{"paper_id":"d18e40e9-b06b-44f2-a2cb-81ec0fab314d","body_text":"1 \n \nHuman Rhinovirus 16 impacts cilia structure in 3D cultured primary bronchial 1 \nepithelial tissue through alternative splicing of host cilia RNAs.  2 \nChristoforos Rozario1, 2*, Quan Gu1,2, Andrew Stevenson1, Rose A Maciewicz3, Sheila V 3 \nGraham1,4 4 \n 5 \n1MRC-University of Glasgow Centre for Virus Research, School of Infection and Immunity, 6 \nCollege of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, G61 7 \n1QH, Scotland, UK. 8 \n2 these authors contributed equally to the work. 9 \n3 GLAZgo Discovery Centre, School of Infection and Immunity, College of Medical, 10 \nVeterinary and Life Sciences, University of Glasgow, Glasgow, G12 8TT, Scotland, UK.   11 \n*Current address: Department of Life Sciences, Program in Biological Sciences, European 12 \nUniversity Cyprus, 6, Diogenous Str, Nicosia, Cyprus 13 \n4 Corresponding author: Address: Rm 03 Botham Building, Garscube Estate, Glasgow, G61 14 \n1QH, Scotland, UK. Tel +44 141 3306256. Email address: Sheila.Graham@glasgow.ac.uk. 15 \n 16 \nShort title: Human rhinovirus controls host cilia function through altered splicing. 17 \nKey words: Human rhinovirus, respiratory epithelial cell, SR proteins, SRPK1, cilia 18 \n 19 \nData availability statement: The underlying data for the graphs shown in this manuscript can 20 \nbe accessed at https://doi.org/10.5525/gla.researchdata.1946. The transcriptomic data can 21 \nbe found at RBI/ENA with the accession ID PRJEB88791. 22 \n23 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n2 \n \nAbstract 24 \nHuman rhinoviruses (HRV) are a leading cause of the common cold but can often lead to 25 \nrespiratory complications such as wheeze in young children. In a transcriptomic study of 26 \nrespiratory nasal swab specimens from children hospitalised with acute wheeze, a significant 27 \nalteration was found in the expression of the serine/arginine rich splicing factor (SRSF) 28 \nkinase, SRPK1, between HRV positive children with acute exacerbations and HRV negative 29 \ncontrols. As this kinase can regulate host RN A splicing, we hypothesised that HRV infection 30 \ncould dysregulate the expression of host mRNAs to affect antiviral functions or to alter the 31 \nmorphological features of the infected resp iratory epithelium. Here, we show that 32 \npharmacological inhibition of SRPK1 in prim ary bronchial epithelial cells resulted in 33 \nincreased HRV16 replication while overexpressi on of SRPK1 reduced viral replication. In a 34 \nprimary bronchial epithelial 3D model infe cted with HRV16 decreased phosphorylation of 35 \nSRSF1, 3 and 6 was observed. Furthermore, transcriptomic and alternative splicing (AS) 36 \nbioinformatic analysis revealed the significantly altered AS of 1228 host genes during 37 \ninfection. Subsequent pathway analysis revealed the enrichment of most of these genes in 38 \nnetworks related to cilia development and function. HRV16 infection led to significantly 39 \ndecreased cilia length and total cilia numbers in  the primary bronchial epithelial 3D model 40 \ntogether with changes to selected cilia proteins. Overall, this investigation has unravelled 41 \nnovel cellular networks implemented during HRV infection that may lead to acute 42 \nexacerbations of respiratory infections. 43 \n 44 \nAuthor summary 45 \nHuman rhinoviruses cause the common cold. In immunocompetent individuals this is usually 46 \na self-limiting infection. However, in young children and the elderly, infection can lead to 47 \ncomplications such as bronchiolitis, croup, and wheezing. Rhinovirus infection can 48 \nexacerbate chronic conditions such as cystic fi brosis, chronic obstructive pulmonary disease 49 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n3 \n \nand asthma. Understanding the molecular pathol ogy of this exacerbation could lead to new 50 \navenues for therapy. In this study, we discovered that a multifunctional cellular enzyme 51 \ncalled serine arginine protein kinase 1 (SRPK1) is a restriction factor for human rhinovirus 16 52 \n(HRV16) infection. One key cellular function of  SRPK1 is to regulate RNA splicing through 53 \nmodifying the SR proteins that normally enhanc e splicing. In three dimensional tissues 54 \ngrown from human bronchial epithelial cells, we found that HRV16 infection led to decreased 55 \nlevels of modified SR proteins. This change resulted in significant alterations in RNA 56 \nexpression in the infected cell. Most of these alterations affected production of the correct 57 \nversions of cilia proteins resulting in reduced cilia numbers and cilia blunting. This type of 58 \ndamage due to HRV infection would result in inefficient clearance of subsequent viral 59 \ninfections prolonging the viral infection leading to lower respiratory tract infection and to 60 \nexacerbations of existing chronic disease. 61 \n 62 \n 63 \n 64 \n65 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n4 \n \nIntroduction 66 \nHuman rhinoviruses (HRVs) are positive-sense single stranded RNA enteroviruses within 67 \nthe Picornaviridae family, collectively comprising more than 160 genotypes grouped into 3 68 \nspecies (A, B and C) [1]. HRVs are placed amongst the most frequent human infectious 69 \nagents, causing more than half of upper respiratory tract infections globally, though evidence 70 \nnow includes the lower respiratory tract in t heir niche [2]. They are causative agents of the 71 \ncommon cold, a self-limiting infection with mild symptoms in immunocompetent individuals. 72 \nHowever, HRV infection can lead to severe respiratory complications in 73 \nimmunocompromised groups such as pre- school children and the elderly. These 74 \ncomplications are usually associated with the migration of the infection into the lower 75 \nrespiratory tract and include bronchiolitis, croup,  wheezing as well as the exacerbation of 76 \nchronic conditions such as cystic fibrosis, chronic obstructive pulmonary disease and asthma 77 \n[2, 3].  78 \n 79 \nStrong evidence associating HRV and asthma exacerbations has been accumulating, with 80 \nHRV shown to account for more than 50% of total exacerbations and common cold 81 \ncomplications in asthmatics costing about 60 billion USD annually [1]. Additionally, HRV-82 \ninduced wheezing in early age is linked with asthma development in adulthood, while 83 \noffspring of atopic (hyperallergic) mothers are more susceptible to HRV infections [4]. 84 \nDespite all the evidence associating HRV with asthma, molecular mechanisms underlying 85 \nthis pathophysiology remain unclear.  86 \n 87 \nThe Mechanisms of Acute Viral Respiratory Infections in Children (MAVRIC) study 88 \nconducted in Perth, Australia, aimed to investigate further the molecular mechanisms 89 \nunderlying asthma pathogenesis in the context of HRV infections. In this study, nasal swabs 90 \ncontaining nasal epithelial cells along with a variety of immune cells including neutrophils 91 \nand peripheral blood mononuclear cells were coll ected from pre-school children hospitalised 92 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n5 \n \nwith acute wheezing; these were subjected to microarray analysis comparing expression 93 \nprofiles between uninfected controls and HRV infected patients [5-8]. Surprisingly, for an 94 \nRNA virus infection operating in the cytoplasm, genes involved in the pre-mRNA splicing 95 \nprocess showed altered RNA expression.  