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Selective and ATP‐Competitive Kinesin KIF18A Inhibitor Suppresses Received: 16 October 2019 | Revised: 13 February 2020 | Accepted: 6 March 2020 DOI: 10.1111/jcmm.15200 ORIGINAL ARTICLE Selective and ATP-competitive kinesin KIF18A inhibitor suppresses the replication of influenza A virus Yong-Bin Cho1 | Sungguan Hong2 | Kyung-Won Kang3 | Ji-Hun Kang1 | Sang- Myeong Lee3 | Young-Jin Seo1 1Department of Life Science, Chung-Ang University, Seoul, South Korea Abstract 2Department of Chemistry, Chung-Ang The influenza virus is one of the major public health threats. However, the devel- University, Seoul, South Korea opment of efficient vaccines and therapeutic drugs to combat this virus is greatly 3Division of Biotechnology, College of Environmental and Bioresources, Jeonbuk limited by its frequent genetic mutations. Because of this, targeting the host factors National University, Iksan, South Korea required for influenza virus replication may be a more effective strategy for inhibit- Correspondence ing a broader spectrum of variants. Here, we demonstrated that inhibition of a motor Sang-Myeong Lee, Division of protein kinesin family member 18A (KIF18A) suppresses the replication of the influ- Biotechnology, College of Environmental and Bioresources Sciences, Jeonbuk enza A virus (IAV). The expression of KIF18A in host cells was increased following National University, Gobong-ro 79, Iksan-si, IAV infection. Intriguingly, treatment with the selective and ATP-competitive mitotic Jeollabuk-do 570-752, Korea. Email: [email protected] kinesin KIF18A inhibitor BTB-1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression Young-Jin Seo, Department of Life Science, College of Natural Sciences, Chung-Ang of KIF18A enhanced the replication of IAV. Importantly, BTB-1 treatment attenuated University, 221 Heukseok-dong, Dongjak-gu, the activation of AKT, p38 MAPK, SAPK and Ran-binding protein 3 (RanBP3), which Seoul 156-756, South Korea. Email: [email protected] led to the prevention of the nuclear export of viral ribonucleoprotein complexes. Notably, administration of BTB-1 greatly improved the viability of IAV-infected mice. Funding information National Research Foundation of Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and Korea (NRF), Grant/Award Number: thus, KIF18A could be a potential therapeutic target for the control of IAV infection. NRF-2018R1A5A1025077 and NRF- 2018R1D1A1B07043782 KEYWORDS influenza virus, KIF18A, RanBP3 1 | INTRODUCTION every several decades. For example, the swine influenza (H1N1) pandemic in 2009 led to 151 000 to 575 000 deaths globally,5 which The influenza virus is a major cause of serious respiratory infections, has continuously raised concerns about the emergence of next po- with systemic manifestations that include fever, coughs, diarrhoea tential influenza pandemic.6,7 Although antiviral drugs such as osel- and headaches, and it is associated with significant morbidity and tamivir and amantadine are currently available, multiple strains that mortality.1-3 Despite vaccination, 5%-15% of the population suffer exhibit resistance to these drugs have been reported.8-12 Thus, the from influenza virus infections annually due to frequent mutations in development of novel therapeutics that can protect the host from a the viral RNA genome.4 Moreover, influenza pandemics occur once broad range of influenza variants is needed. Cho and Hong contributed equally to this work. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd J Cell Mol Med. 2020;24:5463–5475. wileyonlinelibrary.com/journal/jcmm | 5463 5464 | CHO ET AL. The influenza virus is a member of the Orthomyxoviridae fam- human lung epithelial (A549) cells were maintained in Dulbecco's ily, and its genome consists of eight single-strand RNA segments, modified Eagle's medium (DMEM, Welgene) supplemented with encoding 11-12 proteins.13 The influenza virus life cycle is divided penicillin/streptomycin and 10% FBS. In order to infect the cells, into multiple steps including entry, replication of the viral genomic MDCK, HEK293 or A549 cells were incubated for 1 hour with the RNAs, export of viral ribonucleoprotein (vRNP) complexes from the influenza virus in the presence of MEM or DMEM (Welgene) con- 14 nucleus, assembly and release. Each step in this process strongly taining 0.3% bovine serum albumin (BSA) and TPCK-trypsin (2 μg/ depends on specific interactions with host cellular elements. For mL, Sigma-Aldrich). example, PI3K/AKT/mTOR signalling pathways are activated during viral entry,15 and the transcription and replication of viral RNAs are 16 also known to require the