Reversible disruption of XPO1-mediated nuclear export inhibits respiratory syncytial virus (RSV) replication

Respiratory syncytial virus (RSV) is the primary cause of serious lower respiratory tract disease in infants, young children, the elderly and immunocompromised individuals. Therapy for RSV infections is limited to high risk infants and there are no safe and efficacious vaccines. Matrix (M) protein is a major RSV structural protein with a key role in virus assembly. Interestingly, M is localised to the nucleus early in infection and its export into the cytoplasm by the nuclear exporter, exportin-1 (XPO1) is essential for RSV assembly. We have shown previously that chemical inhibition of XPO1 function results in reduced RSV replication. In this study, we have investigated the anti-RSV efficacy of Selective Inhibitor of Nuclear Export (SINE) compounds, KPT-335 and KPT-185. Our data shows that therapeutic administration of the SINE compounds results in reduced RSV titre in human respiratory epithelial cell culture. Within 24 h of treatment, RSV replication and XPO1 expression was reduced, M protein was partially retained in the nucleus, and cell cycle progression was delayed. Notably, the effect of SINE compounds was reversible within 24 h after their removal. Our data show that reversible inhibition of XPO1 can disrupt RSV replication by affecting downstream pathways regulated by the nuclear exporter.

where Fn/c is the nuclear/cytoplasmic ratio, Fn is the nuclear fluorescence, Fc is the cytoplasmic fluorescence, and Fb is the background or autofluorescence). Data shown are mean ± SEM, n ≥ 15. Statistical significance was determined using two-way ANOVA with Tukey's post hoc test with GraphPad Prism v8.4.3. ns: non-significant; ***p < 0.001, ****p < 0.0001. Data is representative of three independent experiments. www.nature.com/scientificreports/ XPO1-mediated nuclear export is vital for normal cell functioning 8 . Therefore, it is important to determine if the expression of XPO1 would be restored upon removal of SINE compounds. Uninfected A549 cells were treated with 1.5 μM of KPT-185 or KPT-335 for 48 h, the medium was replaced with tissue culture medium, and cells incubated further for another 24 or 48 h (Fig. 2c,d). XPO1 expression in KPT-185-treated cells recovered within 24 h post removal (h.p.r) to levels similar to DMSO-treated cells. Removal of KPT-335 also resulted in an increase in XPO1 but to a lesser extent and the recovery of XPO1 expression required more time in KPT-335-treated cells. This suggests the effect of KPT-335 treatment lasts longer than KPT-185. Our data agrees with previous literature that treatment with SINE compounds would cause minimal effects on non-infected cells 23 . 50 and low IC 50 values. The CC 50 values for KPT-185 and KPT-335 were 86.03 µM and 44.9 µM, respectively. The percentage cytotoxicity induced by KPT-301 treatment did not exceed 10% even after treatment with the highest dose of 100 µM (Fig. 3a). The anti-RSV efficacy of the SINE compounds was evaluated using immunoplaque assays. A549 cells were infected with RSV for 1 h and treated with increasing doses of SINE compounds from 2 h post infection (h.p.i.) to 48 h.p.i. The percentage reduction in the number of plaques in the SINE-treated A549 cells in comparison to non-treated cells is shown in Fig. 3b. The IC 50 values for KPT-185 and KPT-335 were 1.3 µM and 0.96 µM at 48 h.p.i, respectively. 100% inhibition of RSV replication was observed following treatment with ≥ 10 µM of KPT-185 and KPT-335. KPT-301 was ineffective against RSV-A2 at all the tested doses (0.1-100 μM), indicating that it is ineffective against RSV. Viral plaque morphology in human cell lines can gauge infectivity, cytopathic effects, and is used to assess viral fitness 24,25 . The immunoplaque assay used in this study is similar to a quantitative comet assay used to evaluate efficacy of antiviral drugs or antibodies 26,27 . Comet shaped plaques indicate the sample contains infectious particles capable of spreading in contiguous cells 27 . Change in plaque morphology to smaller, blunted cometshaped plaques or rounder, pin-point plaques indicates presence of fewer infectious particles 27   www.nature.com/scientificreports/ shown in Fig. 3c. Plaque morphology in KPT-185-and KPT-335-treated cells had fewer comets and scattered pin-point plaques in comparison to DMSO-or KPT-301-treated cells (Fig. 3c). Decrease in viral titre and change in plaque morphology indicate disruption of XPO1 function as a promising post-infection therapy against RSV.