96 \n 97 \nIt is well-established that HRV proteases 2A pro and 3C pro can degrade nuclear proteins and 98 \nthat proteases from the three different HR V species degrade different substrates [9]. Key 99 \nsubstrates for HRV proteases are the nucleoporins that make up the nuclear pore complex 100 \n(NPC) [9-11]. Degradation of nucleoporins re sults in mis-localisation of heterogenous 101 \nribonucleoprotein particles (hnRNPs) and SR sp licing factors from the nucleus to the 102 \ncytoplasm [12, 13]. It has been suggested that this virus-mediated relocation of splicing 103 \nfactors aids viral replication by supplying RNA genome-binding proteins [14] or by regulating 104 \nvirus mRNA translation [13, 15]. 105 \n 106 \nThere are nine classical SR proteins (serine-arginine-rich splicing factors (SRSFs) 1-9). 107 \nThey all contain an N-terminal RNA rec ognition motif (RRM) and a C-terminal serine-108 \narginine-rich (RS) domain [16]. Some SR proteins such as SRSF1 and SRSF6 possess an 109 \nadditional pseudo-RRM [17]. SRSFs are found predom inantly in the nucleus but some (e.g. 110 \nSRSF1) can dynamically shuttle to and from  the cytoplasm [16]. SRSFs are essential 111 \nregulators of constitutive and alternative sp licing. They bind exonic or intronic sequence 112 \nenhancers to define exon-intron boundaries and stabi lise formation of the spliceosome at 113 \nthese boundaries to enhance splicing [18, 19]. A role for virus-associated splicing regulation 114 \nin respiratory disease was strengthened when the SRSF6 gene was found to be upregulated 115 \nin equine airway smooth muscle cells from asthmatic horses [20]. Several viruses of different 116 \nBaltimore classification groups, for example, human papillomavirus [21], human 117 \nimmunodeficiency virus [22], influenza A viru s [23], and alphavirus [24] have evolved to 118 \nutilise or control SR protein family members and the host splicing machinery. SR proteins 119 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n6 \n \nare also known to exert regulatory functions beyond splicing in the nucleus, including nuclear 120 \nexport, cytoplasmic stability and translation [25, 26]. 121 \n 122 \nSRPK1 is a moonlighting protein involved in nu merous intracellular signalling pathways [27]. 123 \nIt has been shown to regulate innate immunity to viral infection [28]. However, it is a key 124 \nregulator of cellular splicing. The RS domain of SR proteins is subject to phosphorylation by 125 \nkinases including serine/arginine protein kinases (SRPK) and CDK-like kinases (Clk) [29, 126 \n30]. This post-translational modification regulates both the function and subcellular 127 \nlocalisation of SRSFs [31, 32]. SRPK is normally  present in the cytoplasm of cells where it 128 \nphosphorylates newly synthesised SRSFs, to licenc e their entry into the nucleus [33]. There 129 \nare three SRPKs in human cells (SRPK1, 2 and 3). Only SRPK1 and SRPK2 are expressed 130 \nin epithelial cells. In the nucleus, SRPK1 interacts with Clk1 to promote splicing [30]. The 131 \ndynamic phosphorylation of the SRSF RS domains  by SRPK1 governs their levels, activity 132 \nand cellular localisation [31, 34-37]. Not all SR proteins are equally affected by SRPK1 133 \nactivity. For example, SR proteins e.g. SRSF1, with two RRMs may be phosphorylated to 134 \ncontrol splicing in a different manner from t heir single RRM–containing counterparts e.g. 135 \nSRSF3 [38]. 136 \n 137 \nHere we show SRPK1 activity on SR proteins is reduced during HRV16 (a variant of HRV-A) 138 \ninfection of primary epithelial cells and that SRPK1 is a restriction factor for HRV16 infection. 139 \nRNA-Seq analysis revealed significant host transcriptome changes between HRV16-infected 140 \nand mock-infected 3D cultured primary bronchi al epithelial cells similar to those found 141 \npreviously [39-43]. However, alternative spli cing analysis of the RNA-Seq data revealed that 142 \nsplicing of RNAs involved in cilia structure and function was significantly altered. HRV16 143 \ninfection impacted expression of key cilia co mponents. This may indicate a sophisticated 144 \nviral mechanism of host cell disruption duri ng infection. The data suggest that HRV16 may 145 \nregulate splicing of cilia-related RNAs leading to altered mucociliary clearance and ultimately 146 \nprolonging productive viral replication. 147 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n7 \n \n  148 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n8 \n \nMaterials and Methods 149 \nViruses stock generation 150 \nA variant of HRV-A, HRV16, was used for this study. Viral stocks obtained from ATCC 151 \n(ATCC VR-283), were propagated in HeLa Ohio cells at 33°C, seeded at 70-80% confluence 152 \nand grown in Dulbecco's modified Eagle medium (DMEM) with 10% foetal bovine serum and 153 \n1% penicillin/streptomycin (Thermo Fisher Sc ientific, UK). Infected cell lysates were 154 \ncentrifuged at 120 x g for 7 min at 4°C. The supernatant was filtered through a 0.22 μ M pore 155 \nand stored at -80°C.  Viral titres were determined via TCID50 assays using HeLa Ohio cells in 156 \n96-well plates. Cells were inoculated for 4 hours wi th serially diluted virus. Inoculates were 157 \nwashed off and cells were incubated for 5 days at 33°C. Cell death was recorded, with each 158 \ndilution tested in quadruplicate and viral titre calculated using the Spearman & Karber 159 \nmethod [44]. 160 \n 161 \nCell growth 162 \nNormal Human Bronchial Epithelial cells (HBECs) were purchased from Lonza (Basel, 163 \nSwitzerland # CC-2540S). The cell donor was a female Caucasian aged 16 years old, with 164 \nno known disease or smoking history and a BMI of 22. Cells were grown in PneumaCult-Ex 165 \nplus medium (Stem Cell Technologies, Cambridge, UK) in T75 flasks for 2D culture. For air-166 \nliquid interface 3D culture, cells were seeded on 6.5 mm transwells with 0.4 µm pore 167 \npolyester membranes (Corning, Berlin, Germany). When cells reached full confluence, airlift 168 \nwas performed by aspirating the apical medium and replacing the basal medium with 169 \nPneumaCult ALI maintenance medium (Stem Cell Technologies, Cambridge, UK). Medium 170 \nwas replaced 3 times per week, with initial mucus production at approximately 2 weeks post-171 \nairlift. Mucus was washed off once per week and tissues reached full differentiation by 4 172 \nweeks post-airlift. 173 \n 174 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n9 \n \n 175 \nHBEC infections 176 \nA mucus wash and basolateral medium change were performed. Cultures were incubated 177 \nfor one hour at 33 oC while virus stocks were thawed on ice. Based on calculations of an in 178 \nvivo infectious dose [40] the inoculum (3x10 6 pfu) was added in 100 µl medium apically. The 179 \nsame volume of medium was added to the top of mock-infected control cultures. Tissues 180 \nwere incubated for 3.5 hours at 33 oC then the inoculum was aspirated off and tissues were 181 \nwashed apically three times with PBS without Ca 2+ and Mg 2+ to remove non-internalised 182 \nvirus. Tissues were then incubated 33oC for the times stated in the experiments. 183 \n 184 \nSRPK1 overexpression, depletion and SRPIN340 inhibition 185 \nThe SRPK1 (transcript variant 1) human cDNA clone (untagged) (OriGene,Herford, 186 \nGermany, #RC205315) was transfected using Lipofectamine2000 (Thermo Fisher Scientific, 187 \nUK) at a concentration of 200ng/ml for 48 hours. SRPK1 was depleted by transfecting 188 \nDharmacon SMART-Pool siRNAs in RNAiM ax transfection reagent (Thermo Fisher 189 \nScientific, UK) into bronchial epithelial cells. siGLO (Dharmacon, # D-001630-01) was used 190 \nas a non-target siRNA control and to monitor transfection efficiency. SRPIN340 (Sigma 191 \nAldrich, UK, #5.04293) was dissolved in DMSO to 20mM stocks and was administered at 192 \n20μ M for 48 hours. Both treatments were adminis tered to primary bronchial epithelial cells 193 \nseeded at 60% confluence. 