目的：流感RNA聚合酶（FluPol）催化病毒基因组转录和复制的内在机制已得到很大程度的解决。然而,转录和复制如何动态调节的机制仍然难以捉摸。我们最近报道，病毒NS2蛋白的最后一个氨基酸在促进流感微型复制子系统中的病毒基因组复制中起关键作用。这里,我们在病毒解救和连续传代的最后121个残基处进行了20个氨基酸的替换筛选。我们的结果表明，NS2的复制促进功能对于病毒的存活和有效繁殖很重要。我们进一步证明了NS2和NS2-I121适应性突变PA-K19E/PB1-S713N通过促进FluPol二聚化来调节病毒基因组复制的证据。这项工作强调了NS2和FluPol在实现有效基因组复制方面的协调。它进一步促进了我们对甲型流感病毒RNA合成调控的理解。
Both influenza A virus genome transcription (vRNA→mRNA) and replication (vRNA→cRNA→vRNA), catalyzed by the influenza RNA polymerase (
FluPol), are dynamically regulated across the virus life cycle. It has been reported that the last amino acid I121 of the viral NS2 protein plays a critical role in promoting viral genome replication in influenza mini-replicon systems. Here, we performed a 20 natural amino acid substitution screening at residue NS2-I121 in the context of virus infection. We found that the hydrophobicity of the residue 121 is essential for virus survival. Interestingly, through serial passage of the rescued mutant viruses, we further identified adaptive mutations PA-K19E and PB1-S713N on FluPol which could effectively compensate for the replication-promoting defect caused by NS2-I121 mutation in the both mini-replicon and virus infection systems. Structural analysis of different functional states of
FluPol indicates that PA-K19E and PB1-S713N could stabilize the replicase conformation of
FluPol. By using a cell-based NanoBiT complementary reporter assay, we further demonstrate that both wild-type NS2 and PA-K19E/PB1-S713N could enhance
FluPol dimerization, which is necessary for genome replication. These results reveal the critical role NS2 plays in promoting viral genome replication by coordinating with FluPol.IMPORTANCEThe intrinsic mechanisms of influenza RNA polymerase (FluPol) in catalyzing viral genome transcription and replication have been largely resolved. However, the mechanisms of how transcription and replication are dynamically regulated remain elusive. We recently reported that the last amino acid of the viral NS2 protein plays a critical role in promoting viral genome replication in an influenza mini-replicon system. Here, we conducted a 20 amino acid substitution screening at the last residue 121 in virus rescue and serial passage. Our results demonstrate that the replication-promoting function of NS2 is important for virus survival and efficient multiplication. We further show evidence that NS2 and NS2-I121 adaptive mutations PA-K19E/PB1-S713N regulate virus genome replication by promoting FluPol dimerization. This work highlights the coordination between NS2 and
FluPol in fulfilling efficient genome replication. It further advances our understanding of the regulation of viral RNA synthesis for influenza A virus.
PDF(Pubmed)