Temporal regulation of MDA5 inactivation by Caspase-3 dependent cleavage of 14-3-3η

Abstract

The kinetics of type I interferon (IFN) induction versus the virus replication compete, and the result of the competition determines the outcome of the infection. Chaperone proteins that involved in promoting the activation kinetics of PRRs rapidly trigger antiviral innate immunity. We have previously shown that prior to the interaction with MAVS to induce type I IFN, 14-3-3η facilitates the oligomerization and intracellular redistribution of activated MDA5. Here we report that the cleavage of 14-3-3η upon MDA5 activation, and we identified Caspase-3 activated by MDA5-dependent signaling was essential to produce sub-14-3-3η lacking the C-terminal helix (αI) and tail. The cleaved form of 14-3-3η (sub-14-3-3η) could strongly interact with MDA5 but could not support MDA5-dependent type I IFN induction, indicating the opposite functions between the full-length 14-3-3η and sub-14-3-3η. During human coronavirus or enterovirus infections, the accumulation of sub-14-3-3η was observed along with the activation of Caspase-3, suggesting that RNA viruses may antagonize 14-3-3η by promoting the formation of sub-14-3-3η to impair antiviral innate immunity. In conclusion, sub-14-3-3η, which could not promote MDA5 activation, may serve as a negative feedback to return to homeostasis to prevent excessive type I IFN production and unnecessary inflammation.

Fig 1. The full-length 14-3-3η degradation and sub-14-3-3η accumulation observed upon MDA5 activation were associated with Caspase 3 activation.

(A) Huh7 cells were infected with hCoV-229E at 0.1 MOI for 0 to 42 hours. Immunoblotting was performed to detect the viral protein and endogenous 14-3-3η protein abundance. (B) RD cells were infected with EV71 at 1 MOI for 0 to 12 hours. Immunoblotting was used to determine the viral infection and sub-14-3-3η accumulation. (C) Huh7 cells were transfected with 1 μg/mL HMW poly(I:C) in a time course from 0 to 36 hours. The cell lysates were analyzed by immunoblotting. (D) Huh7 cells were co-transfected with Flag-tagged MDA5 and various Myc-tagged 14-3-3 isoforms for 48 hours, followed by anti-Flag immunoprecipitation (IP). Immunoblotting was used to analyze the recovered products. (E) Huh7 cells were mock-treated or pretreated with Z-DEVD-FMK 50 μM for 1 hour and then co-transfected with Flag-tagged MDA5 and different Myc-tagged 14-3-3 isoforms for 48 hours. Anti-Flag immunoprecipitation (IP) was performed to detect the interaction of Flag-MDA5 and Myc-14-3-3 isoforms and the accumulation of Myc-sub-14-3-3. (F) Wildtype or CASP3 KD Huh7 cells were co-transfected with Flag-MDA5 and Myc-14-3-3 isoforms. After 48 hours, cells were harvested and then subjected to anti-Flag immunoprecipitation (IP) to determine the interaction of Flag-MDA5 and Myc-14-3-3 isoforms and the accumulation of Myc-sub-14-3-3 isoforms.

Fig 2. D209 of 14-3-3η has a critical role in sub-14-3-3η accumulation during MDA5 activation.

(A) The constructions of Myc-tagged 14-3-3η, 14-3-3ηΔC and 14-3-3ηΔαI are showed by illustration. (B) Huh7 cells were co-transfected with Flag-MDA5 and Myc-14-3-3η wildtype or truncated mutants for 48 hours, followed by anti-Flag immunoprecipitation (IP) to determine the interactions between Flag-MDA5 and the Myc-14-3-3η constructs. (C) Indicated Myc-14-3-3η constructs were co-transfected with empty vector, Flag-MDA5 or Flag-N-MDA5 into Huh7 cells, and the cell lysates were utilized to determine the IFNβ promoter activities by dual luciferase reporter assay. *: p<0.05, when compared with Flag-MDA5, mock. ##: p<0.01, ###: p<0.001, when compared with Flag-N-MDA5, mock. Ectopic protein expression levels were analyzed by immunoblotting. (D) Indicated Myc-14-3-3η constructs were co-transfected with empty vector, Flag-MDA5 or Flag-N-MDA5 into Huh7 cells. The IFNβ promoter activities were measured via dual luciferase reporter assay. *: p<0.05, **: p<0.01, when compared with Flag-MDA5, mock. Ectopic protein expression levels were analyzed by immunoblotting. (E) Huh7 cells were co-transfected with Flag-MDA5 and Myc-14-3-3η wildtype or D to A mutants for 48 hours, then subjected to anti-Flag immunoprecipitation (IP) to determine the interactions of Flag-MDA5 and Myc-14-3-3η wildtype or D to A mutants.

