ARRDC4 regulates enterovirus 71-induced innate immune response by promoting K63 polyubiquitination of MDA5 through TRIM65

Figures

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  Figure 1. EV71 infection upregulates ARRDC4 expression. (a) Microarray analysis of EV71-infected t-MØ (MOI=50) for indicated time. Heat map represents arrestins family genes expression change (P<0.05). (b) Q-PCR analysis of expression of α-arrestin members in t-MØ treated with EV71 for 24 h or untreated. (c) mRNA expression of ARRDC4 was assayed by Q-PCR in t-MØ infected with EV71 for indicated time. (d) Immunoblot analysis of ARRDC4, EV71-VP1 and β-actin expression in lysates of t-MØ stimulated with EV71 for shown time. Data are shown as mean±S.D. of triplicate samples (b and c), or are representative of three independent experiments with similar results (d)

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  Figure 2. ARRDC4 promotes proinflammatory cytokines production in response to EV71 infection. (a and b) Q-PCR and immunoblot analysis of ARRDC4 knockdown efficiency in t-MØ 48 h after transfected with control siRNA or ARRDC4 siRNA. (c) Q-PCR analysis of IL-6, TNF-α and CCL3 mRNA expression in t-MØ, which were transfected as in (a) and infected with EV71 for 12 h. (d) ELISA of cytokines in supernatants of t-MØ treated as in (c). (e) Q-PCR analysis of EV71-VP1 RNA expression in EV71-infected t-MØ transfected as in (a). (f) Tissue culture infective dose (TCID50) assay in supernatants of t-MØ treated as indicated. Data are shown as mean±S.D. of triplicate samples, *P<0.05 (a, c-f), or are representative of three independent experiments with similar results (b)

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  Figure 3. ARRDC4 expression is positively correlated with serum proinflammatory cytokines concentration in EV71-infected HFMD patients and healthy controls. (a) Q-PCR analysis of ARRDC4 mRNA expression in PBMCs from EV71-infected HFMD patients (n=30) and controls (n=30), which normalized by the mean expression value. Data are shown as mean±S.D., **P<0.01. (b) The correlation between ARRDC4 mRNA expression level (assayed by Q-PCR) and IL-6, TNF-α, CCL3 serum concentration (assayed by ELISA) in patients were evaluated with the Pearson correlation test

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  Figure 4. ARRDC4 enhances EV71-triggered activation of NF-κB and MAPK pathway. The t-MØ were transfected with ARRDC4-specific siRNA or the negative control siRNA. After 48 h, the cells were infected with EV71 for indicated time, then total and phosphor-JNK, -ERK, -p38, -p65, -IRF3 and total IκBα were detected with immunoblot, the expression of EV71-VP1 and β-actin were detected as well. Data are representative of three independent experiments with similar results

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  Figure 5. ARRDC4 interacts with MDA5 via arrestin-like N domain. (a and b) The t-MØ were infected with EV71, immunoprecipitation (IP) with anti-ARRDC4 (a) or anti-MDA5 (b) and control IgG antibodies. Immnoblot analysis of the interaction between ARRDC4 and MDA5; input, the whole-cell lysates without immunoprecipitated. (c and d) HEK293T cells were transfected with Flag- or Myc-tagged ARRDC4 and MDA5 plasmids, after 48 h, IP with anti-Flag antibodies, then detected protein interaction by immunoblot. (e) Schematic structure of ARRDC4 and the derivatives were shown, full-length ARRDC4 (ARRDC4-WT), arrestin-like N domain (ARRDC4-N), arrestin-like C domain (ARRDC4-C), PPXY motif (ARRDC4-PPXY). The IP and immunoblot analysis of the interaction between MDA5 and the indicated ARRDC4 constructs in HEK293T cells. Data are representative of three independent experiments with similar results

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  Figure 6. ARRDC4 mediates K63 ubiquitination of MDA5. (a) Immunoblot analysis of K63 polyubiquitination of the precipitated MDA5 in t-MØ, which were transfected and stimulated as indicated. (b and c) HEK293T cells were transfected with indicated plasmids and infected with EV71 for 12 h, followed by IP with anti-Flag antibody. Immunoblot analysis of ubiquitination of MDA5; Ub-K0, mutant ubiquitin with no lysine residues retained. Data are representative of three independent experiments with similar results

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  Figure 7. ARRDC4 promotes MDA5 K63 ubiquitination through TRIM65. (a) The t-MØ were infected with EV71, and IP with anti-ARRDC4 and control antibodies, followed by immunoblot analysis as indicated. (b and c) IP and immunoblot analysis of protein interaction in HEK293T cells transfected with Flag- or Myc-tagged ARRDC4 and TRIM65. (d) Immunoblot analysis of the association between precipitated TRIM65 and MDA5, in t-MØ, which were transfected with ARRDC4 siRNA and control siRNA for 48 h and stimulated with EV71 for indicated time. (e) IP and immnunoblot analysis of ubiquitination of MDA5 in HEK293T cells transfected with indicated plasmids. (f) HEK293T cells were transfected with NF-κB luciferase reporter plasmid together with the other indicated plasmids, after 48 h, the luciferase activity was determined. Mock means empty vector. Data are representative of three independent experiments with similar results (a-e), or are shown as mean±S.D. of triplicate samples, *P<0.05, **P<0.01 (f)