KPT-335 and KPT-185 have high CC
Treatment with SINE compounds reduced RSV replication with no substantial change in RSV protein expression. We have previously shown that short-term treatment with leptomycin B (LMB) disrupts XPO1 function in the early or late stages of RSV infection and leads to reduced viral replication 11 . To determine the efficacy of SINE compounds against RSV over the same time period, RSV-A2-infected A549 cells were treated with 1.5 µM of KPT-185 or KPT-335, from 6 to 18 h.p.i (early stage of infection), or 18-30 h.p.i (late stage of infection), and DMSO was used as the control. The SINE-containing medium was replaced with DMEM containing 2% FBS and 1 X PSN and incubated up to 30 h.p.i or 48 h.p.i. The viral titer in the lysate was determined using immunoplaque assay (Fig. 4). Short-term treatment (12 h) with KPT-335 was more effective than KPT-185 in both early and late stages of RSV infection. Incubation up to 48 h.p.i (30 h in the absence of SINE compounds) reduced viral titre markedly compared to 12 h of treatment (18-30 h.p.i) (Fig. 4a,b). Relative to DMSO, treatment with KPT-335 in the early stages of infection significantly (p = 0.002) reduced viral titre. At 48 h.p.i, both KPT-335 and KPT-185 had significantly (p < 0.0001; to the same extent) reduced viral titre relative to DMSO. This correlates to higher efficacy of KPT-335 to reduce XPO1 expression and longer recovery period for the treated cells to restore XPO1 (Fig. 2). Treatment in the late stages of infection was effective at 48 h.p.i (p = 0.031), but not at 30 h.p.i (Fig. 4b,d). This suggests SINE compounds disrupt RSV replication after at least two rounds of replication. Reduced plaque formation is evident after short-term disruption (12 h) of XPO1 (Fig. 4a,b). This relates to the indirect reduction of RSV replication because of disruption of the XPO1 function and downstream processes.
The presence of fewer infectious virions in SINE-treated samples is shown in Fig. 4c,d. The number of plaques was substantially lower in SINE-treated A549 cells relative to DMSO-treated cells. Fewer comet-shaped plaques were observed in KPT-335-treated A549 cells compared to KPT-185-treated cells at both time points. Independent of time of addition, continuous treatment with the compounds up to 48 h.p.i showed increased efficacy against RSV relative to DMSO.
Immunofluorescence assays of A549 cells treated during the early and late stages of RSV infection were used to determine the subcellular localization of M protein. Congruent to decreased RSV replication, increased retention of RSV M protein was observed in SINE-treated A549 cells. Partial, yet statistically significant (p < 0.05), retention of M protein in the nucleus was observed following extended incubation up to 48 h.p.i regardless of period of treatment ( Our data is in agreement with our previous study showing significant increase in nuclear localisation of M protein at 48 h.p.i. after treatment with KPT-335 early in infection 12 . Short-term SINE treatment (12 h) following RSV infection is not therapeutically germane and the effects of long-term SINE treatment were examined ( A pattern of reduction in viral titre similar to short-term treatment was observed with continuous SINEtreatment (Fig. 5c,d). 60% reduction in viral titre was observed by 24 h.p.i. with KPT-335 (p = 0.007 compared to DMSO), and at 48 h.p.i. following treatment with KPT-185 (p = 0.018 compared to DMSO) (Fig. 5d). Viral titre was significantly reduced following treatment with KPT-335 (compared to DMSO treatment) at both 24 h.p.i (p = 0.007) and 48 h.p.i. (p = 0.005). These findings suggest KPT-335 is more effective at disrupting XPO1-mediated export of M protein and retarding RSV replication in comparison to KPT-185 (also refer to Figs. 2, 3, 4).
