Figure 1.
MAVS induces JNK activation during viral infection.
(A) HEK293 cells were transfected with the Flag-MAVS plasmid or empty pCDNA3.0 vector (EV) at dose gradient (0, 1 or 2 µg/well in a 12-well plate) for 24 hours, cell lysates were collected for western blot analysis using the indicated antibodies. (B) HEK293 cells were transfected with MAVS siRNA or negative control siRNA and then treated with SeV (MOI = 1) or TNF-α (10 ng/ml) for the indicated times. Whole cell lysates were collected for western blot analysis of p-JNK, JNK, p-ERK, ERK, p-p38, p38 and MAVS. NC, negative control, MOI, multiplicity of infection, p-, phosphorylated. (C) HEK293 cells were transfected with EV or with plasmids of the indicated antiviral signaling components. Whole cell lysates were collected for western blot analysis. Anti-p-JNK antibody was used to measure the level of JNK phosphorylation, and anti-Flag antibody was used to determine the transfecting efficiency. (D, E and F) Wild type (WT), RIG-I knockout (Rig-i−/−), TBK1 knockout (Tbk1−/−) or MAVS knockout (Mavs−/−) MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were immunoblotted for phosphorylated JNK and corresponding deficient protein.
Figure 2.
JNK2, but not JNK1, is essential for virus-induced apoptosis.
(A) Control, JNK1 and JNK2 siRNA knock-down HEK293 cells (left), or wild type, Jnk1−/− and Jnk2−/− MEF cells (right), were treated with SeV (for HEK293 MOI = 1, for MEF MOI = 4) for the indicated times. Cell lysates were analyzed by western blot, probing for ISG15, ISG60, JNK1 and JNK2 with the indicated antibodies. (B) Wild type and Mavs−/− MEF cells were treated with SeV (MOI = 4), or TNF-α (10 ng/ml) plus cycloheximide (CHX, 10 µg/ml) for the indicated times. Cell lysates were collected for western blot analysis using anti-PARP antibody to determine cell apoptosis and using anti-MAVS antibody to measure the deficiency of MAVS protein. (C) HEK293 cells were transfected with the indicated plasmids and 24 hours later, cell lysates were collected for western blot analysis of PARP, phosphorylated JNK, phosphorylated IRF3, Flag-tagged proteins and β-actin. (D) HEK293 cells were treated by SeV (MOI = 1) with or without JNK kinase inhibitor SP600125 (5 µM). Cell lysates were collected for western blot analysis of PARP, cleaved caspase-3 and β-actin to probe for cell apoptosis. (E) Control, JNK1 or JNK2 knocked down HEK293 cells were treated with SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis to measure cell apoptosis using the indicated antibodies. (F) Wild type, Jnk1−/− or Jnk2−/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis.
Figure 3.
MKK7 functionally links MAVS to JNK2 during virus-induced apoptosis.
(A) Wild type, Mkk3/6−/− or Mkk4/7−/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell lysates were collected for western blot analysis using anti-JNK, anti-p-JNK, anti-MKK3, anti-MKK4, anti-MKK6, anti-MKK7 and anti-β-actin antibodies. (B) HEK293 cells were transfected with NC, MKK4 and MKK7 siRNAs respectively for 48 hours and then treated by SeV (MOI = 1) for the indicated times. Cell lysates were collected for western blot analysis of p-JNK, JNK, MKK4, MKK7 and β-actin. (C) Wild type, Mkk3/6−/− or Mkk4/7−/− MEF cells were treated with SeV (MOI = 4) for the indicated times. Cell apoptosis was determined by western blot analysis of activated PARP and cleaved caspase-3. (D) HEK293 cells were transfected with NC, MKK4 and MKK7 siRNAs for 48 hours and then treated with SeV (MOI = 1) for 24 hours. Cell lysates were collected for western blot analysis of PARP and cleaved caspase-3 to measure cell apoptosis. Mkk3/6−/−, Mkk3 and Mkk6 double knockout; Mkk4/7−/−, Mkk4 and Mkk7 double knockout.
Figure 4.
Viral infection triggers MKK7 to bind MAVS on mitochondria.
(A) HEK293T cells were transfected with the indicated combination of plasmids. Cell lysates were immunoprecipitated using anti-Flag antibody. The immunoprecipitated proteins and input were detected by western blot analysis using anti-HA and anti-Flag antibodies. IP, immunoprecipitation. (B) HEK293 cells were treated with SeV (MOI = 1) or TNF-α (10 ng/ml) for 6 hours. Cell lysates were immunoprecipitated using anti-MKK7 antibody or normal rabbit IgG, followed by western blot analysis with anti-MAVS and anti-MKK7 antibodies. The immunoprecipitated MAVS was quantified relatively and the numeric values are shown on the top of the MAVS blot. Asterisk indicates a cross reaction band. (C) HEK293T cells were transfected with the indicated combinations of plasmids for 24 hours. Cell lysates were immunoprecipitated with anti-HA antibody, followed by western blot analysis with anti-HA and anti-Flag antibodies. (D) HA-tagged MKK7 or its truncation mutants were transfected into HEK293T cells along with Flag-MAVS for 24 hours. Cell lysates were immunoprecipitated with anti-HA antibody, followed by western blot analysis with anti-HA and anti-Flag antibodies. Asterisk indicates a cross reaction band. (E) HEK293 cells were transfected with HA-MKK4 or HA-MKK7 plasmids for 24 hours and then stimulated with or without SeV (MOI = 1) for 6 hours. Total cell lysates as well as mitochondrial and cytosolic fractions (obtained using subcellular fractionation) were probed with anti-HA, anti-caspase-3 (full length) and anti-Tom20 antibodies. Full length caspase-3 and Tom20 were applied to indicate the accuracy of fractionation. (F) Wild type or Mavs−/− MEF cells were stimulated with or without SeV (MOI = 4) for 6 hours. Subcellular fractionation was performed as described in E and the fractions were probed with anti-MKK7, anti-MAVS, anti-caspase-3(full length), and anti-Tom20 antibodies. (G) MEF cells were treated with or without SeV (MOI = 4) for 6 hours, The cells were then stained with the indicated antibodies and imaged by confocal microscopy. (H) MEF cells were transfected with either EGFP-MAVS, EGFP-MKK7-Δ3D, or EGFP-MKK7 and then treated with SeV (MOI = 4) for 6 hours. Cells were stained with Mitotracker and DAPI, and then imaged by confocal microscopy.
