (E) Nuclear and cytoplasmic fractions were collected at 0, 3, 6, and 9 hpi (MOI = 0.5) and analyzed by immunoblotting with antibodies against Nrf2, Keap1, histone H3, VP1, and β-actin as an internal control. (F) The relative gray intensity of cytoplasmic Nrf2 and nuclear Nrf2 was normalized against β-actin and histone H3, respectively, using ImageJ. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (G) BHK-21 cells were transfected with HA-Nrf2 or co-transfected with HA-Nrf2 and either GFP, GFP-3C, GFP-3C-H48A, GFP-3C-C160A, or GFP-3C-DM (H48A-C160A). At 24 hpi, samples were processed for immunofluorescence staining using an HA antibody to detect exogenous HA-tagged Nrf2 proteins, followed by observation via confocal microscopy. (H) The graph shows the quantitative analysis of percentage of cytoplasmic Nrf2 from the results in panel G. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (I) Immunoblot was used to analyze the expression of Nrf2 and HO-1 after siRNA transfection-mediated knockdown with antibodies against Nrf2 and HO-1, respectively, with β-actin as an internal control. (J and K) Growth curves of SVV (MOI = 0.5) after transfection with siRNAs, HA-Nrf2, and HA-HO-1 plasmids in BHK-21 cells. At 3, 6, 9, and 12 hpi, the total viruses were titrated with the TCID₅₀ assay. Error bars indicate mean ± SD from three independent infection experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (L and M) Immunoblot was used to analyze VP1 protein production in siRNAs, HA-Nrf2, and HA-HO-1 transfected BHK-21 cells at 6 and 12 hpi (MOI = 0.5).

Fig 3

SVV 3C^pro targets Nrf2/HO-1 for degradation. (A) BHK-21 cells were co-transfected with influenza hemagglutinin (HA)-tagged Nrf2 and HO-1 with different green fluorescent protein (GFP)-tagged SVV protein expression plasmids for 24 h, respectively. Cell samples were subjected to immunoblotting with antibodies against HA, GFP, and β-actin. (B) BHK-21 cells grown on six-well plates were co-transfected with single mutant or double mutants GFP-tagged 3C^pro, including 3C-H48A, 3C-C160A, and 3C-DM (H48A-C160A) with HA-tagged Nrf2, and HO-1 plasmids for 24 h, respectively. Cell samples were subjected to immunoblotting with antibodies against HA, GFP, and β-actin. (C) BHK-21 cells grown on six-well plates were co-transfected with GFP-3C and HA-tagged Nrf2 and HO-1 plasmids for 24 h, respectively, and GFP empty vector with HA as a control. MG132 (10 µM), NH₄Cl (10 mM), Z-VAD-FMK (50 µM), Baf A1 (200 nM), CQ (40 µM), and 3-MA (25 mM) were added to cells at 12 h post-transfection (hpt) with DMSO as a control. Cells were collected at 24 hpt and subjected to immunoblot with antibodies against HA, GFP, and β-actin. (D) BHK-21 cells grown on six-well plates were co-transfected with GFP-tagged SVV protein expression plasmids for 24 h. TBHQ (10 µM) was added to the cells at 12 hpt. Cells were collected at 24 hpt and subjected to immunoblot with antibodies against Nrf2, HO-1, GFP, and β-actin. (E and F) BHK-21 cells grown on six-well plates were transfected with GFP-3C or GFP empty vector for 24 h. TBHQ (10 µM) was added to the cells at 12 hpt. Cells were collected at 24 hpt and subjected to immunoblot with antibodies against Nrf2 (E), HO-1 (F), GFP, and β-actin, respectively. (G and H) BHK-21 cells grown on six-well plates were co-transfected GFP-3C with HA-tagged Nrf2 and HO-1 plasmids for 24 h, respectively, and GFP empty vector as a control. Z-VAD-FMK (50 µM), Z-VDVAD-FMK (50 µM), Z-DEVD-FMK (50 µM), Z-IETD-FMK (50 µM), and Z-LEHD-FMK (50 µM) were added to cells at 12 hpt DMSO as a control. Cells were collected and subjected to immunoblot with antibodies against HA, GFP, and β-actin. (I) Quantitative RT-PCR (qRT-PCR) was performed to analyze the silencing efficiency of caspase-3. BHK-21 cells were transfected with caspase-3-targeting siRNA at a concentration of 20 pmol, while cells transfected with siNC served as negative controls. The graph shows the relative mRNA change from the results in panel I. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (J and K) BHK-21 cells grown on six-well plates were co-transfected with GFP-3C, HA-Nrf2, or HA-HO-1 with siRNA targeting caspase-3. GFP empty vector and siNC were used as controls. Cells were collected at 36 hpt and subjected to immunoblot with indicated antibodies. (L and M) Purified GST-tagged 3C (GST-3C) and His-tagged Nrf2 or His-tagged HO-1 were incubated together and treated with Z-VAD-FMK (50 µM). The GST protein was used as a negative control. After incubation, the complexes were pulled down with glutathione-Sepharose beads and analyzed by Western blotting with anti-His and anti-GST antibodies.