The absence of observable changes in RSV protein expression following treatment with SINE compounds suggests the inhibition of XPO1 does not affect RSV protein expression substantially (Fig. 6). Negligible changes in the level of RSV G, F, N, P or M proteins were detected in RSV-infected cells after treatment with SINE compounds up to 48 h.p.i compared to vehicle control.
Disruption of XPO1 is efficacious for inhibiting RSV replication following either prophylactic 12 or therapeutic treatment with SINE compounds. This is most likely related to the inhibition of XPO1-mediated export and reduction of XPO1 in treated cells. The antiviral effect of SINE compounds could be a cumulative effect of reduced XPO1, disruption of XPO1-mediated export, partial nuclear retention of M, and impact on downstream sequences of other host pathways, including the cell cycle and expression of cytokines and chemokines. Both  www.nature.com/scientificreports/ progression of the cell cycle 28,29 . Previous studies with SINE compounds have shown reduced cell division in cancer cells 30 . RSV subverts the progression of cell cycle at both mRNA and protein levels.

Treatment with SINE compounds delays cell cycle progression and affects expression of IL-8, IFN-λ and IFN-β.
In non-infected cells, the cell cycle distribution in DMSO-treated A549 cells at 24 h.p.i was 73% in the G0/ G1 phase, 10% in the S phase and 17% in the G2/M phase, as expected 31,32 . Treatment with SINE compounds significantly altered the distribution of cells in each phase of the cell cycle (Fig. 7a). Treatment with KPT-185 reduced the percentage of cells in the S phase (84% in G0/G1, 4% in S and 12% in G2/M phase) within 24 h.p.i (p = 0.004 relative to DMSO) while KPT-335-treatment reduced the percentage of cells in the G2/M phase (p < 0.0001 relative to DMSO; 81% in G0/G1, 18% in S and 1% in G2/M) ( Fig. 7a; representative histograms shown on the right). Treatment with KPT-301 had no effect on cell cycle distribution, which was similar to that of DMSO-treated cells (77% in G0/G1, 10% in S and 13% in G2/M). Previous studies in cancer cells have   (Fig. 7b). For RSV-infected A549 cells treated with KPT-185, there was an increase in cells in G0/G1 phase (84%), followed by 10% in S, and 6% in G2/M. Similarly, treatment with KPT-335 increased the proportion of A549 cells in G0/G1 phase (82%), with the remaining cells equally distributed between the S and G2/M phases (9% each) (Fig. 7b). Our data suggests RSV infection resulted in some arrest in the S phase, while treatment with SINE compounds resulted in arrest in the G0/G1 phase.
RSV induces a time-dependent increase in the expression of cytokines and chemokines that are regulated by NFκβ through the Rel/NFκB or p50/p65 pathway, which is actively involved in cell differentiation, host immune response and in the transcription of many inflammatory cytokines 33,34 . Analysis of protein interactions using the STRING database highlights the relationship between IL-8, IFN-β and IFN-λ expression and NFκβ-mediated signalling and the role of XPO1 therein (Fig. 8a,b) [35][36][37] . Chemokine expression is often mediated via NFκβ, JAK-STAT pathways or by activating protein 1 (AP1)-mediated transcription 38 . Exit of NFκB (p105) (NFKB1 in Fig. 8a) via XPO1 pathway leads to increased expression of IFN-β (IFNB1) and IL-8 (CXCL8) (Fig. 8a). Both NFκB (NFKB1 in Fig. 8b) and CXCL8 can interact with IFN-λ1, a critical antiviral and immunomodulatory cytokine in epithelial cells 39,40 .