194 \n 195 \nRNA extraction and RT-qPCR 196 \n2D cultures: Cells grown in 6-well plates were lysed in 500 μ l of Trizol reagent (Thermo 197 \nFisher Scientific, UK) and stored at -20°C. Upon thawing cells were scraped into Trizol and 198 \nRNA was isolated according to the manufacturer’s instructions. 3D cultures: Tissues were 199 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n10 \n \nflash frozen in liquid nitrogen and stored at - 80°C. Upon thawing cells were vortexed to 200 \ndetach from the transwell membrane then tissu es were ground to a fine powder under liquid 201 \nnitrogen using a mortar and pestle and RNA wa s isolated using the RNeasy extraction kit 202 \n(Qiagen, Germany) according to the manufacturer’s instructions. RNA was quantified using a 203 \nNanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, UK, #ND-2000). 204 \ncDNAs were synthesised using the Maxima First Strand cDNA Synthesis kit with DNase 205 \ndigestion according to the manufacturer’s instru ctions (Thermo Fisher Scientific, UK). 20 μ l 206 \nreactions were prepared using the Takyon™ ROX Probe 2X MasterMix dTTP blue 207 \n(Eurogentec, Camberly, UK), primers and probes at 300 and 100 nM respectively and 208 \nnuclease-free water. Primer/probe sets used are listed in Table 1. 209 \nReactions were run on an ABI 7500 thermocycler (Thermo Fisher Scientific, UK) at this 210 \nprofile: 95°C (5 min), 60°C (15 sec), 72°C (3 min), 40 cycles. Data analysis was performed 211 \nusing the 7500 v2.3 (Thermo Fisher Scientific, UK) software. Ct values were determined 212 \nrelative to GAPDH as the reference target gene. 213 \n 214 \nProtein extraction and western blotting 215 \nCells grown in 6-well plates were scraped in 400 μ l 2X Bolt LDS buffer (Thermo Fisher 216 \nScientific) containing PhosphoSTOP (Merck, UK catalogue #  04693116001) and complete 217 \nminiprotease inhibitor cocktail (Merck, UK, catalogue # 200-664-3) in PBS. 20 µl of sample 218 \nwas loaded per lane on Bolt 4-12% Bis-Tris polyacrylamide gels (Thermo Fisher Scientific, 219 \nUK) and electrophoresed at 150 V for 60 minutes. Prot eins were transferred to nitrocellulose 220 \nmembranes using the iBlot2 Dry Blotting System (Thermo Fisher Scientific, UK). Membranes 221 \nwere blocked in 5% (w/v) milk powder in PBS containing 0.01% (v/v) Tween. PBST at room 222 \ntemperature for 1 hour, then washed in PBST  (3 x 7 minutes) and incubated with primary 223 \nantibody in 5% milk powder in PBST for 1 hour at room temperature or overnight at 4°C with 224 \nrotation. Following incubation, membranes were washed in PBST (3 x 7 minutes) and 225 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n11 \n \nincubated with secondary antibody in 5% mi lk powder in PBST for 1 hour at room 226 \ntemperature in the dark. A final round of washes was performed in PBST (2 x 7 minutes) and 227 \nPBS (2 x 7 minutes), and bands were visualised on the LI-COR Odyssey CLx Infrared 228 \nimaging system. Primary antibodies were SRPK1 1:300 (1:500, clone G211-637 BD 229 \nTransduction Laboratories, catalogue #611072), SRSF1 1:2500 1:1000, Mab96, Thermo 230 \nFisher Scientific, catalogue # 32-4500), SRSF 3 (1:300, Life Technologies, UK, catalogue 231 \n#334200 ), SRSF6 (1:300 Abcam, UK, catalogue #ab140623, GAPDH (1:1000, Meridian Life 232 \nSciences, UK, catalogue #H86504M, clone 6C5), HRV16 VP0/VP2 (1:250, QED 233 \nBiosciences, Aachen, Germany, catalogue # 18758). mAb104-detecting phosphorylated 234 \nSRSFs was prepared from hybridoma (AT CC CRL-2067) supernatants and was used neat 235 \nwith 5% milk powder and 0.01% Tween. Secondary antibodies were goat anti-rabbit Dylight 236 \n800 conjugate (1:2000, Thermo-Fisher, UK, catalogue #SA5-35571), goat anti-mouse 237 \nDylight 800 conjugate (1:2000, Thermo-Fisher, UK, catalogue #SA5-35521) and IRDye anti-238 \nmouse 800CW (1:2000, IRDye Licor Biosciences Ltd, UK, catalogue #926-32210). 239 \nMembranes were imaged on an Odyssey Infrared Im ager (LiCOR). The intensity of protein 240 \nbands was quantified using Odyssey Image Studio software. Protein levels were determined 241 \nand normalised to the level of the endogenous control (GAPDH).  242 \n 243 \nFormalin fixed paraffin embedded (FFPE) sample preparation  244 \nPrimary bronchial epithelial 3D cultures were fixed by fully submerging in 10% (v/v) buffered 245 \nformaldehyde (BNF) at room temperature overni ght. The cultures were submitted to the 246 \nVeterinary Diagnostic Services (University of Glasgow) for paraffin embedding, and 247 \nhaematoxylin and eosin (H&E) staining. 248 \n 249 \nImmunofluorescence microscopy 250 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n12 \n \nAntigen retrieval for 4µm sections from formalin-fixed, paraffin-embedded samples was 251 \ncarried out in 10 mM sodium citrate buffer, pH 6.0 using a Menarini Access Retrieval Unit, at 252 \n1100C on full pressure for 10min. (Veterinary Diagnostic Services, University of Glasgow). 253 \nMicroscope slides were washed sequentially six times in PBST. Slides were blocked in 10% 254 \n(v/v) filtered donkey serum in PBS for 1 hour at room temperature. Sections were incubated 255 \nwith primary antibody diluted in 5% donkey se rum in PBS for 2 hours at room temperature. 256 \nSlides were washed and sections were incubated with secondary antibody diluted in 5% 257 \ndonkey serum in PBS for 2 hours at room tem perature under dark conditions. Slides were 258 \nmounted in ProLong Gold Antifade Mountant with DAPI (Thermo Fisher, UK, catalogue # 259 \nP36931) and visualised on a ZEISS LSM 710 conf ocal microscope. Images were acquired 260 \nusing the ZEN Blue software. Primary antibodies were HRV16 VP0/VP2 (1:100, QED 261 \nBiosciences, Aachen, Germany, catalogue # 18758), β -tubulin (1:250,Merck, UK, catalogue 262 \n#AB9354) and TMEM67 (1:250, Proteintech, UK, catalogue #Ag5009). Secondary antibody: 263 \ndonkey anti-mouse Alexa-fluor 555-labelled anti body (1:1000 Thermo Fisher Scientific, UK, 264 \ncatalogue #A-31570). 265 \n 266 \nRNA sequencing, differential expression and pathway analysis  267 \n3D bronchial epithelial cultures were infected (3x10 6 pfu) at four weeks post-airlift and 268 \nharvested 48 hours post-infection. Total RNA wa s prepared from 3 biological replicates per 269 \ncondition and sequenced in-house. Eluted RNA was quantified using a NanoDrop 2000 270 \nSpectrophotometer (Thermo Fisher Scient ific, ND-2000) and quality controlled on a 271 \nTapeStation (Agilent Technologies, G2991AA). All samples had a RIN score of ≥  9. One 272 \nmicrogram of total RNA was used to prepare libraries for sequencing using an Illumina 273 \nTruSeq Stranded mRNA HT kit (Illumina, #20020594) and SuperScript2 Reverse 274 \nTranscriptase (Thermo Fisher Scientific, #18064014) according to the manufacturer’s 275 \ninstructions. Libraries were pooled in equi molar concentrations and sequenced using an 276 \nIllumina NextSeq 500 sequencer (Illumina, #FC-404). RNA-Seq reads were analysed for 277 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n13 \n \nquality using FastQC (version 0.11.9) and reads were trimmed of adaptor sequences and 278 \nlow-quality bases using Trimgalore 279 \n(https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). The trimmed reads were 280 \naligned to the human genome GRCh38 ( Ensembl) using Hisat2 (version 2.2.0) [45]. 