activation of NF-κB. In addition, Raf/ 2.2 | Reagents, plasmids and antibodies MEK/ERK/NF-κB signalling pathways are involved in the nuclear export of vRNP complexes and release processes.17,18 Although in- A KIF18A inhibitor (BTB-1, ≥99%) was purchased from Tocris hibiting these signalling pathways could block influenza virus repli- Bioscience. To treat the infected cells with BTB-1, cells were washed cation, they are essential to host cell survival as well. Therefore, it is with PBS twice after 1-hr incubation with IAV to wash out unbound important to discover host cell factors that regulate influenza viral virus. For KIF18A overexpression, plasmid DNA (pMX229 was a replication, while their inhibition minimally affects the host cells. gift from Linda Wordeman, Addgene plasmid #23002) was used.40 Viruses use the cytoskeleton transport system during their rep- The transfection of cells with the plasmids was performed using lication cycle, with interactions between subviral molecules and Lipofectamine™ 3000 Reagent (Invitrogen) according to the manu- cytoskeleton proteins critical for virus assembly and release.19-24 facturer's instructions. Antibodies used to detect influenza viral M1, A kinesin is a motor protein that moves various substances such as NP and KIF18A were purchased from Abcam. Anti-influenza viral NS1 diverse membranous organelles, mRNAs, intermediate filaments was purchased from Santa Cruz Biotechnology. Antibodies against and signalling molecules within cells along microtubules.25 Kinesins actin, AKT, p-AKT (Ser473), p38, p-p38, SAPK, p-SAPK, RanBP3 and are known to play a role in regulating cell division, cell movement, p-RanBP3 were purchased from Cell Signaling Technology. Anti- spindle assembly and chromosome alignment/segregation.26-28 rabbit IgG, HRP-linked antibody was purchased from Cell Signaling Interestingly, it has been recently reported that kinesin family Technology. Goat antimouse IgG F(ab')2, polyclonal antibody (HRP member proteins regulate the replication of some viruses, includ- conjugate) was purchased from Enzo Life Sciences. ing HIV, 29 HSV30 and the Lassa virus.31 Thus, kinesin family proteins could be potential antiviral targets. KIF18A is a member of the kine- sin motor protein family and known to play a critical role in diverse 2.3 | Trypan blue exclusion assay cellular processes including microtubule dynamics and subcellular organelle transportation.32 While KIF18A is known to be associated Cell viability was analysed by a trypan blue exclusion assay as de- 33-35 41 with several cancers, no role has been previously described for scribed previously. Briefly, 50 μL of cell suspension was mixed with this protein in viral infection. equal parts of 0.4% trypan blue dye (Thermo Scientific). After incu- In this study, we investigated the role of KIF18A in the replication bation for 1 minute, cells were counted under a light microscope. of the influenza A virus (IAV). We discovered that KIF18A is a bene- Cell viability was calculated by dividing the number of viable cells ficial host protein for IAV replication. Importantly, the inhibition of (unstained) by the number of total cells and multiplying by 100. KIF18A by a highly specific small-molecule inhibitor, BTB-1,36-38 sig- nificantly suppressed IAV replication and enhanced the survival rate of IAV-infected mice. Thus, KIF18A could be targeted as a potential 2.4 | Western blot analysis therapy to control IAV infection. Western blotting was performed as described previously.39,42 All proteins were extracted using an NP 40 protein extraction solu- 2 | METHODS tion (Elpisbio) supplemented with a protease/phosphatase inhibitor (Thermo Scientific). Amount of protein was quantified using Pierce 2.1 | Virus, cells and infection BCA protein assay kit according to the manufacturer's instruction (iNtRON Biotechnology). Equal amounts (10-20 μg) of protein were Influenza A/California/04/2009 (H1N1) virus was kindly provided by loaded onto SDS-PAGE gels (10% or 12% or 15%) for separation be- Dr Baik Lin Seong (Yonsei University). Amplification and titration of fore being transferred onto nitrocellulose membranes. After incu- the virus were performed on Madin-Darby Canine Kidney (MDCK) bating the membranes with primary antibodies (1:1,000) overnight cells as described previously.39 MDCK cells were maintained in at 4°C, membranes were incubated with the secondary antibody Eagle's minimum essential medium (MEM, Welgene) supplemented (1:5000) at room temperature for 1 hr Membrane-bound antibodies with penicillin/streptomycin (Welgene) and 10% foetal bovine serum were detected
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