Fig 4

Nrf2 inhibits SVV replication. (A and B) BHK-21 cells were infected with SVV (MOI = 5) for 12 h, followed by treatment with the Nrf2 activator TBHQ (10 µM) or inhibitor ML385 (5 µM). At 12 hpt, intracellular ROS was stained by DCFH-DA probe (10 µM) and detected by flow cytometry and confocal microscopy, respectively, in indicated group. Erastin (10 µM) was included as a positive control for ROS induction. (C) The statistical results of the relative ROS fluorescence from (B) using ImageJ. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (D–G) BHK-21 cells were infected with SVV (MOI = 5) for 12 h, then treated with either TBHQ (10 µM) (D and E) or ML385 (5 µM) (F and G). At 12 hpt, cells were collected and subjected to either Western blot analysis using antibodies against Nrf2, HO-1, p62, LC3, VP1, and β-actin (D and F) or to qRT-PCR to assess changes in Nrf2 and HO-1 mRNA expression levels (E and G). Error bars indicate mean ± SD from three independent infection experiments (**, P < 0.01; ***, P < 0.001). (H–J) BHK-21 cells were infected with SVV (MOI = 5) for 12 h, then treated with either TBHQ (10 µM) (H and J) or ML385 (5 µM) (I and J). At 12 hpt, cells were collected and subjected to either Western blot analysis using antibodies against NF-κB, p-p65, IκBα, p-IκBα, and β-actin (H and I) or qRT-PCR to determine the mRNA expression levels of CCL-20, TNF-α, and IL-6 (J). Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001).

Fig 5

HO-1 agonist inhibits SVV infection. (A) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM). At 12 and 16 hpi, the cell lysates were collected and then analyzed by immunoblotting with indicated antibodies. (B) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP at various concentrations (100, 50, 10, and 1 µM). At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. (C) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM). The transcriptional expression level of the indicated gene was analyzed using qRT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (D and E) BHK-21 cells were treated with erastin or in the presence of HO-1 activator CoPP (50 µM) and inhibitor ZnPP (10 µM) or transfected with siHO-1. ROS was measured using DCFH-DA (10 µM) and examined by flow cytometry (D) and confocal microscopy (E), respectively. (F) Relative fluorescence intensity in BHK-21 cells from panel E. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (G) BHK-21 cells were infected with SVV (MOI = 5) and treated with HO-1 inducer hemin (50 µM). The cell lysates were collected at 12 and 16 hpi and analyzed by immunoblotting with indicated antibodies. (H) BHK-21 cells were infected with SVV (MOI = 5) and treated with varying concentrations of hemin (100, 50, 10, and 1 µM). At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. (I) BHK-21 cells were infected with SVV (MOI = 0.5), followed by treatment with CoPP (50 µM), ZnPP (10 µM), or hemin (50 µM). At 6 and 12 hpi, the viral titers were determined with TCID₅₀ assay. Error bars indicate mean ± SD from three independent infection experiments (*, P < 0.05; **, P < 0.01).

Fig 6

HO-1 metabolite inhibits SVV replication. (A and D) BHK-21 cells were infected with SVV (MOI = 5) and treated with CORM-2 (50 µM) (A) and biliverdin (50 µM) (D), respectively. At 12 and 16 hpi, the cell lysates were collected and analyzed by immunoblotting with indicated antibodies. (B and E) BHK-21 cells were infected with SVV (MOI = 5) and treated with CORM-2 and biliverdin with various concentrations (100, 50, 10, and 1 µM), respectively. At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. (C and F) BHK-21 cells were infected with SVV (MOI = 5) and treated with CORM-2 (50 µM) (C) and biliverdin (50 µM) (F), respectively. The transcriptional expression level of the indicated gene was analyzed using qRT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (*, P < 0.05; ***, P < 0.001). (G) BHK-21 cells were infected with SVV (MOI = 0.5) and treated with CORM-2 and biliverdin at various concentrations (100, 50, 10, and 1 µM), respectively. The virus productions at 12 hpi were titrated by TCID₅₀ assay. (H and I) BHK-21 cells were infected with SVV (MOI = 5) and treated with CORM-2 (50 µM). At 12 hpi, the cells were collected and subjected to immunoblot analysis of NF-κB pathway protein expression using the indicated antibodies (H) or qRT-PCR quantification of CCL-20, TNF-α, and IL-6 mRNA levels (I). Error bars indicate mean ± SD from three independent infection experiments (**, P < 0.01; ***, P < 0.001). (J and K) BHK-21 cells were either exposed to CORM-2 (50 µM), ODQ (10 µM), or KT5823 (1 µM) or left unexposed, followed by SVV infection. At 24 hpi, the VP1 protein and SVV titers were determined by Western blot (J), and TCID₅₀ (K) was determined. (L and M) BHK-21 cells were treated with BR (50 µM), ODQ (10 µM), or KT5823 (1 µM) or kept untreated, and then infected with SVV. At 24 hpi, the VP1 protein and SVV titers were determined by Western blot (L) and TCID₅₀ (M). Error bars indicate mean ± SD from three independent infection experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Fig 7