281 \nFeatureCounts (Version 2.0.1) [46] was used to quantify reads mapping to gene annotation 282 \nfiles. Reads counts were normalized to counts per million (CPM). The edgeR package was 283 \nused to calculate the gene expression level an d to analyse differentially expressed genes 284 \nbetween sample groups. RNA-Seq data sets are freely available from the European 285 \nNucleotide Archive accession number PRJEB88791, and heat maps were generated in 286 \nGraphPad Prism (version 9). Pathway analysis was performed using the Ingenuity Pathway 287 \nAnalysis (IPA) tool using the rat, mouse, human and undefined species data, in all cells and 288 \ntissues. Only experimentally observed data were selected. 289 \n 290 \nOver representation analysis of alternative splicing events. 291 \nBam files from the RNA paired-end sequencing were sorted by co-ordinate, indexed and 292 \nsubject to SplAdder analysis [47] to measure and quantify alternative splicing events. 293 \nPercentage spliced in (PSI) values were quantified for each splicing event, and a two-tailed 294 \nstudent's t-test was performed for values fr om mock-infected and HRV16-infected primary 295 \nbronchial epithelial cells to determine the most significantly differentially spliced genes. 296 \nPathway analysis was performed using Webgestalt ( http://www.webgestalt.org/) [48]. Over 297 \nrepresentation analysis was carried out test for biological processes using Benjamini-298 \nHochberg multiple testing adjustment. Sashimi plots were generated using MISO 299 \n(https://pypi.org/project/misopy/0.5.4/) [49]. 300 \n 301 \nCilia count 302 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n14 \n \nFive sections from three biological replicates per condition were randomly selected and cilia 303 \nwere manually counted from images obtained at x20 magnification from two technical 304 \nreplicates per condition using a tally meter from FFPE H&E-stained samples. Image J (  305 \nhttps://imagej.net/software/fiji) was used to  quantify the cilia length by measuring the 306 \nproportion of distance in pixels. Ten cilia were measured in each section for each condition. 307 \nStatistical analysis 308 \nStatistical analyses were carried out using a students’ t-test. P-values of < 0.05 were 309 \nconsidered statistically significant.  310 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n15 \n \nResults 311 \nSRPK1 restricts HRV16 infection in primary epithelial cell culture. 312 \nThe kinase SRPK1 was found to be up-regulated at the mRNA level in the MAVRIC study 313 \nwhich compared nasal swab samples from HRV-infected young children with wheezing 314 \nexacerbations to HRV-negative controls to [7 , 50]. However, when the study population was 315 \ndivided into two different phenotypes based on  a Th1/type 1 interferon response versus a 316 \nTh2/IFNγ  response, SRPK1 mRNA was significantly downregulated in the second population. 317 \nThe two phenotypes had quite different clinical characteristics. In the case of a Th2/IFN γ  318 \nresponse, illness progressed more slowly but t here was a greater chance of hospitalisation 319 \nand repeat infections/exacerbation of diseas e [7] suggesting that SRPK1 downregulation 320 \nwas associated with more severe disease. 321 \nHowever, SRPK1 activity, as opposed to SRPK1 levels, is regulated by key cell signalling 322 \npathways such as CK2 and Akt, which can be impacted upon virus infection [51]. To find out 323 \nmore about the relationship between HRV infection and SRPK1, we overexpressed the 324 \nkinase in human bronchial epithelial cells (HBECs). We also inhibited the kinase by treating 325 \ncells with 20µM SRPIN340, a specific inhibitor of the kinase activity of SRPK1 [52]. Each 326 \ntreatment was carried out 48 hours prior to HRV16 infection. We examined changes due to 327 \nthe treatments in the logarithmic phase of vi ral production (MOI=3, 16 hours post infection, 328 \nSupplementary Fig. 1A). Supplementary Fig. 1B shows a significant increase in SRPK1 329 \nmRNA levels in overexpressing cells relative to  mock-transfected cells. The effectiveness of 330 \nthe SRPIN340 treatment was shown through decreased phosphorylation of SRSF1 331 \n(Supplementary Fig. 1C) and SRSF6 (Supplementary Fig. 1E) compared to mock-treated 332 \ncells. Levels of phosphorylated SRSF3 were not significantly decreased by SRPIN340 333 \ntreatment (Supplementary Fig. 1D). No significant change in levels of phosphorylated 334 \nSRSFs was detected when SRPK1 was overexpressed (SRPK1 OE) (Supplementary Fig. 335 \n1C-E). 336 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n16 \n \nWe assessed how inhibition and overexpression of SRPK1 impacted HRV16 infection. The 337 \nlevels of viral RNA, tissue-released virus parti cles and expression of viral capsid proteins 338 \nVP0 and VP2 were compared between mock-treated, SRPIN340-treated and SRPK1-339 \noverexpressed HBECs infected with HRV16 for 16 hours at an MOI=3 (Fig. 2). SRPK1 340 \ninhibition led to increased HRV16 replication, as reflected by the increased viral RNA 341 \nproduction (Fig. 2A, SRPIN340) although this wa s not statistically significant (p=0.07). 342 \nHowever, a statistically significant increase was observed upon kinase inhibition in tissue-343 \nreleased virus particles (p<0.05) (Fig. 2B, SRPIN340) and virus capsid VP2 protein 344 \nproduction (Fig. 2C, D, SRPIN340). In cont rast, SRPK1 overexpression did not have a 345 \nsignificant effect on viral genome replication when compared to untreated controls (Fig. 2A, 346 \nSRPK1 OE). SRPK1-overexpressing HBECs showed significantly decreased virus shedding 347 \n(Fig. 2B, SRPK1 OE) and viral capsid protein VP0 production (Fig 2C, D, SRPK1 OE) 348 \ncompared to untreated samples, thus displaying an opposite effect from respective SRPK1-349 \ninhibited samples. Taken together, these data s uggest that SRPK1 is a restriction factor for 350 \nHRV16 infection in primary epithelial cells, wi th the activity of SRPK1 repressing viral 351 \nreplication, assembly and release, at 16 hours post-infection.  352 \n 353 \nHRV16 infection alters phosphorylation of SR proteins in primary human bronchial 354 \nepithelial cells.  355 \nNext, to assess activity of SRPK1 during HRV16 infection we quantified changes in 356 \nexpression of selected substrates of SRPK1 in HBECs. SRSF1, SRSF3 and SRSF6 were 357 \nselected for further study. SRSF1 is the prototypical SRSF protein [37, 53]. SRSF3 was 358 \npreviously shown to be involved with the internal ribosome entry site (IRES)-dependent 359 \ntranslation of picornavirus mRNAs [54] and SRSF6 is known to be upregulated in equine 360 \nairway smooth muscle cells from asthmatic horses [20]. There was no significant change in 361 \ntotal levels of SRSF1 (Fig. 2A, Supplementary Fig. 2A) or SRSF6 (Fig 2C, Supplementary 362 \nFig. 2A) during a 48 hour time course of infection (MOI=3). However, SRSF3 showed 363 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n17 \n \nsignificantly decreased expression at 16, 24 and 48 hours of infection (Fig. 2B, 364 \nSupplementary Fig. 2A). Next, we measured le vels of phosphorylated SR proteins using the 365 \nSR protein phosphor-specific antibody Mab104 [38]. Levels of phosphorylated SRSF1 were 366 \nsignificantly decreased at 4 and 48 hours post infection in HRV16-infected cells compared to 367 \nmock-infected cells (Fig. 2D, Supplementary Fig. 2B). There was no significant change in 368 \nlevels of phosphorylated SRSF3 at any time point in HRV16-infected cells compared to 369 \nmock infected cells (Fig. 2E, Supplementary Fig. 2B). Finally, phosphorylated SRSF6 levels 370 \nwere significantly decreased in HRV16-infected cells at 4 and 24 hours post infection (Fig. 371 \n2F, Supplementary Fig. 2B). Taken together, these data indicate that HRV16 infection 372 \nrepresses the expression of SRSF3 and inhibits phosphorylation of SRSF1 and SRSF6. The 373 \ndownregulated phosphorylation seen for SRSF1 and SRSF6 suggests that the activity of 374 \nSRPK1 is repressed at early and late times of infection during HRV16 infection. 