The Construction Of H5N1Virus-Host Interaction Network And Study On The Interaction Between PB2and RNF31

H5N1highly pathogenic Avian Influenza Virus has emerged zoonoticphenomenon and aroused panic recently. The interactions with the host proteins helpvirus complete the replication and transcription when influenza viruses infected thebody. In order to determine the role of host proteins in the pathogenesis of H5N1,wescreened179host proteins in human spleen library using viral proteins as baits by theyeast two-hybrid assay, and combined bioinformatic methods for a dynamic analysis.Constructing the interaction networks between viral proteins and host proteins, wefound that these interactions are more concentrated in the viral polymerase complex,and mediate apoptosis, immune response, protein transporter, RNA processing,multiple biological processes. Compared with random protein networks, the degree ofconnectivity and referral that H5N1attacks the proteins in the network are higher thanother proteins, and more inclined to be connected together. While gene enrichmentanalysis of the host and it’s neighboring proteins found that these proteins are mainlyfocused on NF-κB, WNT, MAPK, p53and apoptosis pathway. The gene functionanalysis of prey proteins between H5N1and other avian influenza strains and otherpathogens also be carried out, which provided ideas for better explain of differentlyreactions when infected different flu strains or different pathogens.Based on network analysis, we chose the interactions of viral polymerase PB2and human RNF31for deep study. PB2is an important polymerase subunit ofinfluenza virus, which determine the virulence and host range of the influenza virusesand the host ranges can change along with the changing of multiple amino acidresidues of PB2. RNF31is an E3ubiquitin ligase,which plays an important role inregulating NF-κB transcription activity. The study found that the transcriptionalactivity of NF-κB and some downstream target inflammatory genes induced byTNF-α was inhibited when down-regulated RNF31. Moreover, the expression ofpIκBα was suppressed and the cell apoptosis was increased when TNF-α stimulated.Indirect Immunofluorescence and Nuclear and Cytoplasmic Extractionconfirmed the PB2and RNF31localized in the nucleus, and their interactions alsooccurred in the nucleus by Co-IP. Their interaction domains are located in PB2N-terminal1-471and RNF31C-terminal910-1062peptides. The transcriptionalactivity of NF-κB and some downstream target genes was inhibited when over-expressed PB2and the inhibition of PB2on NF-κB activity was disappear whendown-regulated RNF31. Flowing cytometry also detected that PB2can promote cellapoptosis, which is consistent with the result of promoting cell apoptotic whendown-regulated RNF31. These results suggested that PB2on NF-κB transcriptionalactivity and its effects on biological function are mediated by RNF31. In addition, thestability and nuclear Co-IP also found that RNF31not only affected the stability ofexpression of PB2weakly, but also enhanced the interactions between PB1and PB2.These results suggest that RNF31play an important role in the formation of influenzapolymerization, which provides new ideas for the analysis of pathogenic mechanismsinfluenza virus and a way to find new drug targets for Avian Influenza Virus.