A Study On The Influence Of The HA Subtype-specific Noncoding Region Of Influenza A Virus On The Mechanism Of Viral Replication

Influenza A viruses(IAVs)are pathogens that can cause seasonal epidemics and potential pandemics.posing threats to human health.Based on the antigenicity of the surface glycoproteins,haemagglutinin(HA)and neuraminidase(NA),influenza A viruses can be divided into different subtypes.The genome of influenza A viruses consists of eight single-stranded,negative-sense viral RNA(vRNA)segments.Each vRNA segment is composed of the internal open reading frame(ORF)and the bilateral noncoding regions(NCRs).The noncoding region(NCR)at the 3' or 5' end consists of the terminal highly conserved promoter region and the adjacent non-conserved noncoding reg,ion.Previous studies have demonstrated that the non-conserved noncoding regions are highly conserved in length and nucleotide composition among the same segments of different influenza A virus strains.Therefore,the non-conserved noncoding regions are also called segment-specific noncoding regions(ssNCRs).Our previous studies have demonstrated that,in the subtype-determinant segments.encoding haemagglutinin(HA)and neuraminidase(NA),the segment-specific NCRs are also subtype-specific NCRs(ssNCRs),varying significantly in length and sequence at both the 3' and 5' ends among different subtypes while being relatively conserved among the same subtypes.We demonstrated that the length and the sequence of HA ssNCR play a role in influenza A virus replication by regulating the incorporation efficiency of HA segment.In this study,we further explored the effect of the length and the sequence of H1-ssNCR on influenza A virus replication.We progressively truncated the 3' end Hl-ssNCR.By reverse genetics and minireplicon assays,we compared the replication efficiencies of recombinant viruses and the efficiencies in HA vRNA replication and transcription.We demonstrated that truncations of the 3' end H1-ssNCR resulted in recombinant viruses with decreased vRNA replication and attenuated growth phenotype.Although replication of the 3' end HI-ssNCR truncated vRNA templates was not affected in single-template minireplicon assays,the mutant templates were replicated less efficiently than the wildtype HA vRNA template when multiple templates were present.An adaptive mutation G76A was found in the adjacent coding region after we passaged the 3' end H1-ssNCR truncated recombinant viruses.The G76A mutation restored the HA vRNA replication and reversed the attenuated virus growth phenotype.As the nonsynonymous G76A mutation changes the 15th amino acid from threonine(Thr)to isoleucine(lie)in the sequence of HA protein,we constructed codon mutants that contain other codons for isoleucine(Ile15).The 3' end H1-ssNCR truncated codon mutants showed different efficiencies in HA vRNA replication and virus growth phenotype.We also progressively truncated the 5' end H1-ssNCR and showed that truncations of the 5'end Hl-ssNCR also resulted in attenuated virus growth phenotype.The replication efficiencies of recombinant viruses decreased consistently with the H1-ssNCR truncations at the 5' end.In addition to the length,we explored the sequence requirement for H1-ssNCR.By introducing random sequences into H1-ssNCR and constructing random nucleotide selection libraries,we found that H1-ssNCR with high AU content is favourable for virus growth.The requirements for nucleotides at different positions of H1-ssNCR are different.Nucleotides at different positions act synergistically to ensure optimal efficiency of virus replication.Together,this study characterises the role of H1-ssNCR in influenza A virus replication.Specifically for the 3'-end H1-ssNCR,we demonstrate that sequences outside the promoter region are important for HA vRNA replication.We propose that the terminal noncoding region and adjacent coding region act synergistically to ensure optimal levels of HA vRNA replication.Both the length and the sequence of H1-ssNCR are important in regulating virus replication efficiency.These results enrich our knowledge about the function of H1-ssNCR and provide solid foundation to further research on the ssNCRs of other HA subtypes and the noncoding regions of influenza A viruses.