The Role And Regulation Mechanism Of N-myc And STAT Interactor In Influenza A Virus Pneumoina

Influenza A virus（IAV）is a typical pathogen that causes acute respiratory diseases in humans.During the multiple influenza outbreaks,it has caused a large number of morbidities and mortalities worldwide.Clinically,severe influenza virus infection result in bilateral pulmonary infiltration and hypoxemia in patients.They often die from acute lung injury（ALI）or acute respiratory distress syndrome（ARDS）.Influenza A virus has a broad host range and high mutation rate.It is difficult to prevent and control influenza infection due to the limited efficacy of influenza vaccines,antiviral drug resistance and other reasons.The innate immune system is the first line of host defense against viruses.After IAV infection,the host’s pattern recognition receptors（PRRs）can quickly recognize pathogen-associated molecular patterns（PAMPs）of the virus and induce the activation of downstream nuclear factor-kappa B（NF-κB）,Interferon regulatory factor（IRF）3,and IRF7.Then the production of type Ⅰ interferon（IFN-I）,inflammatory cytokines and chemokines promote the killing and lead to kill and eliminating of virus.IFN-I,as the key cytokine in antiviral immunity,can induce the transcription of a series of interferon stimulated genes（ISGs）.The expression of ISGs prompts the host to exert antiviral activity quickly.In the process of antiviral innate immunity,post-translational modifications（PTMs）including ubiquitination,phosphorylation,acetylation,and methylation are an important ways to regulate protein function dynamically.In the PRR signaling pathways,some regulatory proteins can target sensors,receptors,adapter proteins,enzymes,transcription factors,etc.through different post-translational modifications to fine-tuning the antiviral immune response.Therefore,it is of significance to find important regulatory proteins in the signaling pathways of antiviral innate immunity and reveal their regulatory mechanism.It helps to screen the targets of antiviral drug and prevent influenza virus pneumonia.N-Myc and STAT interactor（NMI）is a protein induced after interferon stimulation.It consists of a coiled-coil domain（CC）at the N-terminus and two tandem NMI/IFP35domains（NID1/NID2）at the C-terminus.Previous studies have shown that NMI is homologous to interferon-induced protein 35（IFP35）and enhances the stability of IFP35 through mutual binding.NMI can inhibit cancer cell proliferation,participate in the JAK-STST pathway,act as damage-associated molecular patterns to promote inflammatory response,and is associated with anti-SARS and Sendai virus.Whether NMI has a regulatory role during IAV infection and its regulatory mechanism remain elusive.In this study,we focused on the role and regulatory mechanism of NMI in IAV infection.We found that the transcription level of NMI was significantly increased in peripheral blood leukocytes of patients with IAV infection,compared with normal people.The NMI expression level in lung tissue peaked on the third day after wild-type C57BL/6 mice were infected with PR8.NMI knockout(Nmi^-/-)mice were more resistant to influenza virus infection.Compared with wild-type mice,Nmi^-/-mice have higher survival rates,reduced lung virus titers,and increased expression of IFN-I（IFN-α/IFN-β）and ISGs.These data suggested that NMI may negatively regulate the host antiviral innate immune response.In vitro,we found that NMI deficiency in human lung epithelial cells and mouse macrophages could enhance the expression of IFN-I and inflammatory cytokines after PR8 infection.We used the dual luciferase reporter system to detect the interferon-stimulating response element（ISRE）and NF-κB transcription activity in NMI knockout or over-expressed cells after PR8 stimulation.We found that NMI inhibits ISRE and NF-κB activity.Then we detected the phosphorylation level of signal molecules after PR8 infection by immunoblotting and found that NMI attenuates activation of IRF3,IRF7 and NF-κB.Further,we used NMI to screen the important proteins in the antiviral signaling pathway,including RIG-1,MDA5,MAVS,TRIF,TBK1,IRF3,and IRF7.The results suggested that NMI functions specifically at IRF3 and IRF7 or downstream of them.Then we confirmed that NMI can interact with IRF3 and IRF7 by co-immunoprecipitation and immunofluorescence.The endogenous NMI also interacts with IRF3 and IRF7 increasingly after PR8 stimulation.NMI interacts with IRF3 and IRF7 through its NID1 and NID2 domains by transfection of NMI truncated plasmids and co-immunoprecipitation.Using protein synthesis inhibitors,proteasome inhibitors,and lysosomal inhibitors,we found that NMI promotes K48-linked polyubiquitination of IRF3 and IRF7,leading to their proteasomal degradation.Protein mass spectrometry analysis showed that E3 ubiquitin ligase TRIM21（tripartite motif-containing 21）can interact with IRF3 and IRF7,and TRIM21 reduced the protein expression of IRF3 and IRF7.Our results also revealed that NMI interacts with TRIM21.After overexpression of TRIM21 and NMI,endogenous ubiquitination of IRF3 and IRF7 is enhanced.After knockout of TRIM21,endogenous ubiquitination of IRF3 and IRF7 could even hardly be detected if NMI is overexpressed.These data indicated that NMI promotes the ubiquitination and degradation of IRF3 and IRF7 dependent on TRIM21.In summary,for the first time we identified that NMI negatively regulates antiviral innate immunity after influenza A virus infection.Furthermore,we revealed that NMI promote the ubiquitination and degradation of IRF3 and IRF7 dependent on TRIM21,thereby inhibiting the production of IFN-I and antiviral response.These results have enriched the basic research of the regulation network of ubiquitination,and a new mechanism to regulate the host antiviral innate immune response.Our study provides novel therapeutic targets for the prevention and treatment of viral pneumonia.