A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein.

Michinori Kakisaka,Hirokazu Hikono,Yasumitsu Kondoh,Takehiko Saito,Kentaro Tomii,Yoko Aida,Hiroyuki Osada,Yutaka Sasaki,Kazunori Yamada,Paul G Thomas

doi:10.1371/journal.ppat.1005062

Abstract

Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP.

Highlights

Influenza A virus (IAV) causes periodic and widespread epidemics or pandemics, which take the form of respiratory diseases with cold-like symptoms; the virus can sometimes cause serious disease with high mortality rates [1]
Influenza A virus nucleoprotein (NP) is a highly conserved multifunctional protein that plays an essential role in infection by all subtypes of influenza A virus, making it an attractive target for new antiviral drugs
RK424 inhibits viral genome replication/transcription and nuclear export of NP by destabilizing the NP oligomer and inhibiting the binding of chromosome region maintenance 1 (CRM1) to NP via nuclear export signal (NES) 3, which is located in close proximity to the NP pocket

Summary

Introduction

Influenza A virus (IAV) causes periodic and widespread epidemics or pandemics, which take the form of respiratory diseases with cold-like symptoms; the virus can sometimes cause serious disease with high mortality rates [1]. The only approved classes of anti-influenza virus drugs are viral M2 ion channel inhibitors and neuraminidase (NA) inhibitors [2,3]. These drugs can be effective for treating influenza infection, the emergence of drug-resistant viral strains is a serious problem [4,5,6]. The use of this drug is limited to the treatment of newly-emergent influenza viruses that are resistant to current antivirals; NA inhibitors are the only drugs currently used to treat the majority of influenza virus infections. There is an urgent need for new antiviral drugs with novel mechanisms of action

Results

Discussion

Conclusion