We have previously shown that treatment with KPT-335 up to 24 h.p.i. had no effect on IL-8 or IFNγ levels in RSV infected cells. We were interested to determine if XPO1 inhibition by SINE compounds would affect the expression of IL-8, IFN-β and IFN-λ after longer treatment. Previous studies have shown RSV replication induces a steady state of IL-8 production 41 . Relative to DMSO, treatment with KPT-335 significantly reduced IL-8 expression in infected A549 cells (p = 0.0010 at 24 h.p.i and p = 0.028 at 48 h.p.i; Fig. 8c). Treatment with KPT-185 marginally reduced IL-8 expression (p = 0.009) relative to DMSO at 24 h.p.i but was less effective compared to KPT-335 (Fig. 8c). No significant effect on IL-8 expression was observed after treatment with KPT-185 from www.nature.com/scientificreports/ 2 to 48 h.p.i relative to DMSO (Fig. 8c). The data suggests that treatment with KPT-335 induces an early and consistent decrease in IL-8 expression over 48 h relative to DMSO while KPT-185 has a short-term effect. Our data are somewhat in contradiction to our previous findings 12 and may be due to the higher dose of KPT-335 used in this study (1.5 μM compared to 1 μM in the previous study). RSV infection of primary airway epithelial cells in vitro and nasopharyngeal samples from infants infected with RSV results in dysregulated expression of IFNs including IFN-β and IFN-λ that provide antiviral resistance to host cells 42,43 . No significant effect on IFN-β expression was detected following treatment with SINE compounds at 24 h.p.i. relative to DMSO-treated cells (Fig. 8d). However, there was a significant difference in IFN-β expression between SINE treated cells, with a lower expression in KPT-335-treated cells relative to KPT-185 (p = 0.038; Fig. 8d). At 48 h.p.i, a marginal yet significant change was observed in SINE-treated cells relative to DMSO (p = 0.012 for KPT-185 and p = 0.030 for KPT-335; Fig. 8d). A marginal, yet significant, reduction in www.nature.com/scientificreports/ IFN-λ was also observed at 48 h.p.i relative to DMSO-treated cells (p = 0.0283 for KPT-185 and p = 0.030 for KPT-335), but not 24 h.p.i. (Fig. 8e). The small effect of SINE treatment on IFN production suggests the involvement of alternate pathways affecting their expression 44 . For example, RSV induces the expression of IL-1β (IL-1B; Fig. 8f) which can increase the expression of IL-8 and IFN-β and in turn IFN-λ, bypassing the NFκβ-mediated signalling pathway 45,46 . Also, transient inhibition of XPO1 may have a short-lived effect which was evident in short-term treatment, but this temporary disruption may not be sufficient to have an overall impact on interferon production.

Discussion
The therapeutic administration of KPT-185 and KPT-335 significantly reduced RSV replication in a dose-and time-dependent manner in cell culture, while KPT-301 was ineffective against RSV. Both KPT-335 and KPT-185 had low cytotoxicity, disrupted XPO1-mediated export, reversibly reduced the amount of XPO1 in treated cells and delayed cell cycle progression within 24 h of treatment. KPT-301 had low cytotoxicity but showed no inhibitory effects against RSV, did not reduce XPO1 levels and had no effect on cell cycle progression. SINE compounds have been extensively characterized as chemotherapeutics for various solid and hematologic malignancies. Over-expression of XPO1 enables cancer cells to prematurely export Tumour Suppressor Proteins (TSPs) such as p53, p73, p21, p27, FOXO (1, 3a, and 4), PP2A, BRCA1 and BRCA2, and E2F4 and evade apoptosis and promote malignancy 14,23,47,48 . Inhibition or reduction of XPO1 expression forces the nuclear retention, accumulation, and functional activation of TSPs to limit oncogenesis [49][50][51] . The mechanism of action of SINE compounds is similar to LMB, binding to the Cys528 residue located within the Nuclear Export Signal (NES) groove of XPO1. Unlike LMB, SINE compounds are smaller, bind to the NES groove exclusively through hydrophobic interactions and are not hydrolyzed after conjugation. These attributes lead to the reversible nature of interaction and improved tolerance in non-malignant cells 52 . SINE compounds bind to XPO1 long enough to kill cancer cells, but their reversible nature allows them to be released in time to spare normal cells 52 .
