BPR2-D2 targeting viral ribonucleoprotein complex-associated function inhibits oseltamivir-resistant influenza viruses

Objectives: The emergence of oseltamivir-resistant viruses raised the global threat with regard to influenza virus infection. To develop alternative antiviral agents against influenza virus infection is significant and urgent. Methods: A neutralization test was applied as a screening assay and a plaque reduction assay was used for confirmation. Expression plasmids for viral ribonucleoproteins (RNPs) and a plasmid that allowed expression of a pseudoviral reporter RNA were transfected into cells to investigate the effects of a novel antiviral compound on viral RNA synthesis. Results: BPR2-D2 was identified as a novel inhibitor against influenza virus from a hit obtained from high throughput screening of 20000 or more compounds. BPR2-D2 exhibited an excellent antiviral efficacy for the oseltamivir-resistant virus (EC 50 ranging from 0.021 to 0.040 μM). No resistant virus was produced throughout 20 passages in the presence of BPR2-D2, whereas oseltamivir-resistant virus was generated at passage 8 using the same experimental system. A molecular target other than neuraminidase (NA) was found because BPR2-D2 inhibited the synthesis of viral RNA that was driven by influenza viral RNP in a transfection assay. BPR2-D2 also exhibited a broad antiviral spectrum against various strains of influenza A and influenza B viruses. Conclusions: BPR2-D2 was identified as a novel inhibitor of influenza virus. It may target viral RNPs that are responsible for viral RNA synthesis. Targeting different molecules compared with NA allows BPR2-D2 to inhibit oseltamivir-resistant viruses.