key: cord-0682146-ilf4nftp
authors: Chutiwitoonchai, Nopporn; Mano, Takafumi; Kakisaka, Michinori; Sato, Hirotaka; Kondoh, Yasumitsu; Osada, Hiroyuki; Kotani, Osamu; Yokoyama, Masaru; Sato, Hironori; Aida, Yoko
title: Inhibition of CRM1-mediated nuclear export of influenza A nucleoprotein and nuclear export protein as a novel target for antiviral drug development
date: 2017-04-08
journal: Virology
DOI: 10.1016/j.virol.2017.04.001
sha: 65a34d151b5bc8e1e83e1def7b51b3680d321a2e
doc_id: 682146
cord_uid: ilf4nftp

An anti-influenza compound, DP2392-E10 based on inhibition of the nuclear export function of the viral nucleoprotein-nuclear export signal 3 (NP-NES3) domain was successfully identified by our previous high-throughput screening system. Here, we demonstrated that DP2392-E10 exerts its antiviral effect by inhibiting replication of a broad range of influenza A subtypes. In regard to the molecular mechanism, we revealed that DP2392-E10 inhibits nuclear export of both viral NP and nuclear export protein (NEP). More specifically, in vitro pull-down assays revealed that DP2392-E10 directly binds cellular CRM1, which mediates nuclear export of NP and NEP. In silico docking suggested that DP2392-E10 binds at a region close to the HEAT9 and HEAT10 domains of CRM1. Together, these results indicate that the CRM1-mediated nuclear export function of influenza virus represents a new potential target for antiviral drug development, and also provide a core structure for a novel class of inhibitors that target this function.

Influenza virus, which belongs to family Orthomyxoviridae, replicates its single-stranded negative-sense genomic RNAs in the host nucleus. The viral genomic RNAs are then packed into viral ribonucleoproteins (vRNPs) with viral RNA-dependent RNA polymerase and nucleoproteins (NPs) and exported into cytoplasm for further viral particle assembly and budding at the plasma membrane (Eisfeld et al., 2015) . vRNP nuclear export is mediated by the cellular chromosome region maintenance 1 (CRM1) pathway, with assistance from viral matrix protein 1 (M1) and nuclear export proteins (NEP or NS2) (Akarsu et al., 2003; Huang et al., 2013; O'Neill et al., 1998) . The vRNP-M1-NEP-CRM1-RanGTP complex is then exported through the nuclear pore to the cytoplasm, where the viral cargo is spontaneously released upon hydrolysis of RanGTP to RanGDP (Eisfeld et al., 2015) .

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 cells (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) .

Here, we extended our study of DP2392-E10 to characterize its broad range of anti-influenza effects, cytotoxicity, and efficiency of nuclear export inhibition. In addition, we investigated the molecular mechanism of inhibition by DP2392-E10 and found that it targets the CRM1 protein to block CRM1-mediated nuclear export of NP and NEP, thereby preventing vRNP nuclear export and viral replication.

We previously succeeded in identifying a chemical compound, DP2392-E10 (Fig. 1A) , that inhibits the nuclear export function of NP-NES3 domain and decreased replication of influenza A/WSN/1933 (Kakisaka et al., 2016) . To further characterize the broad-range inhibitory effect of DP2392-E10, we performed plaque assays to monitor the replications of seasonal influenza A H1N1 and H3N2 including other subtypes, H6N2, H8N4, H9N2, H14N5, and H15N8 in the presence ,,,,,,of the compound. The results revealed that DP2392-E10 decreased replication of all tested subtypes in a dose-dependent manner at the half-maximal inhibitory concentration (IC 50 ) range between 12.63 and 34.57 µM, depending on the viral strains ( Fig. 1B and C). Cytotoxicity tests were evaluated in Madin-Darby canine kidney (MDCK) and A549 (a human lung adenocarcinoma epithelial) cells indicating that the compound modestly affected cell viability at the half-maximal cytotoxic concentration (CC 50 ) of 61.25 ± 2.67 and 94.49 ± 7.56 µM, respectively (Fig. 1C ). Viral replication kinetic in the presence of DP2392-E10 showed that the compound decreased replication throughout the assay course (Fig. 1D ). These results demonstrate that DP2392-E10 inhibits replication of a broad range of influenza A subtypes.

