Effects of R108K and G189D mutations in the NS1 protein of influenza A virus on viral replication and transcription

Abstract:

Background The non-structural protein 1 (NS1) of the influenza A virus is a key factor in antagonizing the host’s innate immune response. Its amino acid variations may significantly influence viral replication fitness and immune evasion capabilities.Objective To construct back-mutant viruses at specific sites of NS1 to investigate the effects of these mutations on the replication and transcription processes of the influenza virus across different cell lines.Methods With the A/California/07/2009 (H1N1) strain as the backbone, recombinant viruses carrying R108K, G189D, and double mutation were constructed. The replication kinetics of the mutant strains were compared with the wild-type (WT) virus in MDCK and A549 cell lines. Furthermore, the dynamics of viral vRNA, cRNA, and mRNA were detected by RT-qPCR.Results Sequence analysis revealed that K108R and D189G mutations occurred in the NS1 of H1N1 subtype influenza viruses around the 2009 pandemic. The constructed mutant strains carrying R108K, G189D, and double mutation showed no significant differences in proliferation levels compared with the WT virus in MDCK cells. However, in A549 cells, the replication capacity of the mutant strains was significantly weaker than that of the WT. The G189D mutant exhibited higher levels of vRNA and cRNA than the WT, suggesting that this mutation specifically enhanced the efficiency of viral genome replication. Conversely, R108K and the double mutation primarily promoted mRNA synthesis.Conclusion The effects of back-mutations at positions 108 and 189 of NS1 on viral replication efficiency were host-dependent. Although these mutations enhanced RNA synthesis, the overall yield of the viral life cycle was inhibited within the specific host cell environments tested. This suggests that the evolutionary selection at these sites may be influenced by interactions with specific internal factors in host cells.