RND3 restricts encephalomyocarditis virus replication by promoting IKKε ubiquitination and type I interferon production

Figures

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  Fig 1. Screening of restrictive factors against EMCV in vitro. (A) HEK293T cells were transfected with plasmids pcDNA3.1-Myc-IFI44 (1 μg), pcDNA3.1-Myc-ZFP36 (1 μg), pcDNA3.1-Myc-ZDHHC1 (1 μg), pcDNA3.1-Myc-NEURL3 (1 μg), and pcDNA3.1-Myc-RND3 (1 μg) for 24 h. Then infected with EMCV at MOI = 0.1 and harvested cells after 24 h, qRT-PCR detected EMCV-VP1 gene mRNA. (B–E) HEK293T cells infected with different strains of EMCV at MOI = 0.1 were harvested after 0 h, 12 h, 24 h, 36 h, and 48 h, qRT-PCR detected RND3 mRNA. Protein level measured using anti-RND3 antibodies. β-actin was used as a loading control. The results are presented as means ± standard deviations. ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs control.

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  Fig 2. Overexpression of RND3 inhibits virus replication. (A) HEK293T cells were transfected with pcDNA3.1-Myc-hRND3 (1 μg) or pcDNA3.1-Myc (1 μg) for 24 h and then infected with EMCV at MOI = 0.1 were harvested at different points in time, qRT-PCR detected EMCV-VP1 gene mRNA. (B) Virus titers in the supernatants were analyzed by a TCID50 assay. (C) HEK293T cells were transfected with pcDNA3.1-Myc-hRND3 (1 μg), pcDNA3.1-Myc-hRND3 (2 μg), or pcDNA3.1-Myc (1 μg) for 24 h and then infected with EMCV at MOI = 0.1 were harvested at 24 post hours infection, virus titers in the supernatants were analyzed by a TCID50 assay. (D) HEK293T cells were transfected with pcDNA3.1-Myc-hRND3 (300 ng), pcDNA3.1-Myc-hRND3 (500 ng), or pcDNA3.1-Myc (300 ng) for 24 h, the cells then were infected with VSV-GFP at MOI = 0.01 for 18 h, the cells were observed microscopically. (E) VSV mRNA level was detected by qRT-PCR. (F) MEF cells were transfected with pcDNA3.1-Myc-mRND3 (1 μg) or pcDNA3.1-Myc (1 μg) for 24 h and then infected with EMCV at MOI = 0.1 were harvested at different points in time, qRT-PCR detect EMCV-VP1 gene mRNA. (G) Virus titers in the supernatants were analyzed by a TCID50 assay. (H) MEF cells…

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  Fig 3. RND3 activates type I interferon production. (A and B) HEK293T cells were co‐transfected with pcDNA3.1‐Myc‐RND3 (0.5 μg), pcDNA3.1‐Myc‐RND3 (1 μg), pIFN-β‐luc (200 ng), pISRE‐luc (200 ng), and pRL‐TK (10 ng). After 24 h, the cells were stimulated with poly(I:C) for 12 h. Then, luminescence was detected using dual‐luciferase report. (C and D) HEK293T cells were co‐transfected with pcDNA3.1‐Myc‐RND3 (0.5 μg), pcDNA3.1‐Myc‐RND3 (1 μg), or pcDNA3.1-Myc (1 μg). After 24 h, the cells were stimulated with poly(I:C) for 12 h. Then, IFN-β, CXCL10, RANTES, OASL, and ISG56 mRNA levels were detected using qRT-PCR. (E and F) HEK293T cells were co‐transfected with pcDNA3.1‐Myc‐RND3 (0.5 μg), pIFN-β‐luc (200 ng), pISRE‐luc (200 ng), and pRL‐TK (10 ng). After 24 h, the cells were stimulated with SeV at MOI = 1 for 12 h. Then, luminescence was detected using dual‐luciferase report. (G and H) HEK293T cells were co‐transfected with pcDNA3.1‐Myc‐RND3 (0.5 μg), or pcDNA3.1-Myc (1 μg). After 24 h, the cells were stimulated with SeV at MOI = 1 for 12 h. Then, IFN-β, CXCL10, RANTES, OASL, and ISG56 mRNA levels were detected using qRT-PCR. (I) HEK293T cells were transfected with pcDNA3.1‐Myc‐RND3 (1 μg)…

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  Fig 4. Silencing RND3 inhibits type I interferon production. (A) HEK293T cells were transfected with negative control siRNA (siRND3-ctrl) or siRND3 against RND3. The protein expression was analyzed by Western blotting with anti-RND3 and anti-β-actin antibodies. The mRNA of RND3 was quantified by qRT-PCR. (B and C) HEK293T cells were transfected with negative control siRNA (siRND3-ctrl), siRND3-1 for 24 h and then co‐transfected with pIFN-β‐luc (200 ng), pISRE‐luc (200 ng), and pRL‐TK (10 ng). After 24 h, the cells were stimulated with poly(I:C) for 12 h. Then, luminescence was detected using dual‐luciferase report. (D and E) HEK293T cells were transfected with negative control siRNA (siRND3-ctrl), siRND3-1 for 48 h. The cells were stimulated with poly(I:C) for 12 h. Then, IFN-β, CXCL10, RANTES, OASL, and ISG56 mRNA levels were detected using qRT-PCR. (F and G) HEK293T cells were transfected with negative control siRNA (siRND3-ctrl), siRND3-1 for 24 h and then co‐transfected with pIFN-β‐luc (200 ng), pISRE‐luc (200 ng), and pRL‐TK (10 ng). After 24 h, the cells were stimulated with SeV at MOI = 1 for 12 h. Then, luminescence was detected using a dual‐luciferase report. (H and I) HEK293T…

