Selective and ATP‐competitive kinesin KIF18A inhibitor suppresses the replication of influenza A virus

Abstract The influenza virus is one of the major public health threats. However, the development of efficient vaccines and therapeutic drugs to combat this virus is greatly limited by its frequent genetic mutations. Because of this, targeting the host factors required for influenza virus replication may be a more effective strategy for inhibiting a broader spectrum of variants. Here, we demonstrated that inhibition of a motor protein kinesin family member 18A (KIF18A) suppresses the replication of the influenza A virus (IAV). The expression of KIF18A in host cells was increased following IAV infection. Intriguingly, treatment with the selective and ATP‐competitive mitotic kinesin KIF18A inhibitor BTB‐1 substantially decreased the expression of viral RNAs and proteins, and the production of infectious viral particles, while overexpression of KIF18A enhanced the replication of IAV. Importantly, BTB‐1 treatment attenuated the activation of AKT, p38 MAPK, SAPK and Ran‐binding protein 3 (RanBP3), which 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. Collectively, our results unveiled a beneficial role of KIF18A in IAV replication, and thus, KIF18A could be a potential therapeutic target for the control of IAV infection.

The influenza virus is a member of the Orthomyxoviridae family, and its genome consists of eight single-strand RNA segments, encoding 11-12 proteins. 13 The influenza virus life cycle is divided into multiple steps including entry, replication of the viral genomic RNAs, export of viral ribonucleoprotein (vRNP) complexes from the nucleus, assembly and release. 14 Each step in this process strongly depends on specific interactions with host cellular elements. For example, PI3K/AKT/mTOR signalling pathways are activated during viral entry, 15 and the transcription and replication of viral RNAs are also known to require the activation of NF-κB. 16 In addition, Raf/ MEK/ERK/NF-κB signalling pathways are involved in the nuclear export of vRNP complexes and release processes. 17,18 Although inhibiting these signalling pathways could block influenza virus replication, they are essential to host cell survival as well. Therefore, it is important to discover host cell factors that regulate influenza viral replication, while their inhibition minimally affects the host cells.
Viruses use the cytoskeleton transport system during their replication cycle, with interactions between subviral molecules and cytoskeleton proteins critical for virus assembly and release. [19][20][21][22][23][24] A kinesin is a motor protein that moves various substances such as diverse membranous organelles, mRNAs, intermediate filaments and signalling molecules within cells along microtubules. 25 Kinesins are known to play a role in regulating cell division, cell movement, spindle assembly and chromosome alignment/segregation. [26][27][28] Interestingly, it has been recently reported that kinesin family member proteins regulate the replication of some viruses, including HIV, 29 HSV 30 and the Lassa virus. 31 Thus, kinesin family proteins could be potential antiviral targets. KIF18A is a member of the kinesin motor protein family and known to play a critical role in diverse cellular processes including microtubule dynamics and subcellular organelle transportation. 32 While KIF18A is known to be associated with several cancers, [33][34][35] no role has been previously described for this protein in viral infection.
In this study, we investigated the role of KIF18A in the replication of the influenza A virus (IAV). We discovered that KIF18A is a beneficial host protein for IAV replication. Importantly, the inhibition of KIF18A by a highly specific small-molecule inhibitor, BTB-1, [36][37][38] significantly suppressed IAV replication and enhanced the survival rate of IAV-infected mice. Thus, KIF18A could be targeted as a potential therapy to control IAV infection.

| Trypan blue exclusion assay
Cell viability was analysed by a trypan blue exclusion assay as described previously. 41 Briefly, 50 μL of cell suspension was mixed with equal parts of 0.4% trypan blue dye (Thermo Scientific). After incubation for 1 minute, cells were counted under a light microscope.
Cell viability was calculated by dividing the number of viable cells (unstained) by the number of total cells and multiplying by 100.

| Western blot analysis
Western blotting was performed as described previously. 39,42 All proteins were extracted using an NP 40 protein extraction solution (Elpisbio) supplemented with a protease/phosphatase inhibitor (Thermo Scientific). Amount of protein was quantified using Pierce BCA protein assay kit according to the manufacturer's instruction (iNtRON Biotechnology). Equal amounts (10-20 μg) of protein were loaded onto SDS-PAGE gels (10% or 12% or 15%) for separation before being transferred onto nitrocellulose membranes. After incubating the membranes with primary antibodies (1:1,000) overnight at 4°C, membranes were incubated with the secondary antibody (1:5000) at room temperature for 1 hr Membrane-bound antibodies were detected using SuperSignal West Pico PLUS-enhanced chemiluminescent substrate (Thermo Scientific).