In a high-throughput screen using MDCK cells stably expressing AcGFP-NP-NES3, we identified DP2392-E10 as a compound that could inhibit the CRM1-dependent export of the fusion protein (Kakisaka et al., 2016) . To validate the mechanism of inhibition by DP2392-E10, we monitored the intracellular localization of intact NP and another CRM1-dependent nuclear export protein, NEP (O'Neill et al., 1998) , in infected cells treated with the compound for 2, 4, or 6 h. In the dimethyl sulfoxide (DMSO) control cells, NP and NEP were localized in the nucleus at 2 h post-infection (hpi), but most of the NP and some NEP had been observed in the cytoplasm at 4 and 6 hpi (Fig. 2 , left panel), reflecting nuclear export observation of NP and NEP in the infected cells. In the DP2392-E10-treated cells, NP and NEP were still localized in the nucleus at 4 and 6 hpi, indicating that the compound prevented nuclear export of both proteins (Fig. 2, right panel) . Together with the results of the plaque assays described above, these data suggested that DP2392-E10 reduced virus replication by inhibition of NP and NEP nuclear export.

Influenza virus hijacks the cellular CRM1 nuclear export machinery to facilitate nuclear export of NP and NEP via the NP-NES3 and NEP-NES2 domains, respectively (Eisfeld et al., 2015; Huang et al., 2013; Yu et al., 2012) . To further confirm that DP2392-E10 inhibits nuclear export of NP-NES3 and NEP-NES2 via the CRM1 pathway, well-known CRM1-dependent nuclear export marker, HIV-1 Rev (Dong et al., 2009; Fornerod et al., 1997) , and a specific inhibitor of CRM1 nuclear export, LMB (Kudo et al., 1999) , were used. MDCK cells stably expressing Aequorea coerulescens green fluorescent protein (AcGFP)-NP-NES3, AcGFP-NEP-NES2, or AcGFP-Rev-NES domain ( Fig. 3A ) (Kakisaka et al., 2016) were treated for 8 h with DMSO, LMB, or DP2392-E10, and the nuclei were stained with Hoechst 33342. CELAVIEW quantification of the green fluorescence intensity from AcGFP-tagged NES domain protein in the nuclear region revealed that DP2392-E10 inhibited cytoplasmic localization of the NP-NES3 and NEP-NES2 domain proteins in a similar manner to LMB ( Fig. 3B and C). As expected, DP2392-E10 also inhibited nuclear export of the positive control, the Rev-NES domain protein (Fig. 3D ). The inhibition efficiency of DP2392-E10 against nuclear export of NP-NES3, NEP-NES2, and Rev-NES were at the half-maximal effective concentration (EC 50 ) of 7.42 ± 0.01, 12.33 ± 2.74, and 9.93 ± 0.92 µM, respectively. The comparable EC 50 values suggest that DP2392-E10 targets the CRM1 protein to inhibit nuclear export of different viral NES domains. This result confirmed that DP2392-E10 inhibits CRM1-dependent nuclear export of NP and NEP via the NP-NES3 and NEP-NES2 domains, respectively, thereby decreasing virus replication.

The nuclear export function of the NP-NES3 domain is CRM1dependent , and NP directly binds CRM1 (Chutiwitoonchai et al., 2014; Elton et al., 2001; Kakisaka et al., 2016) . To verify that DP2392-E10 inhibits nuclear export by blocking the NP/CRM1 (and NEP/CRM1) interaction, we performed in vitro pull-downs of NP and CRM1 proteins in the presence of the compound. For this purpose, NP-FLAG protein was immobilized on anti-FLAG monoclonal antibody (mAb)-conjugated agarose beads and incubated with purified CRM1-HA protein in the presence of DMSO or DP2392-E10 at 3, 10, 30, or 100 µM. Western blot analysis of the pull-down samples indicated that DP2392-E10 inhibited NP/CRM1 binding in a dose-dependent manner (Fig. 4A) .

We next investigated whether CRM1 is the target of DP2392-E10 by generating DP2392-E10 photo-crosslinked Sepharose beads and subjecting them to the pull-down assay. Specifically, the beads were incubated with purified NP-FLAG or FLAG-CRM1 protein overnight, and then pulled down. Western blots revealed that DP2392-E10 directly bound CRM1, but not NP (Fig. 4B) .

