PA protein x PB1

In this study, we report the rational design and synthesis of 34 novel quinazoline analogs targeting the influenza virus ribonucleoprotein (RNP) complex. These compounds, categorized into four structural classes, were evaluated for anti-influenza activity. Lead compounds 26 and 30 demonstrated potent antiviral efficacy against A/WSN/33(H1N1), with EC₅₀ values of 5.09 ± 0.21 μM and 3.63 ± 0.06 μM, respectively, and high selectivity indices (SI > 19.7 and > 27.5). Surface plasmon resonance (SPR) experiments confirmed dual binding to nucleoprotein (NP; K_D = 6.72 μM for 26, 10.1 μM for 30) and the PA C-terminal domain (PAc; K_D = 3.94 μM for 26, 0.857 μM for 30), key components of the viral RNP complex. Molecular docking and dynamics simulations revealed critical interactions: disruption of the NP Glu339-Arg416 salt bridge (essential for oligomerization) and binding to the PA-PB1 interface (residues Lys643, Glu623, Trp706), destabilizing polymerase assembly. RNP inhibition assays further validated suppression of viral transcription/replication (56.8-68.5 % inhibition at 12.5 μM). Despite favorable potency, solubility optimization remains necessary for improved drug-like properties. By integrating static docking poses with MD-derived dynamic correlations (DCCM), principal component analysis (PCA), and FEL-quantified energy basins, this study revealed transient yet mechanistically vital ligand-protein interaction nodes. This study establishes quinazoline-based dual-targeting inhibitors as promising anti-influenza agents, providing a foundation for further development against resistant strains.

The influenza A virus (IAV), renowned for its high contagiousness and potential to catalyze global pandemics, poses significant challenges due to the emergence of drug-resistant strains. Given the critical role of RNA polymerase in IAV replication, it stands out as a promising target for anti-IAV therapies. In this study, we identified a novel C-3-substituted oleanolic acid benzyl amide derivative, A5, as a potent inhibitor of the PA_C-PB1_N polymerase subunit interaction, with an IC₅₀ value of 0.96 ± 0.21 μmol/L. A5 specifically targets the highly conserved PA_C domain and demonstrates remarkable efficacy against both laboratory-adapted and clinically isolated IAV strains, including multidrug-resistant strains, with EC₅₀ values ranging from 0.60 to 1.83 μmol/L. Notably, when combined with oseltamivir, A5 exhibits synergistic effects both in vitro and in vivo. In a murine model, dose-dependent administration of A5 leads to a significant reduction in IAV titers, resulting in a high survival rate among treated mice. Additionally, A5 treatment inhibits virus-induced Toll-like receptor 4 activation, attenuates cytokine responses, and protects against IAV-induced inflammatory responses in macrophages. In summary, A5 emerges as a novel inhibitor with high efficiency and broad-spectrum anti-influenza activity.

The H5N6 avian influenza virus, a highly pathogenic strain, poses a significant threat to poultry production and public health. The RNA-dependent RNA polymerase (comprising PB1, PB2, and PA proteins) and nucleoprotein of highly pathogenic avian influenza viruses interact with avian host proteins, influencing the efficiency of viral RNA synthesis and severity of infection. To comprehensively understand how H5N6 avian influenza virus interfaces with host cellular mechanisms during infection and replication, it is essential to identify which host proteins physically associate with viral polymerase proteins. In this study, affinity purification mass spectrometry was used to identify physical interactions between H5N6-JX polymerase proteins (PB1, PB2, PA, and nucleoprotein) and host proteins in chicken DF-1 cells. We identified 455 H5N6-chicken interacting proteins and successfully cloned 231 of these genes. Overexpression experiments revealed several host proteins involved in viral replication in DF-1 cells. Specifically, nine host genes were found to promote avian influenza virus proliferation, whereas 20 inhibited it. Furthermore, we demonstrated that avian NUP93 interacts with the viral PB1 protein, enhancing polymerase transcriptional activity and promoting viral proliferation. These findings provide a more comprehensive understanding of how host mechanisms are manipulated during H5N6 avian influenza viral infection and replication, providing insights into the mechanisms of avian influenza virus cross-species transmission.

The RNA-dependent interaction of RNA polymerase with avian host protein determines the efficiency of viral RNA synthesis and the severity of infection. However, the strain-specific interactions of the avian influenza virus (AIV) remain unclear. In this study, we identified 455 H5N6-chicken interacting proteins and successfully cloned 231 of them. Nine host genes that promote the proliferation of avian influenza virus and 20 host genes that inhibit the proliferation of avian influenza virus were identified through overexpression experiments. In addition, we demonstrated that avian NUP93 interacts with viral PB1 protein to enhance polymerase transcriptional activity and promote viral proliferation. This study contributes to a more comprehensive and detailed understanding of the molecular mechanisms of host utilization during the H5N6 highly pathogenic avian influenza virus infection and replication.