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

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.

Intrinsically disordered region of influenza A NP regulates viral genome packaging via interactions with viral RNA and host PI(4,5)P 2

“…Acti-stain 670 fluorescent phalloidin and cytochalasin D were purchased from Cytoskeleton and Sigma, respectively. NP proteins and GST-PLCδpH were expressed using the GST gene fusion system in Escherichia coli strain BL21 CodonPlus (DE3)-RIL (Stratagene) and purified using the Glutathione Sepharose 4FF bead system (GE Healthcare) as previously described (Kakisaka et al, 2015). For removal of GST-tag, GST-NP proteins were digested with PreScission Protease (GE Healthcare).…”

“…Plasmids NP/pHH21 and NP/pCAGGS harboring site-specific mutations (D72A, E73A, R74A, R75A, K77A, Y78A, E80A, P83A, G86A, K87A, D88A, P89A, K90A, K91A, and T92A) were generated using Prime STAR Max DNA Polymerase (Takara Bio) as described previously (Kakisaka et al, 2015). Plasmids NP/pHH21 and NP/ pCAGGS were kind gifts from Dr. Yoshihiro Kawaoka (University of Tokyo).…”

“…Recombinant viruses were generated by DNA transfection as described previously (Kakisaka et al, 2015). Briefly, a co-culture of MDCK (4 Â 10 5 ) or HEK-293T (6 Â 10 5 ) cells was transfected with the eight viral genome-expressing plasmids and four viral proteinexpressing plasmids.…”

Current and novel antiviral strategies for influenza infection

“…Naproxen (Figure 4), a non-steroidal anti-inflammatory drug that inhibits cyclooxygenase I and II, was identified through virtual screening to disrupt the NP-RNA interaction with its antiviral activity validated in vitro and in the mouse model [77]. In addition, RK424 (Figure 4) was identified from high throughput screening that binds to a NP functional domain critical for NP-NP and NP-RNA interactions as well as the NES3-CRM1 interaction for nucleus exportation [78].…”

Rapid Screening by Cell-Based Fusion Assay for Identifying Novel Antivirals of Glycoprotein B-Mediated Herpes Simplex Virus Type 1 Infection

“…Identification of novel antiviral compounds should be dependent on an in vitro primary assay for high-throughput screening (HTS) of the compound libraries. 30) In addition, structure-based drug design (SBDD) could be a powerful tool, 31) especially for identifying novel inhibitors of viruses. 32) We are currently performing this cell-based fusion assay for the screening of small chemical compounds from the library against HSV-1 infection, in combination with SBDD.…”