Together, these observations indicate that DP2392-E10 targets the CRM1 protein to inhibit the NP/CRM1 interaction, which is necessary for virus replication.

2.5. DP2392-E10 is predicted to interact with CRM1 in the region near the HEAT9 and HEAT10 repeats

The in vitro affinity binding assays described above indicated that DP2392-E10 directly binds to CRM1 protein. To obtain structural insights into the interaction between CRM1 and DP2392-E10, we performed an in silico study involving molecular modeling and docking simulations. Because our experimental assays used both human (293 T, for CRM1 protein purification) and canine (MDCK, for replication and immunofluorescence) cell lines, we constructed molecular models of unliganded CRM1 from both human and canine. The human CRM1 model retains the basic structural fold of twenty-one HEAT repeats, a common characteristic of CRM1 proteins (Fig. 5A) . We then used Molecular Operating Environment (MOE) to search for small-molecule binding pockets in these models. A total of 45 and 40 pockets were found in the human and canine CRM1, respectively, and the data suggested slight differences in the surface structures of the two proteins ( Fig. 5B and C, left panels) . Next, we evaluated the binding affinities and conformations of DP2392-E10 and each pocket, as reflected by U_dock docking scores. The highest U_dock score was identified at a pocket near the HEAT9-HEAT10 repeat regions in both human and canine CRM1s (human CRM1: −34.8 kcal/mol., canine CRM1: −31.0 kcal/mol) ( Fig. 5B and C, right panel) . These results suggest that the optimal binding sites for DP2392-E10 on human and canine CRM1 proteins are near the HEAT9 and HEAT10 repeats.

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 . 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 structureactivity relationship analysis, making it available for use in combination with, or as an alternative to, currently approved anti-influenza drugs. Nuclear export mediated by CRM1 is initiated by an interaction between RanGTP and the HEAT9 loop of CRM1, triggering a conformational change in the NES binding site that permits NES-cargo protein binding (Dong et al., 2009; Fung and Chook, 2014) . Our docking simulation results predicted that DP2392-E10 binds at the region around the HEAT9 and HEAT10 repeats of CRM1. This may directly cause a conformational change of the NES binding site of CRM1, or indirectly affect the folding of the HEAT9 loop that is critical for RanGTP binding. Thus, these unusual conformational changes of CRM1 decrease the affinity of binding with the NP-NES3 domain, interfere with the nuclear export function of NP, and inhibit viral replication. This proposed mechanism of inhibition by DP2392-E10 differs from that of LMB or Verdinixor, which directly bind the C258 of CRM1 NES binding site to prevent the CRM1/NES-cargo protein interaction (Kudo et al., 1999; Perwitasari et al., 2014) .

Our CELAVIEW analysis indicated that DP2392-E10 also inhibits nuclear export of HIV-1 Rev-NES, which plays a well-defined role in nuclear export of HIV-1 unspliced and singly spliced RNAs for viral protein production and viral particle assembly (Fornerod et al., 1997) . Antiviral activity against intact HIV-1 replication is further worthily verification that will provide a new class of anti-HIV-1 inhibitor based on the Rev nuclear export function for the highly active antiretroviral therapy. In addition, DP2392-E10 is expected to exert an antiviral effect against a broad range of viruses that exploit the cellular CRM1 nuclear export machinery, including severe acute respiratory syndrome coronavirus (Freundt et al., 2009) , hepatitis viruses (Cerutti et al., 2011; Forgues et al., 2001) , and herpes simplex virus (Williams et al., 2008) .

In conclusion, we demonstrated that DP2392-E10 inhibits replication of a broad range of influenza A subtypes, including H1N1, H3N2, H6N2, H8N4, H9N2, H14N5, and H15N8. The compound reduces viral replication by directly targeting the CRM1 protein in the predicted region near the HEAT9 and HEAT10 domains, thereby preventing the CRM1/NP and CRM1/NEP interactions. These effects result in inhibition of the nuclear export functions of NP and NEP, which are important for vRNP nuclear export and viral assembly.