375 \n 376 \nHRV16 infection in 3D cultured primary bronchial epithelial tissues inhibits SRPK1 377 \nprotein levels and phosphorylation of SR proteins. 378 \nNext, we evaluated levels and activity of SRPK1 in HRV16 infection of air-liquid interface 3D 379 \ntissue cultures of HBECs. 3D cultures enable the differentiation of these cells, providing 380 \nincreased physiological relevance in parameters such as cell polarisation, cilia formation, cell 381 \nto cell interactions and nutrient access [55]. HBECs were grown at the air-liquid interface in 382 \ntranswell cultures over a four-week period (Supplementary Fig. 3 compare A to B). After this 383 \ntime, the tissues were fully differentiated as  indicated by cilia development (Supplementary 384 \nFig. 3B, arrowhead) and a multi-layered pseudostratified epithelium was developed. The 385 \ntissues also produced mucus as indicated by periodic acid-Schiff (PAS) staining of the 386 \ntissues which detects polysaccharides (Suppl ementary Fig. 3, compare C to D). This 387 \nsuggests the presence of goblet cells (S upplementary Fig. 3D, arrowheads) further 388 \nconfirming tissue differentiation. HRV16 (3x10 6 pfu) was applied to the surface of the 389 \nepithelium and infection was allowed to proceed for 48 hours. Infection induced evident 390 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n18 \n \nmechanical damage to the structure of the epithelium (Supplementary Fig. 3 compare E and 391 \nF, arrows in F shows regions of damage). However, the extent of damage may not be due 392 \nonly to HRV16 infection but also due to tissue handling for preparation of formalin-fixed 393 \nparaffin-embedded blocks. Immunofluorescence staining to detect the virus capsid protein 394 \nVP2 indicated that infection was established in all the layers of the epithelium 395 \n(Supplementary Fig. 3 compare G and H). West ern blot analysis indicated a significant 396 \ndecrease in SRPK1 and phosphorylated SRSF1, SRSF3, and SRSF6 protein levels in 3D 397 \nHBEC tissues infected with HRV16 (3x10 6 pfu) for 48 hours compare to mock-infected 398 \ntissues (Fig. 3). 399 \n 400 \nTranscriptomic analysis confirms increased expression of innate immune genes 401 \nduring HRV16 infection of 3D cultured primary bronchial epithelial tissues. 402 \nRNA sequencing was performed on HRV16 and mock infected primary bronchial epithelial 403 \n3D cultured tissues. Tissues were mock-infected or infected (3x10 6 pfu) for 48 hours at 4 404 \nweeks post-airlifting. Total RN A extracts were prepared from  triplicate HRV16-infected and 405 \nmock-infected HBEC 3D air-liquid interface cultures. The counts per million (CPM) of 406 \nindividual transcripts were similar between infected (median=33.1 CPM per transcript) and 407 \nmock infected tissues (median=30.2 CPM per tr anscript). 82.2% of bases achieved a quality 408 \nscore of Q30 and Illumina software was used to assign sequencing reads to their 409 \ncorresponding samples. Differentially expressed genes (DEGs) between infected and mock-410 \ninfected samples were identified by mappi ng sequencing reads to the reference human 411 \ngenome and quantifying the mapped reads of individual transcripts. 412 \nAnalysis indicated that there were 4034 DEGs due to HRV16 infection (false discovery rate 413 \n(FDR)<0.05, Benjamini-Hochberg correction). 2143 genes were up-regulated, and 1891 414 \ngenes were down-regulated in infected when compared to mock-infected tissues 415 \n(Supplementary Table 1). The most statistically  significant differences in gene expression 416 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n19 \n \nduring infection were seen in up-regulated genes as illustrated in the volcano plot in Fig. 4A 417 \n(red dots). Key innate immune gene changes are indicated similar to those found by Ong et 418 \nal [43]. The violin plot in Fig. 4B shows t hat the distribution of gene expression was similar 419 \nbetween infected and mock-infected samples. However, more genes showed a higher level 420 \nof expression (increased reads per million) in  control mock-infected (Ctrs) compared to 421 \ninfected (HRV16) samples (Fig. 4B). 422 \nFig. 4C shows a heat map of the top 20 genes that were up- or down-regulated due to 423 \nHRV16 infection. The most up-regulated genes were involved in anti-viral defence, primarily 424 \nthe type I interferon signalling pathway (Fig. 2A arrows, Fig. 4C, Table 2), demonstrated by 425 \nthe strong up-regulation of these genes in HRV 16-infected samples, across all biological 426 \nreplicates as previously reported [39, 43, 56,  57]. On the other hand, the functions of the 427 \nmost down-regulated genes during infection were very variable, but included genes involved 428 \nin epithelial homeostasis, metabolic processes, and cell signalling (Fig. 4C). Although 429 \nSRPK1 was significantly downregulated at the protein level in HRV16 infected primary 430 \nbronchial epithelial 3D cultured tissues (Fig . 3), SRPK1 mRNA expression was found to be 431 \nsignificantly upregulated by 1.3-fold. Protein- protein interaction networks were constructed 432 \nusing Gene Ontology (GO) enrichment analysis  of up- (240) or down-regulated (9) DEGs 433 \nwith log2 fold change >2 or <-2 (Fig. 4D). The antigen presentation pathway was the most 434 \nenriched biological process. ISG15 protein conjugation, apoptosis and type 1 interferon 435 \nsignalling were also identified as biological pathways induced by HRV16 infection. Molecular 436 \nfunction enrichment indicated HRV16-inducti on of antiviral OAS activity, chemokine 437 \nreceptors, and antigen processing (Fig. 4D). 438 \nOver-representation analysis was performed to identify cellular pathways affected by HRV16 439 \ninfection. A subset of DEGs was selected for this pathway analysis, with log2-fold change 440 \nvalues within the range of 1 to -1. This cut-off point included 655 DEGs of which 466 were 441 \nup-regulated and 189 down-regulated during infection (Supplementary Table 2). Pathway 442 \nanalysis mapped the vast majority of the DEGs in innate immune pathways, with the 443 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n20 \n \ninterferon signalling pathway scoring the highes t enrichment value (Supplementary Fig. 4). 444 \nHowever, genes involved in epithelial cell junctions (claudins and cadherins) and the 445 \nepithelial barrier (repetin, involucrin) were also downregulated as expected [58] 446 \n(Supplementary Table 2). Table 2 lists the to p thirty upregulated protein-coding genes of 447 \nknown antiviral functions. 