Foot-and-mouth disease virus non-structural protein 2B downregulates the RLR signaling pathway via degradation of RIG-I and MDA5

Abstract

Foot-and-mouth disease virus (FMDV) is a single-stranded, positive-sense RNA virus containing at least 13 proteins. Many of these proteins show immune modulation capabilities. As a non-structural protein of the FMDV, 2B is involved in the rearrangement of the host cell membranes and the disruption of the host secretory pathway as a viroporin. Previous studies have also shown that FMDV 2B plays a role in the modulation of host type-I interferon (IFN) responses through the inhibition of expression of RIG-I and MDA5, key cytosolic sensors of the type-I IFN signaling. However, the exact molecular mechanism is poorly understood. Here, we demonstrated that FMDV 2B modulates host IFN signal pathway by the degradation of RIG-I and MDA5. FMDV 2B targeted the RIG-I for ubiquitination and proteasomal degradation by recruiting E3 ubiquitin ligase ring finger protein 125 (RNF125) and also targeted MDA5 for apoptosis-induced caspase-3- and caspase-8-dependent degradation. Ultimately, FMDV 2B significantly inhibited RNA virus-induced IFN-β production. Importantly, we identified that the C-terminal amino acids 126-154 of FMDV 2B are essential for 2B-mediated degradation of the RIG-I and MDA5. Collectively, these results provide a clearer understanding of the specific molecular mechanisms used by FMDV 2B to inhibit the IFN responses and a rational approach to virus attenuation for future vaccine development.

Keywords: 2B; MDA5; RIG-I; RNF125; foot and mouth disease virus (FMDV).

Figure 1

FMDV 2B negatively regulate RNA virus-mediated innate immune responses. PK-15 cells (A, B) and HEK293T cells (C, D) were transiently transfected with the control vector or FMDV 2B and infected with VSV-GFP. GFP expression, GFP absorbance, and virus titer was taken at 12 and 24 hpi (A, C). The concentration of secreted IFN-β and IL-6 in supernatants was determined at 12 and 24 hpi by ELISA (B, D). Control vector and FMDV 2B stably expressing RAW264.7 cells were infected with VSV-GFP (E, F) and PR8-GFP (G, H). GFP expression, GFP absorbance, and virus titer was taken at 12 and 24 hpi (E, G). The concentration of IFN-β and IL-6 secreted in supernatants were determined at 12 and 24 hpi by ELISA (F, H). Results representative of at least two independent experiments, each with similar results, and the values are expressed as mean ± SD of three biological replicates. Student’s t-test; *p < 0.05; **p < 0.01; ***p < 0.001; ns, not significant.

Figure 2

FMDV 2B inhibit the transcription of antiviral genes and type-I IFN signaling. Control vector and FMDV 2B stably expressing RAW264.7 cells were infected with PR8-GFP. Cells were harvested at indicated time points after the infection of PR8-GFP. Total and phosphorylated TBK1, IRF3, and p65 were measured by immunoblotting. β-actin was used as the loading control (A). Control vector and FMDV 2B stably expressing RAW264.7 cells were infected with VSV-GFP. At 0 and 24 hpi, cells were harvested, and quantitative real-time PCR was performed to analyze the levels of antiviral genes (B). HEK293T cells were transfected with interferon-β promoter encoding firefly luciferase plasmid, TK-Renilla plasmid, increasing dose of Flag-FMDV 2B plasmid and stimulated with H1N1, NDV infection, Poly(I:C) treatment (C) or transfected with MDA5, RIG-I, MAVS, TRIF, and TBK1 encoding plasmids (D) for 24 hours. Results are expressed relative to those of Renilla luciferase alone (internal control). Results representative of at least two independent experiments, each with similar results, and the values are expressed as mean ± SD of three biological replicates. Student’s t-test; **p < 0.01; ***p < 0.001.

