Kinase Inhibitor Roscovitine As A PB2 Cap-binding Inhibitor Against Influenza A Virus Replication

Background:Because of infectiousness,rapid mutation rate,high morbidity and mortality,influenza A virus becomes one of the diseases that seriously threatens human health and social stability.Currently,vaccination is still the principal strategy for controlling influenza infection.However,influenza vaccines are not always available or efficacious because of continuous antigenic drift and antigenic shifts in the influenza viral surface glycoproteins.Therefore,the use of antiviral drugs is regarded as the first line to defense the influenza pandemic.Currently,M2-ion channel inhibitor and the neuraminidase inhibitors are the FDA-approved antiviral drugs.However,the approved antiviral drugs are not widely used due to their resistance and adverse effects.Thus,despite the utility of these antiviral drugs,novel antivirals with novel mechanism are in demand.PB2cap domain of influenza is highly conserved among all strains and subtypes during viral evolution.Thus PB2cap has been one of the important targets for antiviral drug.However,drug development is an expensive and time-consuming process.It is feasible and economical to research the antivital ability of drugs that are approved for other diseases with known safety.The cyclin-dependent kinase inhibitors(CDKI)have been shown inhibitory effect on a variety of viruses,such as human immunodeficiency virus,herpes simplex virus and influenza virus.This study started with performing cell-level antiviral activity assay to find novel anti-influenza compound.We found that roscovitine is a promising compound against influenza A virus replication.As a cyclin-dependent kinases inhibitor,roscovitine is already in the phase I/II clinical trials,and it is currently evaluated as a potential drug to treat cancers,neurodegenerative diseases and viral infections.Objectives:We explore the anti-influenza activity and mechanism of roscovitine in order to find anti-influenza virus drug from existing compounds,and provide new strategies for the development of safe and effective antiviral drugs.Methods:1.The MTT-based assay was used to detect the cytotoxicity of roscovitine,MTT and plaque assay were evaluated its antiviral activity in different subtypes influenza strains and the production of progeny viral particles.2.Western blotting,qRT-PCR,immunofluorescence microscopy assay were confirmed the inhibition effect of roscovitine on virus replication.3.Haemagglutination inhibition assay,pseudovirus neutralization assay and neuraminidase inhibition assay were explored to confirm whether roscovitine has effect on viral entry or release.Time-of-addition assay and mini-replicon saasy were used to detect which specific stage of virus life cycle was interfered by roscovitine.4.Fluorescence polarization assay,surface plasmon resonance assay and molecular docking assay were used to investigate the specific binding ability between roscovitine and PB2cap protein.Results:1.Roscovitine prevented the different influenza virus strains replication,including A/WSN/1933(H1N1),A/Aichi/2/68(H3N2)and A/FM1/47(H1N1)with IC50 value of 3.35±0.39,7.01 ±1.84 ? 5.99±1.89 ?M,respectively.And roscovitine reduced progeny viral load in a concentration-dependent manner but did not disturb host cell viability in MDCK or Beas-2B at 100%of the antiviral effects.2.Roscovitine could not diminish the blood cell agglutination and the activity of roscovitine against H5N1 pseudovirus was weakly.Besides,the effect of roscovitine on enzyme activity of NA was not obvious.3.Roscovitine could obviously decrease the expression of viral protein and mRNA at 2-5 h and 5-8 h post infection.4.Roscovitine exerted its anti-influenza activity through disturbing viral RNA polymerase activity and binding with the PB2cap domain.5.Roscovitine interacted with PB2cap domain via the amino residues His-357,Phe-404,Phe-323,Met-431 and Lys-376.6.Roscovitine decreased the expression of JAK/STAT signaling pathway.Conclusions:1.Roscovitine possessed antiviral activities against different subtypes of influenza A viruses strains.2.Roscovitine had no effect on viral entry or release.3.Roscovitine suppressed the viral genome transcription and replication step in the viral life cycle.4.Roscovitine exerted its anti-influenza activity through binding specifically to the PB2cap protein.5.Roscovitine reduced excessive inflammatory immune response by acted on JAK/STAT signaling pathway.