MDCK, A549, and human embryonic kidney (HEK) 293 T cells were maintained in Dulbecco's Modified Eagle's Medium (Gibco, Beijing, China) supplemented with 10% fetal bovine serum (HyClone Laboratories, Logan, UT, USA). MDCK stably expressing AcGFPtagged influenza NP-NES3, AcGFP-NP-NES3 or HIV-1 Rev-NES, AcGFP-Rev-NES were established as previously described (Kakisaka et al., 2016) . The influenza NEP-NES2 domain was derived from annealing of two oligonucleotides, 5'-CCGGTCAATGGAATAATAA-CACAGTTCGAGTCTCTGAAACTCTACAGATAAGC-3' and 5'-GGCCG-CTTATCTGTAGAGTTTCAGAGACTCGAACTGTGTTATTATTCCATTG-A-3', to establish stable AcGFP-NEP-NES2-expressing MDCK cells, as described (Kakisaka et al., 2016) .

A plasmid set for reverse genetics of infectious influenza A/WSN/ 1933 (H1N1) virus was gift from Dr. Yoshihiro Kawaoka (Institute of Medical Science, University of Tokyo, Japan) (Neumann et al., 1999) . The pCAGGS/NP-FLAG, pCAGGS/FLAG-CRM1, and pCAGGS/ CRM1-HA plasmids were constructed as described previously (Chutiwitoonchai and Aida, 2016; Chutiwitoonchai et al., 2014) .

DP2392-E10 was provided by the Drug Discovery Initiative at the University of Tokyo (http://www.ddi.u-tokyo.ac.jp/en/) or synthesized by Vitas-M Laboratory, Ltd. (Mongkok, Kowloon, Hong Kong) and GreenPharma (Allée du Titane, Orléans, France). DMSO was purchased from Nacalai Tesque, Inc. (Kyoto, Japan) and used to dissolve DP2392-E10 including normalization of the compound volume at each tested concentration.

MDCK cells (5×10 5 /well) were seeded overnight onto 6-well plates before infection with various influenza A subtypes, and then subjected to plaque assays as previously described (Chutiwitoonchai et al., 2014) in the presence of DMSO (0.5% total volume) or 10, 30, or 50 µM DP2392-E10. The IC 50 was calculated from linear regression of virus titer vs. compound concentration. DMSO was defined as 100% infection.

Replication kinetic assay was performed as described previously (Chutiwitoonchai et al., 2014) in MDCK cells infected with influenza A/ WSN/1933 virus at a multiplicity of infection (MOI) of 0.0001 and treated with DMSO or DP2392-E10 at 10, 30, or 50 µM. Viruses in supernatant at 12, 24, and 36 hpi were titrated by plaque assay.

MDCK or A549 cells (3×10 4 /well) were seeded overnight in 96-well plates and treated with DMSO (1% total volume) or 1, 10, 30, 50, 70, or 100 µM DP2392-E10 for 48 h. Water Soluble Tetrazolium salt (WST-1) assays were performed as described previously (Chutiwitoonchai and Aida, 2016) . The CC 50 was calculated from linear regression of OD 450 vs. compound concentration. DMSO was defined as 100% cell viability.

MDCK cells (4×10 4 cells) were seeded overnight on cover glass in 12-well plates before infection with influenza A/WSN/33 virus at MOI of 2, and then treated with DMSO (0.3% total volume) or 30 µM DP2392-E10 for 2, 4, or 6 h. The infected cells were subjected to immunofluorescence staining with anti-NP mouse mAb (Santa Cruz Biotechnology, Dallas, TX, USA), anti-NEP rabbit polyclonal antibody (GeneTex, Irvine, CA, USA), Alexa Fluor 594 goat anti-mouse IgG (Invitrogen, Waltham, MA, USA), and Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen), as described previously (Chutiwitoonchai and Aida, 2016) .

MDCK cells (2×10 4 /well) stably expressing AcGFP-NP-NES3, AcGFP-NEP-NES2, or AcGFP-Rev-NES (Kakisaka et al., 2016) were seeded overnight in 96-well plates before treatment for 8 h with DMSO, 10 nM LMB (Sigma-Aldrich, Saint Louis, MO, USA; as positive control), or DP2392-E10 at 2.5, 5.0, 7.5, 10.0, 12.5, or 15.0 µM. Nuclear localization of the AcGFP-NES proteins was quantitated on a CELAVIEW RS100 (Olympus) as previously described (Kakisaka et al., 2016) . The mean fluorescence intensity of nuclear NES was subtracted with DMSO control and normalized against the value from the LMB control. The EC 50 was calculated from linear regression of mean fluorescence intensity vs. compound concentration. LMB was defined as 100% inhibition.