Figure 3

FMDV 2B targets RIG-I and mediates degradation of RIG-I via K48-linked polyubiquitination. HEK293T cells were transfected with Flag-hRIG-I plasmid, and increasing doses of Strep-FMDV 2B plasmids (A), PK-15 cells were transfected with increasing amounts of FMDV 2B and 24 hrs post-transfection cells were infected with SeV [1 multiplicity of infection (MOI)] (B). Cells were harvested at 24 hrs post-infection, and RIG-I expression level was measured by immunoblotting. HEK293T cells were transfected with Flag-hRIG-I together with Strep-FMDV 2B and treated with two doses of MG132, chloroquine, NH₄Cl, or z-VAD for 6 hours before harvesting the cells. Whole-cell lysates were subjected to immunoblotting with indicated antibodies (C). PK-15 cells were transfected with Strep-FMDV 2B, and 24 hrs post-transfection cells were infected with SeV (1MOI). Cells were treated with increasing doses of MG132 at 18 hours hrs post-infection and harvested at 24 hrs post-infection. The RIG-I expression level was measured by immunoblotting (D). HEK293T cells were transfected with Strep-FMDV 2B (E) or Flag-RIG-I together with Strep-FMDV 2B (F) and treated with MG132 for 6 hours before harvesting the cells. Cell lysates were subjected to Strep pull-down and immunoblotted with the indicated antibodies. HeLa cells were transfected with Flag-FMDV 2B and treated with MG132 for 6 hours before fixing the plate, followed by confocal microscopy with anti-Flag (red) and anti-RIG-I (green) antibodies. Nuclei were stained with DAPI (blue) (G). HEK293T cells were transfected with Flag-RIG-I, HA-ubiquitin together with increasing doses of Strep-FMDV 2B plasmids and treated with MG132 for 6 hours before harvesting the cells. Whole-cell lysates were subjected to Flag immunoprecipitation and immunoblotted with indicated antibodies (H). HEK293T cells were transfected with HA-ubiquitin together with increasing doses of Strep-2B plasmids and treated with MG132 for 6 hours before harvesting the cells. Whole-cell lysates were subjected to immunoprecipitation with RIG-I antibody and immunoblotted with indicated antibodies (I). LFBK cells were infected with FMDV Asia1/Shamir strain and treated with MG132 for 6 hours before harvesting the cells. Cells were harvested at indicated time after infection and whole-cell lysates were immunoprecipitated with anti-RIG-I antibody and immunoblotted with indicated antibodies (J). Results representative of at least two independent experiments, each with similar results. (pRIG-I: porcine RIG-I, hRIG-I: human RIG-I).

Figure 4

FMDV 2B recruit RNF125 to mediate K48-linked polyubiquitination and degradation of RIG-I. HEK293T cells were transfected with Strep-FMDV 2B. Cell lysates were subjected to Strep pull-down assay and immunoblotted with indicated antibodies (A). HeLa cells were transfected with Flag-FMDV 2B, followed by confocal microscopy assay with anti-Flag (red) and anti-RNF125 (green) antibodies. Nuclei were stained with DAPI (blue) (B). HEK293T cells were transfected with Myc-RNF125 together with increasing doses of Strep-FMDV 2B. The whole-cell lysate was immunoblotted with indicated antibodies (C). LFBK cells were infected with FMDV O1Manisa strain, and cells were harvested after indicated time period post-infection. The whole-cell lysate was immunoprecipitated with FMDV 2B rabbit polyclonal antibody and immunoblotted with indicated antibodies (D). HEK293T cells were transfected with si-control or si-RNF125 together with Flag-RIG-I and different doses of Strep-2B plasmids. The whole-cell lysate was immunoblotted with indicated antibodies (E). (F, G) RAW264.7 cells were transfected with si-control or si-RNF125 together with Flag-FMDV 2B and infected with VSV-GFP. Viral replication was measured by fluorescence microscopy, GFP absorbance, and plaque assay at indicated time points. The concentration of secreted IFN-β in supernatants was determined at 12 and 24 hpi by ELISA. HEK293T cells were transfected with si-control or si-RNF125 together with Flag-RIG-I, HA-ubiquitin, indicated doses of Strep-FMDV 2B and treated with MG132 6 hours before harvesting the cells. Whole-cell lysates were subjected to Flag-immunoprecipitation and immunoblotted with indicated antibodies (H). HEK293T cells were transfected with Strep-RIG-I wild-type or RIG-I K181R mutant together with HA-ubiquitin, Myc-RNF-125, indicated doses of Flag-FMDV 2B and treated with MG132 6 hours before harvesting the cells. Whole-cell lysates were subjected to Strep pull-down and immunoblotted with indicated antibodies (I). Results representative of at least two independent experiments, each with similar results, and the values are expressed as mean ± SD of three biological replicates. Student’s t-test; **p < 0.01; ***p < 0.001.

