Mechanism Of Innate Immune Pathway Regulated By Influenza Virus M2 And PB1-F2 Proteins Through Inducing Autophagy

Influenza A virus?IAV?is an important pathogen that is seriously harmful to the health of humans and animals.IAV infection induces rapidly host innate immune response via a variety of regulatory mechanisms which is essential for pathogen clearance and host survival.However,excessive inflammatory response defined as“cytokine storm”leads to host immunopathology.Influenza A M2?matrix protein 2?protein,one of the best characterized viroporin to date,plays a well-established biological role in viral pathogenesis.IAV M2 protein is required for the activation of NLRP3?NLR family pyrin domain containing 3?inflammasome.Autophagy is a cytostic process that sequesters long-lived proteins,damaged organelles and protein aggregates into the autophagosome and its subsequent fusion with lysosome leads to the degradation of the cargo.Autophagy is classified into two processes:the autophagosome formation and the autolysosome formation.The IAV M2 protein not only is involved in the initiation of the autophagosome formation,but also inhibiting the fusion of autophagosome with lysosome.However,the mechanism how M2 initiates the autophagy process and whether and how M2 protein is involved in modulating host innate immunity remains unclear.In addition,IAV can evade host innate immune response that is involved in several viral proteins with complicated mechanisms.Among the proteins encoded by IAV,the PB1-F2 protein derived from PB1?Polymerase basic protein 1?was reported to lead to the reduction of mitochondrial membrane potential and interact with MAVS?mitochondrial associated protein?,thereby resulting in the inhibition of innate antiviral response.However,the detailed mechanisms how PB1-F2 modulates the innate immune response is not completely clear.Mitophagy is a process of the selective engulfment of mitochondria with low membrane potential by autophagosomes and their subsequent degradation by lysosomes,which functions as selective removal of damaged mitochondria.Mitophagy is documented to negatively regulate the innate immune response.It also has been proposed that A/Puerto-Rico/8?A/PR8?H1N1??influenza virus infection can induce mitophagy.However,the role that PB1-F2 plays in mediating IAV-induced mitophagic process remains unclear.In addition,whether mitophagy is associated with PB1-F2-suppressed innate immunity is obscure.In this study,we unveiled the molecular mechanisms of M2 and PB1-F2 regulation in the innate immune response through investigating the effect of M2 and PB1-F2 on autophagy process.The main research contents are as follows:1.M2 protein regulates MAVS-mediated signaling pathway through interacting with MAVS and increasing ROS production1)M2 protein is able to anchor to mitochondria,promote mitochondria fusion and increase the mitochondrial numberSeveral viroporins have been reported to be partially colocalized with mitochondria and alter the normal morphology of mitochondrial network.We confirmed that M2 protein was able to localize to mitochondria and decrease the expression of mitochondrial fission proteins which lead to increase of the mitochondrion fusion both under IAV infection and M2 overexpression by SIM,immunofluorescence analysis and mitochondria/cytosol fractionation.Furthermore,M2 expresion increased the mitochondrial number by facilitating expression of proteins mediating the mitochondrial biogenesis.However,overexpression of the M2 protein ion channel activity-abolished mutant M2^H37G or treatment with its ion channel activity inhibitor?amantadine?lost or attenuated the above effects,indicating that the function of M2 protein described above is dependent on its ion channel activity.2)M2 protein antagonizes the autophagy pathway to enhance the host innate antiviral immune responseIn this study,we also showed that M2 protein not only blocked the autolysosome formation,but also induced the PI3K-AKT-MTOR dependent autophagosome formation via both western blot and immunofluorescence analysis.Further studies showed that M2-triggered Ca²⁺and ROS production were involved in M2-induced autophagic process.SDD-AGE results demonstrated that M2 increased the ROS-dependent MAVS aggregates formation and dependent on its ion channel activity.Furthermore,M2 antagonisted autophagy process and sequestered the interaction of MAVS with ATG5 or LC3B,thereby reducing the ATG5-MAVS and LC3B-MAVS complexes formation and decreasing the degradation of MAVS aggregates,thus leading to the enhancement of MAVS-mediated innate immune response.2.Influenza A virus protein PB1-F2 impairs innate immunity by inducing mitophagy1)PB1-F2 induces TUFM-mediated mitophagyThe mitochondrial protein M of HPIV3 can mediate the mitochondrial protein TUFM-?Tu translation elongation factor,mitochondrial?dependent mitophagy.Similarly,we also showed that PB1-F2 protein mediated IAV-infection induced mitophagy.Intriguingly,we observed that PB1-F2 carried a typical conserved LIR?LC3 interacting region?motif?W?61?××L?64??,and all of its LIR mutants(PB1-F2^W61A,PB1-F2^W61A+L64A61A+L64A and PB1-F2?LIR)failed to interact with LC3B and induce mitophagy,suggesting that the LIR motif of PB1-F2 is essential for its interaction with LC3B and the induction of mitophagy.Furthermore,we constructed the TUFM mutants?TUFM?domain I,TUFM?domain II and TUFM?domain III?and showed that the domain I of TUFM is critical for its binding to PB1-F2.Thus,PB1-F2 acted as an autophagy receptor to interact with both LC3B and TUFM,thereby mediating the mitophagy induction.2)PB1-F2 inhibits the mitophagy-dependent type I IFN productionWe showed that inhibiting mitophagy by knockdown TUFM and ATG5?autophagy related 5?or expressing the PB1-F2 LIR mutants depressed the negative regulation of PB1-F2 on the type I IFN production and degradation of MAVS protein,suggesting that PB1-F2 inhibits the innate immune response depending on its mitophagy induction.This study unveiled the detailed mechanisms of M2-enhanced MAVS-meidated innate immune response by antagonizing the autophagy pathway,and the mechanisms of PB1-F2 inhibiting the type I IFN production by mediating the sequestering of mitochondria into autophagosome.Our studies provided novel insights into the interplay between M2 and PB1-F2 proteins of influenza virus and host innate immune response and offered poteintial target for development of novel antiviral therapeutics for influenza virus infection.