Screening And Preliminary Function Analysis Of AIV-M2 Interacting Cellular Proteins

Avian Influenza Virus(AIV)genome encodes at least fifteen viral proteins.AM2/M2 is a multi-functional,proton-selective protein which has roles both in promoting the formation of virus particles,equilibrating the pH of the Golgi apparatus as well as induceing the production of antibody.In addition,AM2 plays acrucial role in affecting the function of autophagy cells and activating NLRP3 inflammasome.The host cellular systems are formed by highly complicated and sophisticated networks that consist of interactions between numerous cellular components;viruses hijack these host cellular systems,leading to the re-construction of different networks to meet their need to replicate.Analysis of the interaction between AM2 protein and cellular proteins will shed light on the function of AM2 protein in the process of Avian Influenza Virus infection.Using AM2 protein as the research object,this study aims to obtain interacting host proteins by yeast two hybrid screening and verify the effects of protein interactions between AM2 protein and cellular protein on viral proliferation.Yeast cells were co-transformed with human lymphatic cDNA library plasmids and bait plasmid pGBKT7-AM2 without toxicity and self-activation.After further screening and retransformation assay,two host proteins could interact with AM2 protein in yeast cells.Sequencing analysis of host protein genes revealed that two protein genes respectively encoded cytochrome c oxidase subunit II(COXII)and G protein beta subunit(Gβ2).The heterotrimeric GTP-binding proteins(G proteins)are the important proteins used with the approximately 700 G protein-coupled receptors(GPCRs)to transduce extracellular signals across the plasma membrane,which can regulate cell proliferation,transformation,apoptosis and chemotaxis and also play an important role in inflammatory diseases and tumors.For a long time,the alpha subunit of the G protein is thought to play a dominant role in receptor signaling so that the role of the Gβγ subunit is neglected.Gβγ can also activate multiple downstream signaling pathways,including Phosphatide-ylinositol 3-kinase(PI3K)and MAP kinase(MAPK).Although much attention has been paid to Gβ,it has not been reported in the case of influenza virus infection.The Gβ2 protein was chosen as the research object in many Gβγ subunits.The interaction between AM2 and Gβ2 was confirmed in vitro and in vivo respectively by using GST pull-down and co-immunoprecipitation.Immuno-fluorescence assay also demonstrated that Gβ2 could specifically interact with AM2 and co-localize in the cytoplasm.In addition to the viral proteins,there are numerous cellular proteins involved at each stage of the influenza virus life cycle.Consequently,We carried out futher research into the effect of Gβ2 protein on viral proliferation.The results show that the expression level of Gβ2 protein was significantly enhanced after influenza virus infection.Furthermore,when the Gβ2 protein was specifically overexpressed,the level of viral replication increased significantly.However,the level of viral replication was significantly decreased by pretreating the host cell with Gallein,an inhabitor that blocks Gβγ-dependent cellular activities,which suggests that AM2 protein may regulate viral infection and proliferation utilizing G protein signaling pathway.As a whole,the host Gβ2 proteins can interact with AIV-AM2 protein using yeast two-hybrid system.Further studies revealed that Gβ2 protein can promote the viral reproduction.The finding of the interaction between AM2 and Gβ2 explore the new function of AM2 protein at the molecular level,which provides the necessary basis for the research on the correlation between G protein signaling pathway and the pathogenesis of influenza virus.