Screening Of Host Proteins That Interact With Influenza A Virus PB1-F2 Protein Using Yeast Two-hybrid System

As we all known,lnfluenza A viruse is one of the most important pathogens that is responsible for the health of humans as well as animals. In recent years, the widespread pandemic raises concern about the potential threat caused by influenza A virus. PB1-F2 is discovered an important virulence factors of influenza A virus in 2001, many scientists believe that the high mortality rate caused by influenza viruses may be associated to the PB1-F2 protein. In some strains,the protein is translocated to mitochondria,which sensitizes cells to death through interactions with two mitochondrial proteins,the inner mitochondrial membrane adenine nucleotide translocator 3 (ANT3) and the outer membrane voltage-dependent anion channel 1 (VDAC1),these interactions promote the permeabilization of the mitochodria and facilitate the release of cytochrome c that trigger cell apoptosis.PBl-F2 is able to promote inflammation and increase an opportunistic secondary bacterial infection, and finally it up-regulates viral polymerase activity by its interaction with the PB1 subunit. However, the molecular mechanism of protein function and its interaction with the host has remained elusive.Therefore, further study the interaction of PB1-F2 and host proteins is the main aspects of protein function.Yeast two-hybrid system is an important tool for the study of protein-protein interactions, protein functions and screening new proteins. To discover new proteins that can interact with PB1-F2.We firstly amplified the full-length PB1-F2 gene by PCR, and then cloned it into bait vector pGBKT7-PBl-F2 as a target gene used in screening.The constructed vector was transformed into yeast AH 109 cells to detect self-activation of pGBKT7-PB1-F2. The purchased human leukocyte cDNA library was tittered and amplified.After that, pGBKT7-PB1-F2 and the human leukocyte library plasmids were transformed sequentially into yeast strain AH109. The transformants were plated on SD agar to screen for interacting partners according to the manufacturer's instructions. Nine proteins were identified as the candidate interacting proteins of PB1-F2. Preliminary positive clones were further checked by restriction enzyme digestion, yeast two-hybrid cotransformation,and a-galactosidase activity detecting to exclude the false positive clones.Finally,the sequencing results of the candidate clones were analyzed using the BLAST database to identify the potential interaction of proteins and their homology analysis,the results are as followed,SNRK(Homo sapiens SNF related kinase),ANXA11(Homo sapiens annexin A11),PASK (Homo sapiens PAS domain containing serine/threonine kinase), ADRBK1 (Homo sapiens adrenergic, beta, receptor kinase 1),FTL (Homo sapiens ferritin, light polypeptide,mRNA),UGP2(Homo sapiens UDP-glucose pyrophosphorylase 2), Gβ2 (Homo sapiens guanine nucleotide binding protein (G protein)beta polypeptide 2), PLAUR(Homo sapiens plasminogen activator,urokinase receptor),PLSCR1 (Homo sapiens phospholipid scramblase 1).G proteins,a group of the activity of GTP binding and hydrolysis,are important intracellular signal transduction proteins and play an intermediary role in the transmission of information between the cell membrane receptor and effector proteins.G proteins are the major signaling transduction components and they are involved in the development of the disease during virus infection.Guanine nucleotide binding protein-βsubunit 2 (Gβ2),a member of the P-subunits of the heterotrimeric G proteins with WD40 repeats multifunction protein, has a crucial function in mitochondrial fusion.Although more and more current study reports on the GP2, none of those reports is relevant to influenza virus infection.We chose Gβ2 as the objective protein for future study,the interaction was further confirmed by GST pull-down and immunoprecipitation(IP)studies.The in vitro interaction assays of Gβ2 and PB1-F2 mutants indicated of PB1-F2,not only the C-terminus but also the N-terminus,were able to interact with Gβ2.The study of interaction between PB1-F2 and Gβ2 could provide important information for clarifying the biological function of PB1-F2, which lays the foundation on the pathogenic mechanism of influenza A viruse infection.