NAC blocks SVV replication via inhibiting autophagy. (A) BHK-21 cells were infected with SVV (MOI = 5) and treated with NAC (5 mM). At the indicated times, the cell lysates were collected and analyzed by immunoblotting with antibodies against Nrf2, HO-1, Keap1, p62, LC3, VP1, and β-actin. (B) BHK-21 cells were infected with SVV (MOI = 5) and treated with NAC at various concentrations (10.0, 5.0, 1.0, and 0.1 mM). At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. (C and D) BHK-21 cells were infected with SVV (MOI = 5) and treated with NAC (5 mM). At 12 hpi, the cells were collected and analyzed by immunoblotting for NF-κB signaling proteins (C) or qRT-PCR quantification for cytokine mRNAs (CCL-20, TNF-α, IL-6, IL-1β, and IL-18) (D). Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (E) BHK-21 cells were infected with SVV (MOI = 0.5) and treated with NAC at various concentrations (10.0, 5.0, 1.0, and 0.1 mM). At 12 hpi, the viral titers were determined with TCID₅₀ assay. Error bars indicate mean ± SD from three independent infection experiments (**, P < 0.01; ***P < 0.001). (F and G) GFP-LC3 was transfected into BHK-21 cells, and treatment with NAC (5 mM) was conducted for 24 h. Fluorescent microscopy was employed to detect LC3 puncta (left), while the quantification of LC3 puncta per cell is displayed on the right. Error bars indicate mean ± SD from three independent infection experiments (***P < 0.001).

Fig 8

HO-1 inhibits SVV replication through the IFN-I pathway. (A and G) BHK-21 cells and PK-15 cells were transfected with HA-HO-1 and infected with SVV (MOI = 5), respectively. At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. (B) BHK-21 cells were transfected with HA-HO-1. The transcriptional expression level of the indicated gene was analyzed using qRT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (C and H) BHK-21 cells and PK-15 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM), respectively. At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. (D and F) BHK-21 cells were infected with SVV (MOI = 5) and treated with CoPP (50 µM) and biliverdin (50 µM), respectively. The transcriptional expression level of the indicated gene was analyzed using qRT-PCR and normalized to β-actin mRNA. Error bars indicate mean ± SD from three independent infection experiments (***, P < 0.001). (E) BHK-21 cells were infected with SVV (MOI = 5) and treated with biliverdin (50 µM). At 12 hpi, immunoblot was used to analyze the protein expression with indicated antibodies. (I) PK-15 cells grown on six-well plates were co-transfected with GFP-3C and HA- HO-1 for 24 h, respectively, and GFP empty vector was used as a control. At 24 hpt, cells were collected and subjected to immunoblot with indicated antibodies. (J and K) HEK-293T cells plated in 24-well plates were transfected with 250 ng of IFN-β-Luc and ISRE-Luc separately, along with 25 ng of pRL-TK and either 250 ng of siHO-1 or siNC. After 24 h, HEK-293T cells were treated with poly(I:C) (2 ug/ml). Then the samples were prepared and analyzed by luciferase assays. (L) HEK-293T cells plated in 24-well plates were transfected with siHO-1 or siNC. After 24 h, the cells were treated with poly(I:C) (2 μg/mL). Then the samples were prepared and analyzed by qRT-PCR.Error bars indicate mean ± SD from three independent infection experiments. (*, P < 0.05; **, P < 0.01; ***, P < 0.001). (M) PK-15 cells grown on six-well plates were co-transfected with HA-Nrf2 and siRNA targeting HO-1; the GFP empty vector and siNC were used as controls. At 36 hpt, cells were collected and subjected to immunoblot with indicated antibodies.

Fig 9

Proposed model for SVV 3C proteinase (3C^pro) targeting Nrf2/HO-1 to facilitate viral replication. SVV infection elicited cellular oxidative stress through the induction of reactive oxygen species (ROS) production, glutathione depletion, and the suppression of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) pathway. Mechanistically, the viral 3C^pro targets the Nrf2/HO-1 axis for degradation via caspase pathway, thereby promoting viral replication. The overexpression of Nrf2/HO-1 exerted antiviral activity against SVV, whereas inhibition of this pathway significantly enhanced viral proliferation. HO-1 metabolic products carbon monoxide (CO) and biliverdin (BV) exhibited inhibition of SVV replication. Additionally, HO-1 promotes IFN-I response and ISG expressions, which contribute to its antiviral mechanism.