448 \n 449 \nHRV16 infection of 3D cultured primary bronchial epithelial tissues leads to changes 450 \nin the splicing of cilia-related genes 451 \nSince we had shown that HRV16 infection alters splicing factors, we wanted to assess 452 \nwhether the splicing of host pre-mRNA transcripts was altered during infection. We used the 453 \nalternative splicing toolbox SplAdder [47] to perform further analysis on our RNA-seq data. 454 \nUsing SplAdder, we identified the total number of single splicing events in our dataset, along 455 \nwith the alternative splicing (AS) mechanism of each particular event. We identified 10952 456 \ngenes with alternative splicing events, with each event categorized by its splicing 457 \nmechanism (e.g., exon skipping, intron retention etc.). The differential splicing between 458 \ninfected and mock-infected samples was determined by comparing the percentage spliced-in 459 \n(PSI) output values given for each transcript. PSI indicates the efficiency of splicing of a 460 \nparticular exon into the transcript population of a gene, with values ranging from 0 to 1. 461 \nTherefore, different PSI values for a give n transcript between infected and mock-infected 462 \nsamples, indicate a different AS pattern for that respective gene during infection. 10514 out 463 \nof 10952 genes (96%) had a different PSI value in infected compared to mock-infected 464 \nsamples. Following a 2-tailed student’s t-test analysis, this number was limited to 1228 465 \ngenes that were significantly differentially spliced due to HRV16 infection (11% of all the 466 \nalternatively spliced genes in the dataset). 467 \n1469 single splicing events specifically associated with HRV16 infection were identified as 468 \nbeing involved in producing mRNAs as some were alternatively spliced via more than a 469 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n21 \n \nsingle mechanism, of which about 40% were exon skipping events (Fig. 5A). The rest of the 470 \nsplicing events identified included alternative 3’ sp lice sites (23%), alternative 5’ splice sites 471 \n(18%), intron retention (10%), multiple exon skipping (8%) and mutually exclusive exons 472 \n(2%) (Fig. 5A). To interpret the biological significance of this HRV16-induced effect on 473 \nsplicing, we performed a pathway analysis using WebGestalt on the 1228 differentially 474 \nspliced genes. While response to interferon was identified as a major pathway altered by 475 \nHRV16 infection through alternative splicing, the functional enrichment analysis indicated 476 \nthat most differentially spliced genes during HRV16 infection were involved in the 477 \nmicrotubule cytoskeleton required for cilia structure, ciliogenesis and cilia function (Fig. 5B). 478 \nTo verify specific alternative splicing changes  due to HRV16 infection seen in the functional 479 \nenrichment analysis we analysed three cilia-related genes whose alternative splicing is 480 \nknown to be related to ciliopathies [59]. T hese were radial spoke head protein 1 (RSPH1), 481 \nwhich is located in the spokes of cilia and controls cilia motility [60]; intraflagellar transport 482 \nprotein 74 (IFT74), which is involved in protein transport within cilia and is required for 483 \nciliogenesis [61]; and transmembrane protein 67 (TMEM67), a ciliary transition zone protein 484 \nrequired for cilia structure [62]. Figure 6A indicates the positions of these proteins in cilia. 485 \nSashimi plots showing the exon coverage from the RNA-Seq data were generated for the 486 \nthree selected genes using MISO (Fig. 6B-D). There was a small decrease in gene coverage 487 \nfor RSPH1 and TMEM67 indicating reduced transcription of the genes upon HRV16 488 \ninfection.  More importantly, clear differenc es in exon inclusion can be seen for each gene 489 \n(see arrowheads indicated on the maps below  the gene coverage profiles) comparing mock-490 \ninfected to HRV16-infected cells suggesting that HRV16 infection alters mRNA splicing of 491 \nthese RNAs leading to mutations in the encoded proteins. 492 \n 493 \nHRV16 infection leads to impaired cilia in 3D cultured primary bronchial epithelial 494 \ntissues 495 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n22 \n \nBased on the finding that HRV16 infection induced changes in the alternative splicing of 496 \ngenes involved in cilia development and function, we wanted to assess whether infection 497 \naffected the numbers and morphology of cilia in our primary bronchial epithelial 3D model 498 \n(Fig. 7). Figure 7A shows a representative image of an H&E stained, mock-infected 3D 499 \nculture of HBECs. Figure 7B shows a repres entative image of an H&E stained HBEC tissue 500 \ninfected with HRV16 (3x106 pfu) for up to 48 hours. HRV16-infected tissues showed reduced 501 \ncilia density, and the cilia appeared shorter than those on mock-infected tissues (enlarged 502 \nimages in A and B). Cilia numbers were manually counted and, compared to mock infected 503 \ntissues treated in exactly the same manner, in fected tissues showing significantly decreased 504 \ncilia numbers at all infection timepoints investigated (Fig. 7C). The largest decrease was 505 \nrecorded at 48 hours post infection, where infe cted samples had 4.5-fold less cilia compared 506 \nto mock-infected samples. Additionally, the average cilium length in these respective 507 \nsamples was quantified (Fig. 7D). This analysis  indicated that the average cilium length was 508 \nsignificantly decreased in infected compared to mock-infected samples at 24 and 48 hours 509 \npost infection. As with cilia numbers, the average cilium length showed the largest difference 510 \nat 48 hpi, where the average cilium length in infected samples was decreased by 1.5-fold 511 \ncompared to mock-infected samples (Fig. 7D). 512 \nTo confirm these data, we carried out immunofluorescence staining of sections of 3D 513 \ncultures of HBECs either mock-infected or HRV16-infected. We chose to use antibodies 514 \nagainst β -tubulin, a core cilium structural protein and TMEM67, a ciliary transition zone 515 \nprotein located at the junctions of cilia to the plasma membrane. β -tubulin was observed 516 \nalong the lengths of the cilia in mock-infected cells (Fig. 8 A, B) but levels of the protein were 517 \nreduced in HRV16-infected cells and cilia were not clearly seen (Fig. 8 C, D). Quantification 518 \nof β -tubulin staining showed a significant decrease at all time points, and this was especially 519 \nsignificant at later times of infection (Fig. 8 I). TMEM67 staining for mock-infected tissues 520 \nwas found in cells in the mid layers and on the outer surface of the tissue, in discrete sub-521 \ncilia regions (Fig. 8 E, F). The location of staining was similar for HRV16-infected tissues, but 522 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n23 \n \nthe staining was more diffuse (Fig. 8 G, H). Q uantification of staining showed a statistically 523 \nsignificant decrease in TMEM67 levels in infected tissues at all time points (Fig. 8 J). Taken 524 \ntogether these data suggest that HRV16 infection causes a reduction in cilia density and 525 \nchanges in cilia structure in the respiratory epithelium. 526 \n  527 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n24 \n \nDiscussion 528 \nHuman rhinovirus (HRV) is the most common respiratory virus infecting humans. Repeat 529 \ninfections in childhood can lead to wheezing and asthma, and these symptoms are 530 \nexacerbated by subsequent HRV infections. In addition, in individuals with chronic 531 \nobstructive pulmonary disease (COPD), HRV infe ction can lead to severe disease requiring 532 \nhospitalisation. Discovering cellular factors that lead to exacerbations of HRV infection is key 533 \nto designing novel therapies against these respiratory infections and conditions. This study 534 \nhas revealed two novel aspects of the cellular response to HRV16 infection. First, we have 535 \nshown that the cellular splicing kinase SRPK1 is a restriction factor for HRV16 (a variant of 536 \nHRV-A) infection in primary bronchial epithelial cells. Second, changes in alternative splicing 537 \ncause by an HRV16 infection-induced down regulation of SRPK1 impact cilia structure and 538 \nfunction in 3D cultured primary bronchial epithelial tissues. 