M2 Protein Transmembrane Region Of Influenza A Virus Inhibits Autophagosome Maturation

Influenza A virus is an important zoonotic pathogen,not only caused huge economic losses,some subtypes even broke through the interspecies barrier,obtaining the ability to infect human.Due to the great threat on public health imposed by influenza A virus,it is of great significance to develop effective antiviral strategy.The virus infection involves a series of complex physiological and biochemical process.Autophagy,as a highly conserved cellular degradative pathway,plays an important role in the antiviral process.Autophagy also induces protective innate and adaptive immunity in addition to its direct elimination by phagorytosis.Virus evades the antiviral mechanism of autophagy to guarantee their own survival.In view of this,more and more attentions are paid to the interaction study between viral infection and antiviral mechanism of autophagy.The M2 protein of influenza A virus is encoded by a spliced mRNA derived from genomic RNA segment 7.Its ion channel activity facilitates the dissociation of vRNPs from the matrix protein M1 at the early stage of the life cycle,and functions in virus assembly and budding.In order to further study the interactions between the M2 protein and autophagy,the GFP-LC3 and pCAF-M2 recombinant plasmids were constructed and transfected into 293 T cells,then the autophagosome level was detected by laser confocal and western blot method.Autophagy flux was also detected through further LC3-II turnover test,mRFP-GFP-LC3 plasmid,GFP-LC3 and LAMP1 colocalization observation.The results indicated that M2 protein could induce the accumulation of autophagosomes by blocking their fusion with lysosome which inhibited the lysosomal degradation of autophagosomes.Mammalian cells have three distinct Beclin1 complexes and function at different stages of autophagy,among them,Beclin1-UVRAG complex mediates the fusion of autophagosome with lysosome.To explore whether the M2 protein blocked autophagosome fusion with lysosome by the interaction with Beclin1 protein which affected Beclin1-UVRAG complex formation,recombinant plasmids of the autophagy-related proteins Beclin1 and UVRAG were constructed and transfected into 293 T cells together with M2 recombinant plasmid,the interactions of the three proteins were confirmed by co-immunoprecipitation experiments.The results showed that the interaction between Beclin1 and M2 protein existed,but gradually increasing the amount of M2 recombinant plasmid in transfection had no obvious effect on the formation of UVRAG-Beclin1 complex,implying that M2 protein might inhibit autophagosome fusion with lysosome by other mechanisms.Then we constructed the truncated M2 recombinant plasmids and assessed the effect of truncated M2 protein on autophagy.The deletion of amino acid 35-45 led to obviously subdued puncta accumulation,indicated that 35-45 amino acid of M2 protein functioned in inhibiting autophagosome maturation.The single deletion of amino acid 35-45 showed that the deletion of 41 tryptophan resulted in the decreased autophagosome accumulation,but the effect was less obvious than 35-45 deletion.