Several studies have described the role of
Several studies have described the role of NP in vRNP nuclear export. For example, leptomycin B (LMB), an inhibitor of the CRM1 nuclear export signal (NES) binding domain (Kudo et al., 1999), inhibits nuclear export of vRNP and NP, but not NEP or M1, in virally infected or transfected 66 8 mg (Elton et al., 2001, Watanabe et al., 2001). In addition, CRM1 overexpression promotes the nuclear export of NP, but not NEP or M1 (Elton et al., 2001), and the interaction of NP with viral genomic RNA, and M1 is sufficient for nuclear export of vRNP (Huang et al., 2001). Moreover, NP directly binds to CRM1 (Chutiwitoonchai et al., 2014, Elton et al., 2001, Kakisaka et al., 2015), and the interaction between NP/CRM1 and a recent identified host factor, nuclear transport factor 2-like export protein 1 (NXT1), promotes nuclear export of NP and vRNA (Chutiwitoonchai and Aida, 2016). NP must also interact with cellular nucleolin, a major component of the nucleolar compartment, to facilitate the interaction between vRNP and the cellular nuclear export machinery (Terrier et al., 2016). In addition to its role in vRNP nuclear export, NP plays other important roles in the viral life cycle, including vRNP nuclear import and transcription/replication of the viral genome (Aida et al., 2012, Cianci et al., 2013, Portela and Digard, 2002, Sasaki et al., 2013). These functional studies support NP as a promising novel target for antiviral drug development.
Inhibition of vRNP nuclear export is an effective strategy for counteracting influenza replication. Treatment with LMB, an inhibitor that irreversibly covalently binds with the C258 of the CRM1 NES binding domain (Kudo et al., 1999), results in retention of vRNPs in the nuclei of infected cells (Elton et al., 2001, Ma et al., 2001, Watanabe et al., 2001). Similarly, Verdinixor, a representative of a new class of CRM1 inhibitor, forms a reversible covalent bond with C258 of CRM1 and thereby inhibits replications of influenza A subtypes H1N1, H5N1, and H7N9 (Perwitasari et al., 2014). Inhibition of vRNP nuclear export by an inhibitor with an unknown target, 14-deoxy-11,12-dehydroandrographolide (Cai et al., 2015), or NP-target inhibitors such as pyrimido-pyrrolo-quinoxalinedione analog (Lin et al., 2015) and 1,3,4,6-tetra-O-galloyl-β-D-glucopyranoside (Chang et al., 2016) can also potentially diminish replication of influenza viruses. Moreover, targeting of the third NES domain of NP (NP-NES3) by an inhibitor we discovered, RK424, results in antiviral effects in vitro and in vivo (Kakisaka et al., 2015). Recently, using our high-throughput CELAVIEW screening system, we identified another potential inhibitor, DP2392-E10, which targets the nuclear export function of the NP-NES3 domain (Kakisaka et al., 2016). Although the molecular mechanism of inhibition by DP2392-E10 was not validated at that time, our preliminary study demonstrated that the compound exerts an antiviral effect against influenza A/WSN/1933 (H1N1) replication (Kakisaka et al., 2016).
Discussion Nuclear export of vRNP was originally thought to be mediated by the NEP/CRM1 interaction (O'Neill et al., 1998), but several later studies indicated a role for NP in vRNP nuclear export: for example, LMB inhibits nuclear export of vRNP in a NEP-independent manner (Elton et al., 2001, Ma et al., 2001, Watanabe et al., 2001), and NES-3–defective NP decreases viral production and replication efficiency (Chutiwitoonchai et al., 2014, Yu et al., 2012). Therefore, we speculate that DP2392-E10 decreases viral replication by interfering with both NP/CRM1– and NEP/CRM1–mediated nuclear export of vRNP. The inhibitory effect of DP2392-E10 on both NP-NES3– and NEP-NES2–mediated nuclear export functions indicates that this molecule represents a promising lead compound for further drug development. Notably, however, DP2392-E10 may not affect the nuclear export function of the M1-NES domain, which is CRM1-independent (Cao et al., 2012). This study demonstrated a broad range inhibitory effect of DP2392-E10 against replication of seasonal influenza A H1N1 and H3N2 including some other subtypes. Because the NP-NES3 domain is conserved among human, avian, and swine influenza viruses (Chutiwitoonchai et al., 2014), we expect that DP2392-E10 will also be able to inhibit pandemic subtypes such as swine influenza H1N1 and avian influenza H5N1. The inhibitory effect of DP2392-E10 could be further improved by structure–activity relationship analysis, making it available for use in combination with, or as an alternative to, currently approved anti-influenza drugs.