Molecular Mechanism Of Influenza A Virus Reassortment And Effect On Viral Biological Characteristics

Reasortantment is one way of variation and evolution of influenza viruses.Reasortantment among different influenza viruses is able to generate novel viruses with better adaptation in host.Most of the pandemics strains in the history were generated by gene rearrangement.However,the molecular factors that influence the Reasortantment of influenza viruses are not well understood.Recently,H17N10 and H18N11 found in bats are two novel influenza viruses.The studies indicated that they have relatively close evolutionary relationships to the known influenza A virus,and H17N10 and H18N11 can reassort,but can't reassort with the conventional IAV.In this study,we used bat influenza virus H17N10 and IAV H9N2 as models to study the molecular factors and mechanism which affect the reasortantment of AIVs.Bird is an important host for IAVs,However,the infection and adaptation of bat influenza viruses in birds remain unclear.In this study,we successfully rescued two modified bat influenza viruses cH9cN2/H17 and cH9cN2/H18 for the first time,in which chimeric HA and NA genes contain coding from those of H9N2 virus and the packaging signal sequence of bat influenza viruses in the background of the H17N10 or H18N11 viruses.The growth characterize of these cH9cN2/H17,cH9cN2/H18 and wild type H9N2?H9N2-wt?in avian cells was compared,the two modified viruses replicated less efficiently than wild type H9N2 virus in cultured chicken cells.The replication and transmissibility of these three viruses was assessed in chickens,the results showed that H9N2-wt virus replicated and transmitted efficiently in chickens,while the cH9cN2/H17 and cH9cN2/H18 viruses barely replicated in chicken with lower titers.Notably,the cH9cN2/H18 transmitted among chickens,but not cH9cN2/H17.Mini-genome assay showed that vRNPs of H9N2 has significantly higher polymerase activity than that of bat influenza viruses in avian cells.The chicken IFN-?antagonism results showed that H9N2 NS1inhibited IFN-?response more efficiently than bat influenza viruses NS1 proteins in chicken cells,Notably,H18N11 NS1 protein inhibited chicken IFN-?response more efficiently than H17N10 NS1protein in avian cells.Taken together,our data indicated that the internal genes of bat-influenza viruses adapted poorly to chickens,while the internal genes of H18N11 seemed to adapt to chicken better than H17N10.To further explore the molecular factors affecting the reassortantment of bat influenza viruses and AIVs,we replaced each gene of the cH9cN2/H17 and H9N2 viruses by reverse genetic technology,but no recombinant virus was rescued,which verified the incompatibility of genes between bat influenza virus and H9N2 AIV.Studies have shown that the packaging signal sequences located at 5'and 3'terminals of viral RNA are critical for the genome selective packaging.In this study,recombinant influenza viruses?cH9cN2/H17 and cH9cN2/H18?could be successfully rescued in which H9N2 HA and NA ORF flanking H17N10 or H18N11 packaging signals,indicated that the packaging signal region was important for influenza virus reassortantment,especially for surface genes.To further investigate the effect of packaging signal on the internal gene reassortantment of bat influenza virus and H9N2 virus,we switched the packaging signal of NP genes of these two viruses,but recombinant virus cannot be rescues in the background of either of cH9cN2/H17 or H9N2 virus,indicating that for internal genes,there are other factors besides the packaging signal that affect viral reassortantment.The 5'and 3'gene promoter regions located in packaging signal region are important for vRNPs to recognize and initialize transcription and replication of the progeny viral genome.To further investigate the molecular mechanisms by which the packaging signal region affects reassortantment of influenza viruses,we constructed mini-genome system containing the PB2,PB1,PA,HA,NA,and NP gene promoters of bat influenza virus and H9N2,and evaluated the transcription efficiency of H17N10 and H9N2 vRNPs on the promoters from different viruses.The results showed that,for most genes,the efficiency of viral vRNPs to identify their own promoter was higher than that of the heterologous promoter,and the difference in efficiency of promoter recognition by vRNPs was one of the factors affecting the reassortantment beween H17N10 and H9N2influenza viruses.Recent studies showed that an interaction network among eight vRNAs is very important for viral genome packaging of AIVs.In order to further study the influence of the interaction among viral RNA sequences on the genome selectively packaging of influenza virus,we analyzed the complementary sequence among eight genes of recombinant cH9cN2/H17 in biological software and mutated synonymously the complementary sequences of NP gene with other fragments but failed to rescue mutant viruses,which showed that complementary sequences of NP with other genes were very important for influenza virus.To illustrate whether mutated NP affects the packaging efficiency of gene fragments,we co-transfected with mutant NP and other seven reverse genetic plasmids and collected virus particles in supernatant to analyze amount of eight vRNAs in virus particles budded from the transfected cells by real time RT-PCR.It showed that compared with wild type virus,amount of PB1,NP,NS vRNAs were significantly less packaged than that of other genes in the NP mutant viral particles,indicating that the complementary sequence of NP gene with other genes was crucial for the packaging of the viral genome.To rescue the mutant virus,then NP mutation was divided into three regions and three NP mutant viruses were rescued successfully.Viral budding and real time RT-PCR results showed that NP mutations located in three regions still affect the packaging efficiency of different genes,especially the NP gene.Comparison of the gene segment packaging ratio of three NP mutant viruses in the same TCID₅₀,the amount of 8 vRNAs packaged in two NP mutant viruses were significantly more than those of wild-type viruses,the potential reason is that the mutated NP influence the packaging efficiency of viral RNA segment,and more“deficient viruses”and less functional viruses were generated than the wild type virus.To reach the same TCID₅₀,the NP mutant viruses contain more virus particles and more genome amount than the wild type viruses.Overall,the interacted sequence between different genes of recombinant bat influenza virus plays an important role in selective packaging of eight vRNAs,the progeny virus prefer to package the RNA segment with stronger interaction with the remaining gene segments during reassortantment.The vRNPs are mainly responsible for the transcription and replication of the influenza virus genome,varied compatibility among the polymerase proteins from different influenza viruses affect the reassortantment,while the underlying molecular mechanism remain unclear.Based on the function domain and crystal structure of the bat influenza virus vRNPs,we investigated the effect of compatibility between vRNPs proteins on viral reassortantment.Mini-genome results showed that exchange of NP protein does not affect the polymerase activity in background of either H17N10 or H9N2 vRNPs,suggesting that NP proteins are compatible between H17N10 and H9N2.While exchange of PB2,PB1,or PA proteins between H9N2 and H17N10 significantly impaired the polymerase activity of H9N2 or H17N10 vRNPs,indicating that low compatibility among polymerase proteins block the reassortantment between H17N10 and H9N2 viruses.In order to explore the potential molecular mechanism,we constructed a series of PB1 hybrid genes between H17N10 and H9N2,the mini-genome results showed that PA-interacting region located at the N-terminus of PB1 protein play a critical role in the compatibility among polymerase proteins.In addition,the vRNA and cRNA binding regions of PB1 also contribute to the compatibility among polymerase proteins,which might be attributed to the low homology of viral RNA promotor sequence between H17N10 and H9N2 viruses.In summary,promoter sequences,packaging signals,interactions between viral gene fragments,interactions between polymerase proteins,and the transcription efficiency of vRNPs on viral RNA promoters are contribute to the reassortantment among influenza viruses.