Fig 3. Sub-14-3-3η affects MDA5 activation through inhibiting MDA5 redistribution.

(A) Wildtype and 14-3-3η KD Huh7 cells were transfected with empty vector, Myc-14-3-3η or Myc-14-3-3ηΔαI and then mock-treated or stimulated with 1 μg/mL HMW poly(I:C) for 42 hours. Cell lysates were analyzed by immunoblotting to determine the protein levels. (B) 14-3-3η KD Huh7 cells were co-transfected with increasing amount of Flag-MDA5 and Myc-14-3-3η or Myc-14-3-3ηΔαI. Cells lysed by Triton X-100 lysis buffer were analyzed by SDD-AGE to determine the MDA5 oligomerization and total protein levels were detected via SDS-PAGE. (C) Huh7 cells were transfected with empty vector, Myc-14-3-3η or Myc-14-3-3ηΔαI for 24 hours, followed by mock-transfection or 1 μg/mL HMW poly(I:C) transfection for 18 hours. Cell lysates were separated into cytosol and mito-MAM fractions. Immunoblotting was utilized for detecting the redistribution of endogenous MDA5. (D) Quantification of MDA5 band intensity in cytosol or mito-MAM fractions with HMW poly(I:C) transfection from 3 independent experiments.

Fig 4. Cells expressing sub-14-3-3η are more permissive to viral infections.

(A) Huh7 cells were transfected with empty vector, Myc-14-3-3η or Myc-14-3-3ηΔαI for 24 hours and subsequently mock-treated or infected with SeV at 100 HAU for 24 hours or hCoV-229E at 1 MOI for 24 or 42 hours. Quantitative RT-PCR was performed to analyze the mRNA expression levels of IFNβ. (*: p<0.05, **: p<0.01; ###: p<0.001) (B) Empty vector, Myc-14-3-3η, Myc-14-3-3ηΔαI or Myc-14-3-3η D209A were transfected into 14-3-3η KD Huh7 cells, followed by mock-treated or infected with hCoV-229E at 1 MOI for 36 or 42 hours. The relative mRNA expression levels of IFNβ were analyzed by quantitative RT-PCR. (***: p<0.001; ##: p<0.01, ###: p<0.001) (C) Empty vector, Myc-14-3-3η, Myc-14-3-3ηΔαI or Myc-14-3-3η D209A were transfected into wildtype or 14-3-3η KD Huh7 cells followed by mock-treated or infected with hCoV-229E at 0.01 MOI for 6 or 12 hours, and the intracellular vRNA levels were determined by quantitative RT-PCR. (*: p<0.05, ***: p<0.001; #: p<0.05, ##: p<0.01) (D) Empty vector, Myc-14-3-3η, Myc-14-3-3ηΔαI or Myc-14-3-3η D209A were transfected into MDA5 KD Huh7 cells followed by mock-treated or infected with hCoV-229E at 0.01 MOI for 18 hours, and the intracellular vRNA levels were determined by quantitative RT-PCR. (E) Huh7 cells were mock-treated or pretreated with Z-DEVD-FMK 50 μM for 1 hour and subsequently infected with hCoV-229E at 0.1 MOI for 0 to 36 hours. Immunoblotting was performed to detect the viral infection and other protein levels. (F) Wildtype and CASP3 KD Huh7 cells were infected with EV71 at 5 MOI for 0 to 24 hours, and immunoblotting was performed to determine the viral infection and other protein levels.

Fig 5. Illustration of the proposed model during viral infection.

Viral infection triggers the activation of MDA5 and RIG-I anti-viral signaling. When MDA5-mediated type I IFN induction is activated, it will lead to the activation of Caspase-3. Then, 14-3-3η is cleaved by active Caspase-3, which results in the formation of sub-14-3-3η. Sub-14-3-3η competes with full-length 14-3-3η to interact with MDA5. Subsequently, MDA5 bound with sub-14-3-3 losses IFNβ-inducing function. Therefore, by forming sub-14-3-3η to block MDA5 signaling as a negative feedback, over-activation of type I IFN induction can be prevented in cells.