| RNA interference
Small interfering RNA (siRNA) targeting KIF18A and scramble siRNA were purchased from Thermo Scientific. The knockdown of KIF18A with a specific siRNA was conducted using Lipofectamine® RNAiMAX Reagent (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293 cells that were seeded in 24-well plate one day earlier were treated with Lipofectamine RNAiMAX reagent/siRNA (si-KIF18A or scramble siRNA) mixtures for 24 hours. RNA interference efficiency was analysed by Western blotting. For each slide, at least three fields were visualized.

| Real-time quantitative PCR
HEK293 cells were infected with IAV at MOI of 3 for 3 hours or 6 hours. Total RNA was extracted using RNAiso Plus reagent (Takara) to synthesize the cDNA using ReverTraAce qPCR RT kit (Toyobo). The quantitative real-time PCR for viral RNA (M1, NP, HA) was performed using a CFX Connect real-time system (Bio-Rad). The following primers were used:

| Animals
Six-to eight-week-old male C57BL/6 mice were used in experi-

| Statistical analysis
All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software). Error bars indicate the standard error of the mean (SEM), and mean values were compared using Student's t test or ANOVA followed by Tukey's post hoc test. P value for mouse mortality was calculated using a log-rank test. All experiments were repeated independently at least three times.

| IAV infection increases the expression of KIF18A
During IAV replication, diverse viral components such as viral proteins and RNAs are transported to specific sites within a host cell.
Thus, it is possible that host cellular motor proteins such as kinesins are required for IAV propagation. This prompted us to investigate the role of KIF18A, a member of the kinesin motor protein family, in IAV propagation. We first measured the expression level of KIF18A in the infected MDCK cells ( Figure 1A), HEK 293 cells ( Figure 1B) and A549 cells ( Figure 1C). After infection (as monitored by M1 expression), KIF18A expression increased in all three cell lines. These results suggest that KIF18A might play a role in IAV replication.

| Treatment with a KIF18A inhibitor reduces the IAV-mediated cytopathic effect
Because IAV infection increased KIF18A expression (Figure 1), we subsequently tested whether the inhibition of KIF18A has any impact on virus propagation. In order to block KIF18A activity, the highly specific small-molecule inhibitor BTB-1 was used. 36

| KIF18A inhibitor treatment decreases IAV propagation
The reduced CPE following treatment with BTB-1 ( The supernatant was harvested to determine the viral titre using a plaque assay. The significant inhibition of infectious virus production was observed when the cells were treated with BTB-1 ( Figure 3G).
Collectively, these results indicate that the inhibition of KIF18A activity interferes with IAV propagation.

| Knock-down of KIF18A decreases IAV replication, while its overexpression increases viral protein expression
To further confirm our observations that the inhibition of KIF18A reduces IAV replication, we utilized siRNA to target KIF18A (si-KIF18A).
The transfection of HEK293 cells with si-KIF18A dramatically de-

| KIF18A inhibition blocks the export of viral RNP complexes from the nucleus
We conducted immunofluorescence analysis to visualize the de-  Figure 5B). This indicates that the inhibition of KIF18A prevents the nuclear export of vRNP complexes.

| Inhibition of KIF18A prevents the activation of MAPK and AKT pathways to block phosphorylation of RanBP3
We next sought to determine whether BTB-1 also displays antiviral activity after viral entry.