Figure 5.
MAVS-MKK7-JNK2 represents a novel apoptotic signaling cascade.
(A) Wild type and Mkk4/7−/− MEF cells were transfected with the indicated combinations of plasmids for 24 hours. Mkk4/7−/− MEFs with HA-MKK4 ectopic-expression were used to mimic Mkk7−/− and Mkk4/7−/− MEFs with HA-MKK7 ectopic-expression were used to mimic Mkk4−/−. Cell lysates were collected for western blot analysis using the indicated antibodies. (B) Wild type, Jnk1−/− or Jnk2−/− MEF cells were transfected with the plasmid Flag-MAVS at a dose gradient (0, 2 and 4 µg/well in a 12-well plate). Cell lysates were collected for western blot analysis using the indicated antibodies. (C) Wild type or truncation mutants of MKK7 were re-introduced into “Mkk7−/− MEF cells” for 24 hours and the cells were then infected with SeV for 48 hours. Cell apoptosis was measured by western blot analysis of PARP and cleaved caspase-3. (D) HA-tagged JNK1, JNK2 and mutated JNK2(183/185A) were re-introduced into Jnk2−/− MEF cells, followed by similar analysis as described in C. (E) Flag-tagged wild type MAVS and truncation mutant MAVS-ΔTM were re-introduced into Mavs−/− MEF cells, followed by similar analysis as described in C.
Figure 6.
JNK2, but not JNK1, protects mice against viral infection in vivo.
(A) Wild type C57BL/6, Jnk1−/− or Jnk2−/− mice (12 mice in each group) were infected with VSV at the indicated doses via nasal dripping (liquid volume≤20 µL). Survival rates of the mice were monitored at 5 days after infection. (B) The mice were intranasally challenged with VSV (106 PFU/g mouse weight). Two days after infection, serum was collected from the mice. Equal volumes (10 µL) of serum were added to HEK293 cells culture media and incubated for 24 hours, followed by plaque assay using crystal violet staining. Viral plaques were observed by microscopy and the viral titer was calculated. (C) Data from B presented as means±SD (n = 3). (D) Wild type C57BL/6, Jnk1−/− or Jnk2−/− mice were infected with NDV-GFP (106 PFU/g mouse weight). Two days after viral challenge, lungs were collected for fluorescence stereomicroscopy to observe NDV-GFP in situ. (E) Serum was collected at 2 days after NDV-GFP infection and equal volumes (100 µL) of serum were added to HEK293 cells culture media. Eighteen hours later, the NDV-GFP infected cells were observed by fluorescence microscopy. (F) GFP-positive cells from E were quantified by flow cytometry.
Figure 7.
JNK2 protects mice against virus-induced organ injury.
(A) BMDMs from wild type, Jnk1−/− or Jnk2−/− mice were treated with or without SeV (MOI = 1) for 18 hours and then IFN-β production was determined by ELISA. Data are presented as means±SD (n = 3). (B) BMDMs from wild type, Jnk1−/− or Jnk2−/− mice were treated with SeV (MOI = 1) for 48 hours, followed by Annexin-V staining analysis. Cells undergoing apoptosis (Annexin-V positive and PI negative) were quantified by flow cytometry. Data are presented as means±SD (n = 3). (C) The indicated siRNAs were transfected into HEK293 cells together with NF-κB-Luc reporter or PRDIII-1-Luc reporter plasmids and 24 hours later, cells were infected with or without SeV (MOI = 1). A luciferase assay was performed 12 hours post-infection. Data are presented as means±SD (n = 3). (D and E) Wild type, Jnk1−/− or Jnk2−/− mice were intranasally challenged with SeV (107 PFU/g mouse weight). Two days later, lungs and livers were harvested for histochemistry analysis by H&E staining and immunohistochemistry analysis by detecting cleaved caspase-3 staining. (F and G) Mice were infected with VSV or NDV as described in Figure 6. Two days later, lungs and livers were harvested for histochemistry analysis by H&E staining and immunohistochemistry analysis by cleaved caspase-3 staining. Tissue sections were visualized by microscopy (20× objective for lung, 40× objective for liver).
Figure 8.
Schematic diagram of the MAVS-MKK7-JNK2 apoptosis signaling pathway.
MAVS is a critical converging point of the host response to RNA virus infection. By engaging RIG-I or MDA5, MAVS forms a mitochondrial signalosome, dynamically composed of TRAF2/3/6, TRADD, TOM70, UXT-V1 and so on. This signalosome leads to the activation of TBK1 and IKK and then the ultimate induction of type 1 interferon and inflammatory cytokines, thereby establishing the antiviral state. In addition, MAVS also recruits MAPK kinase MKK7 onto the mitochondria via protein-protein interaction. MKK7 subsequently induces the phosphorylation of JNK2 at its threonine-183 and tyrosine-185 positions. Finally, JNK2 initiates cell apoptosis to sacrifice the virus-infected host cell, thereby dampening virus-induced inflammatory injury.