Influenza virus contains three integral membrane proteins: haemagglutinin,neuraminidase,and matrix protein (M1 and M2).Among them,M2 protein functions as an ion channel,important for virus uncoating in endosomes of virus-infected cells and essential for virus replication.In an effort to explore potential new functions of M2 in the virus life cycle,we used yeast two-hybrid system to search for M2-associated cellular proteins.One of the positive clones was identified as human Hsp40/Hdj1,a DnaJ/Hsp40 family protein.Here,we report that both BM2 (M2 of influenza B virus) and A/M2 (M2 of influenza A virus) interacted with Hsp40 in vitro and in vivo.The region of M2-Hsp40 interaction has been mapped to the CTD1 domain of Hsp40.Hsp40 has been reported to be a regulator of PKR signaling pathway by interacting with p58IPK that is a cellular inhibitor of PKR.PKR is a crucial component of the host defense response against virus infection.We therefore attempted to understand the relationship among M2,Hsp40 and p58IPK by further experimentation.The results demonstrated that both A/ M2 and BM2 are able to bind to p58IPK in vitro and in vivo and enhance PKR autophosphorylation probably via forming a stable complex with Hsp40 and P58IPK,and consequently induce cell death.These results suggest that influenza virus M2 protein is involved in p58IPK-mediated PKR regulation during influenza virus infection,therefore affecting infected-cell life cycle and virus replication.