Mechanism Of H1N1 Influenza A Virus NS2 And NP Proteins Inhibit The Host Innate Immune Response

Following infection in host cells,the viral RNA of Influenza A virus(IAV)is recognized by pattern recognition receptors and activate the type-I interferon(IFN-I)signaling through signal cascade,and finally initiate the antiviral innate immune response.IAV have evolved various strategies to evade antiviral innate immune responses and promote viral replication.With the deeper insight into the innate immune response mediated by IAV,it has been found that different viral proteins display different regulatory mechanisms against IFN-I signaling.In this study,IAV NS2 and NP proteins were used to explore the mechanisms by which influenza virus proteins regulate the innate immune response.Using luciferase reporter assay,q PCR,ELISA and Western blotting,we confirm that the nonstructural protein NS2 of IAV inhibits IFN-I signaling.We further revealed that different subtypes of IAV NS2 proteins had the same regulatory effects on IFN-I signaling pathway.In order to explore the target of NS2 protein in the IFN-I signaling pathway,we screened the key molecules involved in the IFN-I signaling pathway,using coimmunoprecipitation assay.It was found that NS2 protein interacted with interferon regulatory factors IRF3 and IRF7.Endogenous co-immunoprecipitation assay and si RNA knockdown of host protein IRF7 further identified the functional targets of NS2 protein.To clarify the mechanism of interaction between NS2 and IRF7,we used native PAGE,indirect immunofluorescence and cell fractions isolation assay to explore the mechanism within.NS2 inhibited the formation of IRF7 dimer,blocked the nuclear translocation of IRF7,and reduced the secretion of IFN-β.Our study reveals a novel mechanism by which NS2 negatively regulates IFN-I signaling.In the arm race between virus and host,other proteins of IAV also play important roles in escaping the innate immune response.To explore the effect of IAV NP to the IFN-I signaling,we detected the transcription,and expression level of IFN and the activation level of RIG-I pathway,and found that H1N1 NP protein was a new negative regulator of IFN-I signaling.We screened the target molecules of NP protein,and revealed that NP protein could specifically interact with and degrade MAVS.To investigate the degradation pathway of MAVS,we conducted the pharmacological inhibitor assays,and confirmed that MAVS is degraded by NP proteins via auto phagolysosome pathway.Since MAVS is localized on the outer mitochondrial membrane,it is speculated that the NP-mediated degradation of MAVS may be related to mitophagy.Transmission electron microscopy confirmed that H1N1 NP protein induced typical mitophagy.Co-immunoprecipitation assay revealed the interaction between Tollip and NP protein.MAVS or Tollip knockout cell lines were constructed to confirm that H1N1 NP protein was Tollip-dependent to induce mitophagy,thereby blocking the MAVS mediated antiviral IFN-I signaling.Through sequence analysis and domain mapping of NP protein,it was found that NP protein Y313 F mutation could block NPinduced mitophagy and MAVS degradation.The r PR8-NP-Y313 F mutant virus was rescued by reverse genetics operation system,and the pathogenicity of r PR8-NP-Y313 F mutant virus in BALB/c mice was weakened compared with the wild type strain,which further indicated that position 313 was the key amino acid site of NP protein.In summary,this study broadens the signal transduction network of NS2 and NP proteins in the negative regulation of innate immune response during IAV infection,enriches the understanding of the mechanism of viral protein-induced mitophagy,and provides a theoretical data for the prevention and control of influenza.