Identification And Characterization Of A Novel Protein Encoded By PB2 Gene Of Eurasian Avian-like H1N1 Swine Influenza Virus

As a single-stranded,segmental RNA virus,novel influenza viruses can be generated through high mutation rate and gene rearrangement.It poses a significant threat to public health.As an intermediate host of influenza virus,pigs often play a role of “mixer vessel” of influenza viruses.Different sources of influenza virus can achieve cross-species transmissiblity by infecting pigs and reassortment in pigs,such as the swine-origin H1N1 influenza pandemic that broke out in 2009.Therefore,it is of great significance to study molecular pathogenesis of swine influenza virus.A Eurasian avian H1N1 swine influenza virus was isolated from a diseased farm in our laboratory.A new open reading frame(ORF)was found at the 5' terminal of the PB2 gene by sequence analysis.This ORF encodes a small protein containing 70 Amino acids,tentatively named as PB2-X.To demonstrate whether this coding region is capable of stably expressing PB2-X protein and whether the initiation codon of PB2-X can be recognized and transcribed by viral polymerase vRNPs,we constructed the firefly luciferase reporter gene fused with the PB2-X ORF,and Mini-genome assay was conducted.The results showed that viral vRNPs could recognize and express PB2-X protein.Subsequently,in order to detect whether PB2-X protein is stably expressed in cells,we cloned the PB2-X sequence into a eukaryotic expression vector fused with flag tag,and after transfecting 293 T cells,the expression of the protein was detected.The results showed that PB2-X protein was stably expressed in the transfected cells.To further identify whether the PB2-X protein was expressed during viral replication,we used a synthetic PB2-X peptide to immunize mice to prepare polyclonal antibodies,and the final results showed that PB2-X could be detected during viral replication with significantly lower level.To further explore the function of PB2-X protein,we compared the biological characteristics of viruses containing PB2-X or not.Since the European Avian-like H1N1 isolated in this study was not pathogenic to mice,the swine influenza virus(CH1N1)was used as a background,and PB2-X was artificially introduced without changing the amino acid sequence of PB2,and the CH1N1-PB2-X mutant virus was successfully rescued.By comparing the growth curves of the original strain(CH1N1-WT)and the mutant strain containing PB2-X on ST and MDCK cells,it was found that the CH1N1-PB2-X replicated more efficiently than CH1N1-WT on ST and MDCK cells,indicating that PB2-X is beneficial for replication of the virus.CH1N1-WT and CH1N1-PB2-X were inoculated into mice,and the pathogenicity of CH1N1-WT to mice was significantly stronger than that of CH1N1-PB2-X,indicating that the presence of PB2-X attenuated virus in mice.To further investigate the molecular mechanism by which PB2-X affects viral replication and pathogenicity,we examined the effect of PB2-X on H1N1 viral polymerase activity by using Mini-genome assay,which showed that overexpression of PB2-X protein inhibits polymerase activity.Further studies indicated that PB2-X affects the activity of polymerase indirectly by inhibting the protein expression of the eukaryotic expression plasmid of the polymerase protein(such as PB2,NP)in Mini-genome,and these proteins are utilized the CMV promoter to drive transcription.While overexpressed PB2-X did not change the mRNA level of PB2 gene,suggesting that the PB2-X protein may have a function of inhibiting translation of host mRNA.To further verify this phenomenon,we co-transfected the PB2-X eukaryotic expression plasmid with the EGFP expression plasmid containing the CMV promoter into 293 T cells.It was found that PB2-X protein inhibited the expression of EGFP protein.This indicates that PB2-X has a function of inhibiting the host protein translation system.This study aimed to identify and characterize the novel PB2-X protein found on PB2 gene of European Avian-like H1N1 swine influenza virus which could provide a basis for further study on the pathogenic mechanism of Eurasian avian influenza virus.