539 \nTranscriptomics studies in nasal scrapings [7, 39], bronchial scrapings [63] and nasal and 540 \nlung epithelial air-liquid interface 3D cultures [41, 43, 57] have shown that HRV16 infection 541 \nalters expression of immunity-related genes. Our transcriptomic analysis of HBEC air-liquid 542 \ninterface cultures agrees with the conclusions  of these studies. There was significant up-543 \nregulation of genes encoding proteins involved in anti-viral defence (e.g. OASL, MX2), and 544 \nthe interferon signalling pathway (e.g. IFNL1, INFL2) and several chemokines (e.g. CXCL10, 545 \nCXCL11) known to be induced by HRV infection. Although SRPK1 is well known as a kinase 546 \nthat controls splicing via phosphorylation of SR proteins [29], it also positively regulates 547 \ninnate immunity to viral infections via NF- ĸ B and interferon gamma [28]. Therefore, SRPK1 548 \ndown-regulation during HRV16 infection could be beneficial to the infectious process, as we 549 \nhave demonstrated. 550 \nIn an in vivo study of a mixed nasal cell population from children with wheeze infected with 551 \nHRV, overall SRPK1 mRNA expression was upregulated compared to HRV-negative 552 \ncontrols [7]. SRPK1 mRNA was also upregulated 1.3-fold after a 48 hour HRV16 infection of 553 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n25 \n \n3D HBEC tissues in this study. However, increased SRPK1 expression at the RNA level did 554 \nnot lead to increased levels of SRPK1 protein. In fact, in 3D HBEC tissues infected with 555 \nHRV16 there was a significant decrease in SRPK1 levels and in phosphorylation of selected 556 \nSR proteins compared to mock infected tissues. This result agrees with the decreased 557 \nphosphorylation of SR proteins observed in a study where rhabdomyosarcoma cells were 558 \ninfected with another enterovirus, enterovirus A71 [64]. Our data suggest that HRV16 559 \nrepresses the activity of SRPK1 during infect ion. This could be either through decreased 560 \nprotein levels, as we have observed, or due to changes in cell signalling caused by HRV16 561 \ninfection impacting SRPK1 kinase activity, which is itself controlled by Ck2/Akt-mediated 562 \nphosphorylation [27]. 563 \nHowever, inhibition of SRPK1 kinase activity has  been shown to inhibit replication of viruses 564 \nsuch as HIV, hepatitis C and Sindbis virus [65-68] and it has recently been reported that 565 \nSRPK1 inhibition can reduced IRE-dependent trans lation and replication of enterovirus A71 566 \n[64]. These findings clearly suggest that SRPK1 is not a restriction factor of these viruses as 567 \nit is required for their life cycles. In contra st, both inhibition and overexpression of SRPK1 568 \nhas been shown to reduced Ebola viral replication [69], while SRPK1 is a known restriction 569 \nfactor for hepatitis B virus infection [70]. Thes e diverse findings indicate that viruses may 570 \nutilise SRPK1 in different ways for replication. Further, balancing activity of SRPK1 571 \nthroughout virus infection may be key. For example, repression of SRPK1 stimulation of 572 \ninnate immunity early in infection could facilitate viral infection while upregulation later in 573 \ninfection could be required for viruses such as HIV and that require either cellular splicing 574 \n[22] or enteroviruses that require SRPK1 activi ty for stimulation of viral RNA translation [64] 575 \nto complete their life cycle.  576 \nChanges in expression of genes involved in cilia formation and function were found 577 \npreviously in a study comparing the transcriptome of air-liquid interface cultures established 578 \nfrom the lungs of non-asthmatic versus asthmati c individuals [41] and changes were greater 579 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n26 \n \nfollowing HRV infection. Gene ontology analysis of RNA sequencing data showed that cilia 580 \nfunction was found to be altered in human nasal epithelial cells infected with HRV16 [43]. 581 \nStimulation of the antiviral immune res ponse by addition of poly(I:C) in human nasal 582 \nepithelial stem/progenitor cells also revealed c hanges to genes involved in ciliogenesis and 583 \nfunction [71]. In agreement with these studies, inspection of our DEG data from RNA 584 \nsequencing showed reduced expr ession of genes encoding dynein axonemal assembly 585 \nproteins, Bardet-Bield syndrome (BBS) genes important for cilia development and function, 586 \nintraflagellar transport proteins, and tubulins, structural components of cilia. 587 \nThe hypophosphorylation of SR proteins due to HRV16 infection in 3D HBEC tissues that we 588 \nhave observed would be expect ed to cause changes to cellular splicing [30, 72]. We 589 \nobserved a major impact on alternative spli cing in HBEC tissues. Remarkably, when we 590 \nanalysed changes to gene expression due to alte red splicing events, we discovered that 591 \nmost of the changes affected splicing of mRNAs encoding proteins involved in cilia structure 592 \nand function. Interestingly, tissue-specific alter native splicing of cilia-related RNAs underlies 593 \na wide range of ciliopathies [59]. This suggests that correct alternative splicing is key to 594 \nproduction of normal cilia proteins. Indeed, when we examined the morphology of the air-595 \nliquid interface cultures following HRV16 infection we found a blunting and loss of cilia 596 \ncompared to mock infected tissues. We propose that a combination of transcriptional down-597 \nregulation of cilia-related genes together with specific changes in splicing of cilia-related 598 \nmRNAs could lead to malformed or damaged cili a. These morphological changes could lead 599 \nto increased susceptibility to recurrent HRV infections and/or the development of chronic 600 \ninflammatory disease. 601 \nThere are several limitations to this study. Most importantly, the analyses were carried out 602 \nusing HBECs from a single donor. We cannot rule out the possibility that data from this 603 \ndonor may not reflect the general population. We studied virus infection of HBEC tissues at 604 \n48 hours post infection, and transcriptomic changes  could be significantly different at other 605 \ntime points. As we only analysed transcriptomic changes in bronchial epithelial cells, a 606 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n27 \n \ncomparison of these changes between bronchial and nasal epithelial cells, grown as fully 607 \ndifferentiated 3D tissues would have been interesting to perform. It is known that HRV 608 \ninfection causes redistribution to the cytoplasm of hnRNP proteins, known antagonists of SR 609 \nproteins in alternative splicing [73], and ec topic expression of the HRV16 3C protease has 610 \nalso been shown to mislocate SRSF2, a splicing factor which we have not studied, in the 611 \nnucleus of transfected cells. This means that changes in SR protein phosphorylation may 612 \nonly contribute to, rather than directly caus e, the changes in splicing induced by HRV16 613 \ninfection. Finally, examination of changes in expression of SRPK1, and its effects on its 614 \nsubstrates, and any impact on ciliated epithelial cell function in HRV-C infected cells would 615 \nbe important to consider in the future since HRV-C infection can have more serious 616 \nconsequences, clinically. 