title: A high-throughput screening system targeting the nuclear export pathway via the third nuclear export signal of influenza A virus nucleoprotein.
abstract: Two classes of antiviral drugs, M2 channel inhibitors and neuraminidase (NA) inhibitors, are currently approved for the treatment of influenza; however, the development of resistance against these agents limits their efficacy. Therefore, the identification of new targets and the development of new antiviral drugs against influenza are urgently needed. The third nuclear export signal (NES3) of nucleoprotein (NP) is the most important for viral replication among seven NESs encoded by four viral proteins, NP, M1, NS1, and NS2. NP-NES3 is critical for the nuclear export of NP, and targeting NP-NES3 is therefore a promising strategy that may lead to the development of antiviral drugs. However, a high-throughput screening (HTS) system to identify inhibitors of NP nuclear export has not been established. Here, we developed a novel HTS system to evaluate the inhibitory effects of compounds on the nuclear export pathway mediated by NP-NES3 using a MDCK cell line stably expressing NP-NES3 fused to a green fluorescent protein from aequorea coerulescens (AcGFP-NP-NES3) and a cell imaging analyzer. This HTS system was used to screen a 9600-compound library, leading to the identification of several hit compounds with inhibitory activity against the nuclear export of AcGFP-NP-NES3. The present HTS system provides a useful strategy for the identification of inhibitors targeting the nuclear export of NP via its NES3 sequence.
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Michinori
authors.initials: M
authors.lastname: Kakisaka
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
authors.forename: Takafumi
authors.initials: T
authors.lastname: Mano
authors.affiliation: Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Electronic address: aida@riken.jp.
authors.forename: Yoko
authors.initials: Y
authors.lastname: Aida
mesh_terms: D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology*; D002460:Cell Line; D004285:Dogs; D016503:Drug Delivery Systems; D009980:Influenza A virus / Q000187:drug effects* / Q000378:metabolism*; D061985:Madin Darby Canine Kidney Cells; D008826:Microbial Sensitivity Tests / Q000379:methods*; D049790:Nuclear Export Signals / Q000187:drug effects* / Q000235:genetics; D019590:Nucleocapsid Proteins; D010957:Plasmids; D016601:RNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D014758:Viral Core Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
chemical_list: D000998:Antiviral Agents; C048842:NP protein, Influenza A virus; D049790:Nuclear Export Signals; D019590:Nucleocapsid Proteins; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins
keywords: Compound; HTS; Influenza virus; NES; NP; Nuclear export