Selective inhibition of XPO1-mediated transport is being actively pursued as a promising therapeutic target against viruses 8 , including SARS-CoV-2 53 . Inhibition of nuclear export of M protein by LMB-mediated disruption of XPO1 was shown to reduce RSV titres and site-directed mutation of the XPO1-binding domain in M protein completely inhibited viral replication 13 . This finding highlights the importance of XPO1 in the RSV lifecycle. Inhibition of XPO1-mediated transport was found effective both in vitro and in vivo against influenza A and B strains 17 . Treatment with SINE compounds was also effective against Venezuelan equine encephalitis virus (VEEV) 16 and against opportunistic viruses that commonly affect immunocompromised patients such as Epstein-Barr virus, human cytomegalovirus, adenoviruses in vitro 18 . Recent studies have identified XPO1 as a key host protein modulated by the Nsp-4, -9 and Orf 6 proteins of SARS-CoV-2 and suggest that SINE compounds (ClinicalTrials.gov NCT04349098) could be potentially used for antiviral therapy 54,55 .
Therapeutic administration of KPT-185 or KPT-335 reduced XPO1 expression and partially retained M protein to the nucleus. Transient inhibition of XPO1 has a continued effect in reducing viral titre with successive replication cycles. Short-term treatment (12 h) with SINE compounds in either early or late stages of infection reduced RSV titres even after removing the compound. Continuous treatment with SINE compounds showed a similar reduction of RSV titres within 48 h of treatment. Reduced viral titers could translate to reduced virus spread and decreased lung involvement and disease pathogenesis.
In terms of cytotoxicity and disruption of the XPO1 function, KPT-335 was more effective than KPT-185 while KPT-301 was not effective. KPT-185 was the lead compound designed primarily for in vitro studies, and its orally bioavailable analog, KPT 251 has been successfully shown to have preclinical efficacy against various haematological and solid cancers in mice models 19 . KPT-335 was the first chemotherapeutic agent approved for treatment of canine lymphoma 23,56 . Both KPT-335 and KPT-185 were almost equally effective at disrupting XPO1-mediated export (1.5 µM). A high IC 50 and poor disruption of XPO1 function rules out KPT-301 as a candidate against RSV despite having low cytotoxicity. This is in agreement with other studies that have evaluated its anti-cancer or antiviral activity including Jorquera et al. 12 , Lundberg et al. 16 ; Perwitasari et al. 17 and Widman et al. 18 .
Treatment with KPT-185 or KPT-335 induced a dose-and time-dependent nuclear accumulation of M protein but did not affect the total amount of the protein present in infected cells. The selective disruption of XPO1mediated transport, shown using both pGFP-Rev (NES) transfected cells and nuclear localization of M protein in infected cells, is the primary mechanism of action of SINE compounds against viral replication. Disruption of XPO1 function results in partial M protein-nuclear accumulation and significant reduction in viral titre at 48 h.p.i., suggesting the disruption of M protein has an accumulative effect leading to reduced infectious virions with each replication cycle. Since M protein is conserved across RSV A and B strains and it exclusively traffics between subcellular compartments using XPO1 12,13 , it is probable that administration of KPT-185 and KPT-335 would potentially be effective against all RSV strains by subverting the nuclear export pathway used by the host cell.
In contrast to the marked reduction in viral protein expression following SINE-treatment against influenza, VEEV and opportunistic viruses affecting immunocompromised individuals [16][17][18] , transient inhibition of XPO1 during RSV replication did not detectably affect RSV protein production. This could be due to RSV protein synthesis and viral replication being localized to the cytoplasm as opposed to nuclear-replicating viruses 57 .