| KIF18A inhibitor treatment reduces the morbidity and mortality of IAV-infected mice
Based on the in vitro experiment results, we hypothesized that KIF18A inhibition could protect mice from morbidity and mortality associated with IAV infection. To test this hypothesis, we conducted an in vivo experiment with mice. Mice that were infected with IAV were treated intranasally with PBS or BTB-1 at 3 mpk (mg per kg). BTB-1 treatment significantly reduced IAV titre in the lung as compared to untreated control ( Figure 7A). When bodyweight was monitored, significant weight difference between PBS-and BTB-1-treated groups was not observed at 4 and 9 dpi. However, bodyweight of PBS-treated group (CTR) was significantly lower than BTB-1-treated at 12 and 14 dpi ( Figure 7B), indicating that BTB-1 treatment alleviated IAV-associated morbidity. Furthermore, BTB-1 substantially enhanced the viability of BTB-1-treated mice as compared to the CTR group ( Figure 7C). Thus, these results strongly suggest that inhibition of KIF18A protects mice from the morbidity and mortality induced by IAV infection.

| D ISCUSS I ON
In this study, we investigated the role of KIF18A in IAV replication.
KIF18A expression increased following infection with IAV, which led us to investigate its effect on IAV replication. Our results indicated that the inhibition of KIF18A successfully suppressed IAV replication without significant cytotoxic effects. We also found that the inhibition of KIF18A impaired the nuclear export of vRNP complexes, which is a critical step in viral assembly. Therefore, targeting KIF18A has the potential to be an effective therapeutic strategy for treating IAV infection. in the viral replication process. In our results, although treatment with BTB-1 at 1 hpi produced the strongest suppression response, treatment at 6 or 12 hpi was still able to prevent the expression of viral proteins ( Figure 6A). This indicates that KIF18A plays an important role in viral replication at several different stages of the IAV life cycle.
Interestingly, we observed that the time-point and MOI for highest pathways during human breast carcinogenesis. 35,48 In this study, we found that the inhibition of KIF18A disrupted the infection-induced activation of signalling pathways, including AKT, p38 and SAPK that are necessary for viral replication ( Figure 6). Third, kinesin family members such as KIF18A may play a role in the export of vRNP complexes, a crucial step in IAV assembly. This idea is supported by our results that inhibition of KIF18A blocked the activation of AKT, p38, SAPK and RanBP3 ( Figure 6B and C), which is critical for the nuclear export of vRNP complexes.
KIF18A is also known to be critical for controlling the spindle length and aligning mitotic chromosomes at the spindle equator during cell division. 32 Numerous studies have reported that KIF18A serves as a central regulator in cell transformation and carcinogenesis. [33][34][35] For example, KIF18A is involved in several forms of cancer, including colorectal, breast and hepatocellular cancer. 33,35,49 Thus, KIF18A might be a potential therapeutic target for the treatment of cancer. In this study, we provide strong evidence that KIF18A is a necessary cellular factor for IAV propagation. Although further investigations are required, KIF18A might be an advantageous factor for other viruses as well. Therefore, a single medication targeting KIF18A could have multiple applications in both cancer and infectious diseases, such as influenza.
In conclusion, the results of this study indicate that KIF18A is an important host cell factor required for successful IAV propagation.
F I G U R E 7 KIF18A inhibitor treatment enhances the viability of IAV-infected mice Wild-type C57BL/6 mice were infected intranasally with IAV at 1.5 × 10 3 PFU. At 10 min, 3 h and 6 h after infection, mice were treated intranasally with PBS (CTR) or BTB-1 at 3 mpk (mg per kg). (A) At 3 dpi, lungs were harvested and viral titres were determined by plaque assay. White bar indicates PBStreated group (CTR) (n = 5), while black bar indicates BTB-1-treated group (n = 5). (B) Relative bodyweights that were measured at 0, 4, 9, 12 and 14 dpi are shown. (C) Per cent survival of mice is depicted for each group. Black square indicates BTB-1-treated group (n = 12), while black circle indicates PBS-treated group (CTR) (n = 12). P value was obtained using a log-rank test. The bar graphs (A and B) represent the average of 5 replicates ± SEM. **P ≤ .01. ***P ≤ .001.
Representative data of at least three independent experiments are shown Although further investigation for the efficacy of the treatment with KIF18A inhibitor at later times post-infection in vivo is required, inhibition of KIF18A would be an efficient therapeutic strategy for the treatment of IAV infection.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflicts of interests.

AUTH O R CO NTR I B UTI O N S
YC and YS designed the study and wrote the manuscript. YC, KK and JK performed experiments and analysed data. SL and Y.S supervised the study, and all authors critically revised the manuscript and approved the final version of the manuscript.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data supporting the findings of this study are available from the corresponding author on request.