Figure 5

C-terminal region of FMDV 2B is important for the interaction and proteasomal degradation of RIG-I. Schematic representation of the domain construction of FMDV 2B, RIG-I, and RNF125 (A). HEK293T cells were transfected with GST-RIG-I domain constructs together with Strep-FMDV 2B plasmids and treated with MG132 for 6 hours before harvesting the cells. Whole-cell lysates were subjected to the Strep pull-down assay, followed by immunoblotting with indicated antibodies (B). HEK293T cells were transfected with Flag-RIG-I together with GST-FMDV 2B domain constructs containing plasmids and treated with MG132 for 6 hours before harvesting the cells. Whole-cell lysates were subjected to GST pull-down and immunoblotted with indicated antibodies (C). HEK293T cells were transfected with Strep-FMDV 2B domain constructs together with Flag-RIG-I plasmids and treated with MG132 for 6 hours before harvesting the cells. Whole-cell lysates were subjected to the Strep pull-down assay, followed by immunoblotting with indicated antibodies (D). HEK293T cells were transfected with Strep-RNF125 together with GST-FMDV 2B domain constructs containing plasmids. Whole-cell lysates were subjected to GST pull own and immunoblotted with indicated antibodies (E). HEK293T cells were transfected with Myc-RNF125 together with Strep-FMDV 2B deletion mutant constructs containing plasmids. Whole-cell lysates were subjected to Strep pull own and immunoblotted with indicated antibodies (F). HEK293T cells were transfected with Strep-RNF125 together with GST-RIG-I domain constructs containing plasmids. Whole-cell lysates were subjected to strep pull down and immunoblotted with indicated antibodies (G). (H, I) HEK293T cells were transfected with control vector, FMDV Δ2B (1-114) mutant, FMDV Δ2B (1-125) mutant, and FMDV 2B wild-type plasmids and infected with VSV-GFP. Viral replication was measured by fluorescence microscopy, GFP absorbance, and plaque assay at indicated time points. The concentration of secreted IFN-β in supernatants was determined at 12 and 24 hpi by ELISA. Results representative of at least two independent experiments, each with similar results, and the values are expressed as mean ± SD of three biological replicates. Student’s t-test; **p < 0.01; ***p < 0.001.

Figure 6

FMDV 2B induces apoptosis and apoptosis-mediated caspase-3 and caspase-8 dependent degradation of MDA5. LFBK cells were infected with 0.1MOI of FMDV Asia1/Shamir for 0, 3, 6, 9 hours and whole-cell lysates were immunoblotted with indicated antibodies (A). PK-15 cells were transfected with increasing doses of FMDV 2B, and 24 hrs post-transfection cells were infected with SeV (1MOI). Cells were harvested at 24 hrs post-infection, and the MDA5 expression level was measured by immunoblotting (B). HEK293T cells were transfected with Flag-MDA5 together with Strep-FMDV 2B and treated with two doses of MG132, chloroquine, NH₄Cl, or zVAD for 6 hours before harvesting the cells. Whole-cell lysates were subjected to immunoblotting with indicated antibodies (C). PK-15 cells were transfected with FMDV 2B, and 24 hrs post-transfection, cells were infected with SeV (1MOI). Cells were treated with increasing doses of zVED for 6 hours before harvesting cells. Whole-cell lysates were subjected to immunoblotting with indicated antibodies (D). (E, F) HEK293T cells were treated with Flag-hMDA5, Strep-FMDV 2B plasmids, and treated with increasing doses of zDEVD-FMK (E), or zIETD-FMK (F), 6 hours before harvesting the cells. Whole-cell lysates were subjected to immunoblotting with indicated antibodies. HEK293T cells were transfected with Strep-FMDV 2B wild-type, FMDV Δ2B (1-114) mutant, FMDV Δ2B (1-125) mutant, and control vector. Annexin V apoptosis assay, CCK-8 assay, and MTT assay were conducted at indicated time points post-transfection (G). (H, I) HEK293T cells were transfected with Flag-MDA5, caspase-3 (G), or caspase-8 (H) plasmids together with increasing doses of Strep-FMDV 2B plasmids. Whole-cell lysates were subjected to immunoblotting with indicated antibodies. (J, K) HEK293T cells were transfected with Flag-MDA5, caspase-3 (J), or caspase-8 (K) plasmids together with increasing doses of Strep-FMDV 2B wild-type or FMDV Δ2B (1-125) mutant. Whole-cell lysates were subjected to immunoblotting with indicated antibodies. Results representative of at least two independent experiments, each with similar results. Student’s t-test; **p < 0.01; ****p < 0.0001.