甲型流感病毒NS1蛋白R108K与G189D突变对病毒复制与转录的影响

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

摘要:

背景 甲型流感病毒非结构蛋白1 (nonstructural protein 1, NS1)是拮抗宿主先天免疫应答的关键因子，其氨基酸变异可能影响病毒复制适应性与免疫逃逸能力。目的 构建NS1蛋白特定位点回复突变病毒，探究该位点变异对流感病毒在不同细胞系中复制与转录过程的影响。方法 以A/California/07/2009 (H1N1)株为骨架，构建携带R108K、G189D及双突变的重组病毒。在MDCK及A549不同细胞中比较突变株与野生型病毒的复制动力学；并通过RT-qPCR检测病毒RNA (viral RNA, vRNA)、互补RNA (complementary RNA, cRNA)及信使RNA (messenger RNA, mRNA)的动态变化。结果 序列分析比较发现，在2009年大流行前后H1N1亚型流感病毒NS1蛋白发生K108R、D189G突变。构建的R108K、G189D及双突变病毒株与野生型病毒在MDCK细胞中的增殖水平无显著差异，但在A549细胞中，突变株的复制能力显著弱于野生型。G189D突变株vRNA与cRNA水平高于野生株，提示该突变特异性增强病毒基因组复制效率；R108K突变及双突变株则主要表现为促进mRNA合成。结论 NS1蛋白108/189位点的回复突变对病毒复制效率的影响具有宿主依赖性。虽然该突变能提升RNA合成，但在所测试的特定宿主细胞环境中，其完整生命周期的产出受到抑制。这暗示该位点的进化选择可能受到宿主细胞内部特定因子相互作用的影响。

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.