It is likely that in addition to partial nuclear accumulation of M protein, inhibition of XPO1 may alter other pathways that depend on nuclear export, including cell cycle progression and NFκβ-inflammatory pathways. Both pathways are subverted or manipulated during RSV infection and likely have a direct effect on RSV replication 58,59 . Treatment with SINE compounds could impact downstream pathways regulated by XPO1 and have been shown to cause cell cycle arrest, increase in inflammation and apoptosis in cancer cells 14 www.nature.com/scientificreports/ causes cell cycle arrest of the host cells in the S phase probably to promote its replication 63,64 . Treatment with SINE compounds in non-infected cells caused a delay in cell cycle progression, reducing the percentage of cells entering the S phase at 24 h.p.t followed by recovery by 48 h.p.t. This suggests the reversible interaction of KPT-185 or KPT-335 with XPO1 does not cause a permanent arrest of the cell cycle, rather a delay in progression, causing cells to remain longer in each phase. This data correlates with the longer recovery time for the XPO1 levels, increased nuclear accumulation and larger reduction in virus titre in KPT-335 treated cells. RSV infection induces a strong inflammatory response within the airways 33,34 . This response is induced and stimulated primarily through the Rel/NFκB or p50/p65 pathway, which is actively involved in cell differentiation, host immune response and transcription of many inflammatory cytokines 33,34 . Continuous treatment with KPT-185 or KPT-335 significantly reduced IL-8 by 48 h.p.t. A marginal yet significant change in IFN-β and IFN-λ was also observed at 48 h.p.t. This may be beneficial in vivo since the inflammatory response would be reduced but the antiviral response to detect and eliminate viral particles might not be affected. The limited impact of SINE treatment on the inflammatory markers tested may be because NFκβ-mediated signalling is only one of the ways by which these markers are activated.

Conclusion
In Lactate Dehydrogenase (LDH) Assay. CytoTox96 ® non-radioactive cytotoxicity assay (Promega, Sydney, Australia) was used to determine cytotoxicity following the manufacturer's instructions. Briefly, overnight cultures of A549 cells were treated with increasing concentration of SINE compounds prepared in DMEM containing 2% FBS and 1× PSN. 48 h.p.t, the supernatant was incubated with LDH reagent (Promega) at room temperature for 30 min in the dark, followed by addition of stop solution (Promega) and absorbance was measured at 490 nm. The average optical density (OD) of untreated cells was subtracted from each experimental well. The mean OD values of lysed cells were considered 100% cytotoxic and used to calculate the percent cytotoxicity of SINE compounds. The percent cytotoxicity versus the log10 concentration of SINE compounds was plotted using GraphPad Prism v.8.4.3 and the values were fitted to a non-linear regression curve to determine the 50% cytotoxic concentration (CC 50 ).
Plaque reduction assay. Overnight cultures of A549 cells were infected with RSV-A2 at multiplicity of infection (MOI) = 1 and incubated for 1 h with periodic gentle swirling. The viral inoculum was decanted and replaced with DMEM supplemented with 2% (v/v) heat inactivated FBS and 1× PSN. After 2 h of incubation, the infected cells were treated with increasing doses of SINE compounds up to 48 h.p.i. Cells treated with DMSO alone were used as control. At the end of incubation, the supernatant was decanted, and the cells were fixed with methanol + 2% H 2 O 2 followed by air-drying overnight. The viral titre was determined using an immunoplaque assay with a goat anti-RSV antibody (Merck Millipore, VIC, Australia) and donkey anti-goat IgG-HRP (Thermofisher, VIC, Australia). The percentage reduction in viral titre associated with SINE treatment was determined in comparison to DMSO-treated cells. The values were fitted to a non-linear regression curve using GraphPad Prism v.8.4.3 to determine the IC 50 , the concentration of drug required to induce 50% inhibition of viral replication.
Immuno-plaque assay. The fixed cells were blocked at room temperature for 30 min in 1% BSA (Sigma) prepared in 1× PBS followed by incubation in 1:1000 dilution of goat anti-RSV antibody (Merck Millipore) in Immunofluorescence assay. A549 cells were grown overnight on glass coverslips and infected at MOI = 0.5 or 1 with RSV-A2. The cells were treated with SINE compounds for the specified time interval. Cells treated with DMSO alone were used as controls. At the indicated times p.i., cells were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and immune-stained with a monoclonal antibody specific for RSV M protein (MAbαM) 65 and Alexa Fluor 488-conjugated secondary antibody (Life Technologies). Hoechst 33342 (ThermoFisher) diluted in 1× PBS was used for nuclear staining. The coverslips were mounted on slides using Fluorescence mounting medium (Dako) and analysed using CLSM.
