Molecular Mechanism Of Different PB2 Genes Affecting The Replication Of H5N6 Subtype Avian Influenza Virus

In recent years,H5N6 has gradually replaced H5N1 to become the predominant H5 subtype highly pathogenic avian influenza virus(AIV)in China.With continued viral evolution,the genome composition of H5N6 virus is getting more and more complicated,especially that some circulating isolates bear all their six internal genes from S genotype H9N2(H9N2/S)viruses.However,those presently prevelant H9N2/S viruses in chicken flocks showed a significant feature in molecular evolution that PB2 and M genes of the G1-like having substituted the F/98-like,as compared with the previously prevailing H genotype H9N2(H9N2/H)viruses.Additionally,novel influenza reassortants deriving all their internal genes from H9N2/S,such as H5N6/S,H7N9/S and H10N8/S viruses have already emerged in avian and human cases.Therefore,our study that investigating the influence of G1-like and F/98-like PB2 genes to the replication of H5N6/S reassortants and the relevant mechanism,will provide important reference for further exploration the molecular mechanism that H9N2/S donating the whole set of internal genes for genesis of novel influenza reassortants.1.The effect of different PB2 genes on replication of H5N6 subtype AIV in terms of competitive virus reassortmentIn order to evaluate the effect of G1-like and F/98-like PB2 genes on the replication of H5N6 reassortants in terms of virus competitive reassortment,three H5N6/S circulating isolates of MZ34,YB0597 and JT131 were selected for the following assays in the present study.Based on the reverse genetics technology,a 10-plasmid combination containing the 8 gene plasmids from MZ34 and 2 additional F/98-like PB2 and M gene plasmids was co-transfected for competitive rescue of reassortant H5N6 viruses.Subsequently,rescued H5N6 reassortants after 3 consecutive rounds of plaque purification were used for RNA extraction to sequence the PB2 and M genes.Alternatively,to exclude the possible interference that the 8 gene plasmids from the same parental virus may be prone to self-packaging,we additionally replaced the PB2 and M plasmids of MZ34 with YB0597 or JT131,respectively.The results showed that G1-like PB2 had absolute advantage in quantity in any of the above 10-plasmid transfection groups from the composition of PB2 gene and M gene among the rescued H5N6 reassortants.However,the G1-like M just exhibited advantage in the MZ34(8)+F/98(PB2+M)group.Moreover,comparing with the F/98-like PB2 gene,the H5N6 reassortants containing G1-like PB2 gene increased the replication capability in CEF,A549 and MDCK cells.Therefore,we inferred that in contrast to F/98-like,the G1-like PB2 gene had significant competitive advantage in the reassortment of H5N6 virus to increase the replication of H5N6 AIV.2.Molecular basis of different PB2 genes affecting replication of H5N6 subtype AIV in terms of polymerase activityTo investigate the influence and molecular basis of the advantageous G1-like PB2 gene in the replication of H5N6 subtype AIV in terms of polymerase activity,the H5N6/S representative isolate of MZ34 and H9N2/H representative isolate of SH14 were selected for the following assays in the present study.Their PB2 genes were both divided into three successive fragments according to the protein domain distribution.Subsequently,chimeric PB2 plasmids involving partial G1-like and F/98-like PB2 gene fragments were constructed to identify the critical domain(s)and amino acid(s)of G1-Like PB2 gene that impacting the polymerase activity.The results showed that introducing the N terminal domain(amino acid sites 1-250)of SH14-PB2 gene and the mutation from M to V at site 89 on the backbone of MZ34-PB2 gene both induced significantly lower polymerase activities.Moreover,as compared with the maternal virus MZ34,the mutant virus carrying PB2-M89V variation evidently decreased the mRNA,vRNA,cRNA synthesis of PB2 gene and the expression levels of PB2,PB1,NP proteins.Therefore,we inferred that in contrast to F/98-like,the G1-like PB2 gene could dramatically enhance the polymerase activity of H5N6 subtype AIV.In particular,the N-terminal domain of the G1-like PB2 gene is the key functional domain and 89M is the key amino acid site.3.Mechanism exploration of different PB2 genes affecting replication of H5N6 subtype AIV in terms of cap-binding abilityOn the other hand,to investigate the influence and mechanism of the advantageous G1-like PB2 gene in the replication of H5N6 subtype AIV in terms of cap-binding ability,MZ34 and SH14 strains were still selected for the following assays.Firstly,their PB2 cap-binding domain proteins were expressed prokaryotically and eukaryotically to determine the binding affinity with m7GTP in vitro.Subsequently,sequence alignment of PB2 gene between G1-like and F/98-like lineages was performed to analyze potential amino acids contributing to the cap-binding discrepancy.The results revealed that the binding avidity of eukaryotically expressed MZ34cap with m7GTP was significantly higher than that of the SH14cap,indicating that the bonding performance of cap-binding domain may be a potential mechanism for different replication of H5N6 subtype AIV caused by G1-like and F/98-like PB2 genes.In addition,340R,389K,456N of G1-like PB2 gene might be the key amino acid sites to increase the cap-binding affinity,which is worth of further research.