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  Fig 5. knockdown of RND3 promotes virus replication. (A) HEK293T cells were transfected with negative control siRNA (siRNA-ctrl), siRND3-1 for 48 h and then infected with EMCV at MOI = 0.1 were harvested at 24 post hours infection, qRT-PCR detected EMCV-VP1 gene mRNA. (B) Virus titers in the supernatants were analyzed by a TCID50 assay. (C and D) HEK293T or MEF cells were transfected with pcDNA3.1-Myc-hRND3/mRND3 (1 μg) or pcDNA3.1-Myc (1 μg) for 24 h, the cells then were infected with SeV (MOI = 1) for 24 h. The supernatants were inactivated by ultraviolet radiation and were collected to treat fresh HEK293T or MEF cells for another 24 h. The cells were infected with VSV-GFP (MOI = 0.01) for 18 h, the cells were observed microscopically. The results are presented as means ± standard deviations. **, P < 0.01; ***, P < 0.001 vs control.

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  Fig 6. RND3 enhances adaptor protein mediated the IFN-I response. (A and B) HEK293T or MEFs cells were co-transfected with pcDNA3.1-Flag-hRND3/mRND3 (0.5 μg), pIFN-β‐luc (200 ng), pRL‐TK (10 ng), and TLR3 expressing plasmid for 24 h, followed by poly(I:C) treatment for 6 h, the luminescence was detected using dual‐luciferase report. (C–H) HEK293T cells were co-transfected with the following plasmids: pcDNA3.1-Flag-hRND3 (0.5 μg), pIFN-β‐luc (200 ng), pISRE‐luc (200 ng), and pRL‐TK (10 ng), HA-MDA5 (0.5 μg), HA-RIG-I (0.5 μg), HA-MAVS (0.5 μg), HA-TBK1 (0.5 μg), HA-IKKε (0.5 μg), or HA-IRF3 (0.5 μg). After 24 h, the luminescence was detected using dual‐luciferase report. The protein expression was analyzed by Western blotting with the indicated antibodies. β-Actin was used as a loading control. The results are presented as means ± standard deviations. ns, not significant; ***, P < 0.001 vs control.

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  Fig 7. RND3-specific interaction with IKKε. (A) HEK293T cells were co-transfected with pcDNA3.1-Flag-RND3 (0.5 μg), HA-MDA5 (0.5 μg), HA-RIG-I (0.5 μg), HA-MAVS (0.5 μg), HA-TBK1 (0.5 μg), HA-IKKε (0.5 μg), or HA-IRF3 (0.5 μg). After 24 h, the cell lysate was harvested. Co-immunoprecipitation (Co-IP) was performed, and the precipitated proteins were analyzed by Western blotting using anti-HA antibodies. (B) HEK293T cells were co-transfected with pcDNA3.1-Flag-RND3 (0.5 μg) and HA-IKKε (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed, and the precipitated proteins were analyzed by Western blotting using anti-Flag antibodies. (C and D) HEK293T cells were transfected with HA-IKKε (0.5 μg) or pcDNA3.1-Flag-RND3 (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-RND3 or anti-IKKε antibodies. (E) HeLa cells were co-transfected with peGFP-C1-RND3 and pcDNA3.1-mcherry-IKKε. At 24 h post transfection, co-localization was performed using Confocal fluorescence microscopy. The nuclei were stained by DAPI. The fluorescence intensity profile of DAPI (blue), RND3 (green), and IKKε…

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  Fig 8. RND3 recruits TRIM21 to initiate the ubiquitination of IKKε. (A) HEK293T cells were co-transfected with pcDNA3.1-Flag-hRND3 (0.5 μg), HA-IKKε (0.5 μg), or His-Ub (0.2 μg). After 24 h, the cell lysate was harvested. Co-immunoprecipitation (Co-IP) was performed using an anti-HA antibody (MAb) (1:100). The precipitated proteins were analyzed by Western blotting using anti-His antibodies. (B) HEK293T cells were co-transfected with Flag-hRND3 (1 μg), HA-IKKε (1 μg), and His-Ub or the indicated Ub mutant plasmids (0.5 μg). After 24 h, the cell lysate was harvested. Co-IP was performed and the precipitated proteins were analyzed by Western blotting using anti-His antibodies. (C) HEK293T cells were co-transfected withpcDNA3.1-Flag-hRND3 (2 μg). After 24 h, the cell lysate was harvested. Immunoprecipitation was performed. Subsequently, the interaction proteins of RND3 in cells were analyzed by mass spectrometry. (D) HEK293T cells were transfected with HA-TRIM21 (1 μg) or pcDNA3.1-Flag-hRND3 (1 μg). After 24 h, the cell lysate was harvested. Co-immunoprecipitation (Co-IP) was performed using an anti-HA antibody (MAb) (1:100). The precipitated proteins were analyzed by Western blotting…

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  Fig 9. A working model for the role of RND3 proteins in regulating interferon-mediated signaling to mediate anti-EMCV replication.