NP-FLAG-immobilized agarose beads and purified CRM1-HA protein from transfected HEK293T cells were prepared as previously described (Chutiwitoonchai et al., 2014) . Pull-down assays of NP/ CRM1 interaction with DP2392-E10 were performed by incubation of 15 µl of NP-FLAG-immobilized agarose beads (50% slurry), 2 µg of purified CRM1-HA protein, and either DMSO or 3, 10, 30, or 100 µM DP2392-E10 in bind/wash buffer (10 mM Tris-Cl [pH 7.8], 150 mM NaCl, 0.05% NP-40) at 4°C overnight. The beads were pulled down, washed five times with bind/wash buffer, and subjected to western blot analysis with anti-HA mouse mAb (Medical and Biological Laboratories, Nagoya, Japan) and anti-FLAG rabbit mAb (Sigma-Aldrich). The intensity of CRM1 and NP bands were quantitated by ImageJ 1.50 software (National Institute of Health, Bethesda, MD, USA).

Photo-crosslinked small-molecule affinity binding assay DP2392-E10 was photo-crosslinked onto Sepharose beads as described previously (Kanoh et al., 2005) . The photo-crosslinked Sepharose beads without compound were used as control. NP-FLAG and FLAG-CRM1 proteins were purified as described (Chutiwitoonchai and Aida, 2016; Chutiwitoonchai et al., 2014) from transfected HEK293T cells. Pull-down assays to assess the DP2392-E10/NP or DP2392-E10/CRM1 interaction were performed by incubation of 20 µl of DP2392-E10-photo-crosslinked Sepharose beads (50% slurry) and 2 µg of purified NP-FLAG or FLAG-CRM1 protein in bind/wash buffer at 4°C overnight. The beads were pulled down, washed five times with bind/wash buffer, and subjected to western blot analysis with anti-FLAG rabbit mAb (Sigma-Aldrich).

Molecular models of human and canine CRM1 in the unliganded state were constructed using homology modeling in the MOE (Chemical Computing Group Inc., Montreal, Canada). The X-ray crystal structure of the unliganded CRM1 from Chaetomium thermophilum at a resolution of 2.94 Å (Protein Data Bank accession number 4FGV) was used as the template for homology modeling. Homo sapiens CRM1 (NP_003391) and Canis lupus familiaris (XP_531839) amino acid sequences were retrieved from the NCBI database. After the CRM1 models were constructed, they were refined based on reference to the Ramachandran plot. Potential binding sites on each CRM1 were identified using the site finder application of MOE. Binding affinities and conformation between DP2392-E10 and each pocket of the CRM1s were evaluated using the ASEDock application of MOE (Goto et al., 2008) as described (Yokoyama et al., 2010) .

Forestry and Fisheries of Japan aimed at improving food safety and animal health (grant number 13406421), and by the Japan Society for the Promotion of Science (JSPS) Postdoctoral Fellowship (grant number 15F15416).

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We thank the Platform for Drug Discovery, Informatics, and Structural Life Science of the Ministry of Education, Culture, Sports, Science and Technology, Japan, for providing DP2392-E10. We are also grateful to Dr. Shinji Watanabe (Center for Influenza Virus Research, National Institute of Infectious Diseases, Japan), Dr. Hiroshi Kida, and Dr. Yoshihiro Sakoda (Graduate School of Veterinary Medicine, Hokkaido University, Japan) for giving influenza A subtype viruses, Dr. Yoshihiro Kawaoka (Institute of Medical Science, University of Tokyo, Japan) for providing the plasmids used for reverse genetics, and Ms. Kaori Honda (Bio-Active Compounds Discovery Research Unit, RIKEN CSRS, Japan) for technical support with DP2392-E10 photo-crosslinked Sepharose beads. This work was supported in part by a research project of the Ministry of Agriculture,