| Antiviral responses mediated by innate immune system is the first defense line of human body against viral infection.Deficiency or overreaction of antiviral innate immune responses will lead to related diseases.Thus studying the regulating mechanism of antiviral innate immune responses is of great importance.Post-translational modifications(PTMs)including E3 ubiquitin ligases-mediated ubiquitination play a key role in antiviral innate immune responses,however,the function of RING domain containing E3 ubiquitin ligases(RING E3s)in regulating antiviral innate immune responses has not been fully elucidated yet.Activation of type ? interferon(IFN-?)signaling is the central part of antiviral innate immune responses,the transcription of interferon-stimulated genes(ISGs)mediated by transcription factor signal transducer and activator of transcription 1(STAT1)contribute to the outcome of antiviral responses.However,over-activation of IFN-?/STAT1 signaling not only fails to sustain the effect of antiviral responses,but may also result in the suppression of immune system and chronic viral infection.Thus the negative regulation and related mechanism study of IFN-?/STAT1 signaling is valuable for the clinical application of IFN-?.To find out the new regulators of antiviral innate immune responses,demonstrating the function and mechanism of RING E3 family in antiviral innate immune responses,we screened 115 mouse RING E3 genes through High-content screening system based on siRNAs.Compared to the negative control,we identified 9 positive regulators and 30 negative regulators.To further find out the new negative regulators of IFN-?/STAT1 signaling,we performed the second screening.By adding exogenous IFN-? into cell culture supernatant,we found 9 out of the 30 genes still had negative effects,illustrating that these 9 molecules negatively regulated antiviral responses through IFN-?/STAT1 signaling.Among these 9 molecules,we chose to study ring finger protein 2(RNF2)which had the most effective function.By interfering Rnf2 gene expression in cells,knocking out Rnf2 gene expression in cells and mice,over-expressing of RNF2 in cells,we found that RNF2 negatively regulated VSV or IFN-?/STAT1-induced ISGs expression,suppressed antiviral responses both in vitro andin vivo.Through MS analysis on binding and ubiquitination level,we identified 15 potential substrates of RNF2 in antiviral responses including the transcription factor STAT1.In the meantime,we also identified 12 potential lysine sites of STAT1 which might be regulated by RNF2.By using immunoprecipitation and GST pull down assay,we demonstrated the direct association between RNF2 and STAT1.Besides,by transfecting truncations of RNF2 and STAT1 plus series of ubiquitin mutants followed by immunoprecipitation analysis,we found that upon IFN-? stimulation,RNF2 increased K33-linked polyubiquitination level of STAT1,inhibited the transcriptional activity of ISRE through RING domain.In our mechanism study,we performed chromatin-immunoprecipitation and developed a DNA pull down assay,we found that RNF2 could not affect the activation of IFN-?/STAT1 signaling,but could promote the disassociation of STAT1 with DNA through mediating ubiquitination on K379 of STAT1,indirectly promoting the dephosphorylation of STAT1,by which inhibit the IFN-?/STAT1 signaling in time.Our study systematically analyzed the regulating function of RING E3 family in antiviral responses,we not only found many new regulators of antiviral innate immune responses,but also provided new mechanism on the negative regulation of IFN-?/STAT1 signaling.Hence,our study has important values for clinical instruction and translation.We not only provide new potential targets for treatment of viral infection as well as development of new antiviral drugs,but also provide diagnosing and treating evidence for enhancing the effect of IFN-?,preventing immune tolerance,immune suppression and even immune injuries caused by over activation of IFN-? signaling and long-time application. |
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