CLSM and image analysis. Fixed or live A549 cells were imaged as described previously 13  Analysis of protein expression. A549 cells were infected for 1 h with RSV-A2 (MOI = 1) or left uninfected, and treated with SINE compounds or DMSO from 2 . to 24 or 48 h.p.i. Cells were lysed in freshly prepared RIPA buffer (150 mM NaCl, 1.0% Triton X-100, 0.5% sodium deoxycholate, 10% SDS, 50 mM Tris, pH 8.0; protease inhibitor cocktail, and PhosSTOP inhibitor tablet; (all from Sigma)). The supernatant was collected after centrifugation at 13,000×g, at 4 °C for 30 min, and analysed using Western blotting. The lysates were mixed with 6× Laemmli sample buffer, boiled, and electrophoresed on a 12% polyacrylamide gel; 10 µl of each sample was loaded per lane. The separated proteins were transferred to nitrocellulose and probed with goat anti-RSV antibody, mouse anti-XPO1 (BD Biosciences, NSW, Australia) or mouse anti-α/βtubulin (Genesearch, QLD, Australia) monoclonal antibodies diluted 1:1000 in 1% skim milk in 1× PBS (pH 7.2) containing 0.1% Tween 20 (PBST). Bound antibody was detected with horseradish peroxidase-conjugated secondary antibodies diluted 1:5000 in 1% skim milk in PBST. The bound antibody was detected using Enhanced Chemiluminescence (ECL, Perkin Elmer) and imaged on the LiCor Odyssey ® Fc Imaging System with Image Studio™ Lite software. Wherever required, blots were stripped using stripping buffer (2% SDS, 62.5 mM Tris-HCl (pH 6.8) and 114.4 mM β-mercaptoethanol (all from Sigma)) at 50 °C for 10 min, followed by washing in PBST, blocking in 4% skim milk in 1× PBST and re-probed with primary antibodies overnight as required. Intensity of the bands was measured using Fiji ImageJ (vr. 1.52s). Values were expressed as arbitrary units relative to total protein present (for RSV) or to corresponding intensity of tubulin (for XPO1), used as the loading control.
Cell cycle analysis. A549 cells in 6-well plates were serum starved for 24 h in serum-free DMEM (Sigma).
These synchronized cells were infected with RSV-A2 at MOI = 0.5 for 1 h, as described previously or left uninfected. At 2 h.p.i, cells were treated with 1.5 μM of KPT-185, KPT-335 or DMSO for 24 or 48 h. Cells were trypsinized, fixed in ice-cold 80% ethanol, stained using Tali™ Cell Cycle Kit as per the manufacturer's recommendations and analysed using Tali ® Image-Based Cytometer. The threshold gate for each cell cycle phase was set on the Tali ® Image-Based Cytometer during the analysis of each sample. The percentage of cells in each phase of the cell cycle under different conditions was estimated. The average percentage of cells in each phase of the cell cycle and standard error were plotted using GraphPad Prism v.8.4.3. Significance was determined using two-way ANOVA and Tukey's post hoc test.

Cytokine and interferon expression in infected cells treated with SINE compounds. A549 cells
infected (MOI = 0.5) with RSV-A2 for 1 h were treated with 1.5 μM KPT-185 or KPT-335 or DMSO from 2 to 24 or 48 h.p.i. Culture supernatants were collected and clarified of cellular debris by centrifugation. Interleukin (IL)-8 (R&D Systems), Interferon (IFN)-λ (R&D Systems) and IFN-β (ELISAkit.com) expression was determined by ELISA as per the manufacturer's recommendations. Fold-change in expression in SINE-treated cells was calculated relative to DMSO-treated cells. Significance was determined using two-way ANOVA and Tukey's post hoc test on GraphPad Prism v. 8.4.3. Protein-protein interactions between NFκβ, IFNs, IL-8 and XPO1. Protein-protein interactions based on known functional associations were determined using STRING: functional protein association networks (https:// string-db. org/). The online database depicts a protein network of genome-wide functional connectivity. The relevant proteins were searched under "Multiple Proteins", and "Homo sapiens". The query