Influenza A virus (IAV) continuously poses a considerable threat to global health through seasonal epidemics and recurring pandemics. IAV RNA-dependent RNA polymerases (FluPol) mediate the transcription of RNA and replication of the viral genome. Searching for targets that inhibit viral polymerase activity helps us develop better antiviral drugs. Here, we identified heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB) as an anti-influenza host factor. hnRNPAB interacts with NP of IAV to inhibit the interaction between PB1 and NP, which is dependent on the 5-amino-acid peptide of the hnRNPAB C-terminal domain (aa 318-322). We further found that the 5-amino-acid peptide blocks the interaction between PB1 and NP to destroy the FluPol activity. In vivo studies demonstrate that hnRNPAB-deficient mice display higher viral burdens, enhanced cytokine production, and increased mortality after influenza infection. These data demonstrate that hnRNPAB perturbs FluPol complex conformation to inhibit IAV infection, providing insights into anti-influenza defense mechanisms.

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.