617 \n 618 \nConclusions 619 \nThis study has revealed the cellular kinase SRPK1 as a new restriction factor for HRV16 620 \ninfection in epithelial cells. The mechanism of restriction is not known.  However, since 621 \nSRPK1 can activate production of interfer on, interferon response factors and certain 622 \nchemokines [28], induction of innate immuni ty to viral infection seems likely. Indeed, 623 \ntranscriptomic analysis showed up-regulation by HRV16 of many innate immune factors. 624 \nHowever, a key finding of this study is that HRV16 infection alters expression of splicing 625 \nfactors and their phosphorylation. Importantly, we found major changes in alternative splicing 626 \nof RNAs encoding structural components of cilia. This suggests a molecular mechanism by 627 \nwhich HRV infection might result in damage to the mucociliary compartment which would 628 \nresult in inefficient clearance of subsequent viral infection enabling infection to move to the 629 \nlower airways, which would lead to exacerbations. 630 \n 631 \nAcknowledgements 632 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n28 \n \nThis work was funded by a BBSRC Industrial Case CSV Training Award no. BB/R505341/1. 633 \nQuan Gu is supported by an MRC award #MC_UU_00034/5. We are grateful to Chris 634 \nMcRae at Astra Zeneca for facilitating agreem ents between Astra Zeneca and the University 635 \nof Glasgow and for arranging the industrial pl acement for student Chris Rozario (C.R.) in 636 \nAstra Zeneca. We thank especially Jenny Horndahl, at AstraZeneca, Sweden for hosting 637 \nC.R. during his industrial placement. We would like to thank the staff at the University of 638 \nGlasgow’s Veterinary Diagnostics service for carrying out paraffin embedding, sectioning 639 \nand staining of tissues. Prof Carl Goodyear, Director, the GLAZgo Discovery Centre helped 640 \narrange the studentship and provided critical feedback throughout the study. 641 \n 642 \nDeclaration of Interests  643 \nProf Maciewicz is retired from Astra Zeneca but  at the start of the project she owned shared 644 \nin Astra Zeneca. Astra Zeneca had no input into the design of the study or interpretation of 645 \nthe data. 646 \n 647 \nAuthor contributions 648 \nC.R. Investigation, formal analysis. writing, original draft 649 \nQ.G. Investigation, formal analysis, data curation, visualisation. 650 \nA.S. Data curation, resources. 651 \nR.M. Conceptualisation, supervision, funding acquisition, writing review and editing. 652 \nS.V.G. Conceptualisation, supervision, funding ac quisition, visualisation, writing review and 653 \nediting. 654 \n  655 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n29 \n \n 656 \nPrimer name Sequence \nGAPDH Forward 5’-GAAGGTGAAGGTCGGAGT–3’ \nGAPDH Reverse 5’-GAAGATGGTGATGGGATTTC-3’ \nGAPDH Probe 5’-FAM-CAAGCTTCCCGTTCTCAGCC–TAMRA–3’ \nHRV16 Forward 5’-CCCTGAATGTGGCTAACCTT–3’ \nHRV16 Reverse 5’-ACGGACACCCAAAGTAGTTG–3’ \nHRV16 Probe 5’-FAM-ACAATCCAGTGTGTAGCTGGTCGT-TAMRA–3’ \nSRPK1 Forward 5’-ACCCTCCAGGAATCTCTACTT-3’ \nSRPK1 Reverse 5’-CCATGCTTTGTTCATGCCTAT-3’ \nSRPK1 Probe 5’-FAM-ACTTCACCCTCTTGGGCCTTTCAT-BHQ-3’ \n 657 \nTable 1. List of primers and probes used in RT-qPCR experiments. 658 \n 659 \n  660 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n30 \n \nLog2-fold change Gene name Gene function \n7.434 CXCL11 Chemokine \n7.434 ZBP1 Innate sensor \n7.223 IFNL1 Antiviral response \n6.268 CXCL10 Chemokine \n6.101 IFI6 Induced by interferon \n6.085 OASL Innate immunity to viral infection \n5.971 IFNL3 Antiviral response \n5.912 IFNL2 Antiviral response \n5.705 ISG15 Innate immunity to viral infection \n5.631 MX2 Innate immunity to viral infection \n5.559 IFITM1 Induced by interferon \n5.423 IFIT1 Induced by interferon \n5.239 IFI27 Induced by interferon \n5.229 AIM2 Innate immunity to viral infection \n5.049 IFIT3 Induced by interferon \n5.043 EPSTI1 Induced by interferon \n5.004 CXCL9 Chemokine \n4.810 HERC5 Innate immunity to viral infection \n4.672 DUOXA2 Antiviral protein in  respiratory epithelial cells \n4.447 CMPK2 Innate immunity to viral infection \n4.370 CSF3 Controls production of granuolcytes \n4.303 RSAD2 Antiviral protein \n4.257 OAS3 Innate immunity to viral infection \n4.238 XAF1 Induced by interferon \n4.141 MX1 Innate immunity to viral infection \n4.121 BST2 Innate immunity to viral infection \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n31 \n \n4.007 HERC6 Innate immunity to viral infection \n3.938 OAS2 Innate immunity to viral infection \n3.796 HLA-F Major histocompatability protein \n3.768 IFI44 Induced by interferon \n3.404 IFITM3 Induced by interferon \n 661 \nTable 2. List of top 30 immune-related genes up-regulated by HRV16 infection. 662 \n  663 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n32 \n \nFigure legends 664 \nFigure 1. Changes in SRPK1 levels and activity alter HRV16 infection in primary 665 \nhuman bronchial epithelial cells.  A. RT-qPCR analysis of tissue-associated HRV16 RNA 666 \nlevels in untreated, SRPIN340-treated and (SRPIN340) SRPK1 overexpressing (SRPK1 OE) 667 \nHBECs infected with HRV16 (MOI=3) for 16 hours. The Log2 ΔΔ Ct values were calculated 668 \nusing the values of the housekeeping gene GAPDH as the control target. B. TCID 50 669 \nquantification of tissue-released infective virus using supernatants from untreated, 670 \nSRPIN340-treated (SRPIN340) and SRPK1 overexpressing (SRPK1 OE) HBECs infected 671 \nwith HRV16 (MOI=3) for 48 hours. C. Western blot analysis of the HRV16 capsid proteins 672 \nVP0/VP2 expression in mock-treated and mock-treated SRPIN340-treated or SRPK1 673 \noverexpressed (OE) cells infected with HRV16 (MOI=3) for 48 hours. A GAPDH loading 674 \ncontrol is shown under the virus capsid protein blot. D. Quantification of western blot data 675 \nfrom three separate experiments. All data show the mean and standard error from three 676 \nseparate experiments. ns=non-significant stat istical difference, *=p<0.05, **=p<0.005, 677 \n***=p<0.0005  (student’s t-test, 2-tailed). 678 \n 679 \nFigure 2. HRV16 infection of primary huma n bronchial epithelial cells downregulates 680 \nexpression of SRSF3 and decreases phosphorylation of SRSF1 and SRSF6. 681 \nQuantification of  western blot expression data relative to GAPDH expression of A. total 682 \nSRSF1, B. total SRSF3, C. total SRSF6, D. phosphorylated SRSF1, E. phosphorylated 683 \nSRSF3, and F. phosphorylated SRSF6 in mock-infected (Control) and HRV16-infected 684 \n(HRV16: MOI=3) HBECs at 4, 16, 24 and 48 hours post-infection. The data show the mean 685 \nand standard error of the mean from three separate experiments. Significant statistical 686 \ndifference, *=p<0.05, **=p<0.005, ***=p<0.0005  (student’s t-test, 2-tailed). 687 \n 688 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n33 \n \nFigure 3. HRV16 infection in 3D cultured primary bronchial epithelial tissues 689 \nsignificantly decreases the phosphorylation of SRSF proteins.  A. Western blot analysis 690 \nof levels of SRPK1 and phosphorylated SRSF1, SRSF3, and SRSF6 (detected with 691 \nMab104) in mock-infected (Mock) and HRV16-infected (HRV16) HBEC air-liquid interface 692 \ncultures at 48 hours post-infection (hpi) with 3x10 6 pfu HRV16. GAPDH is shown as a 693 \nloading control and was used to determine relative proteins levels. B. Quantification of data 694 \nfrom three separate experiments. The data s how the mean and standard error of the mean 695 \nfrom three separate experiments. ns=no signifi cant statistical difference. Significant 696 \nstatistical difference,  *p<0.05, **p<0.005 (student’s t-test, 2-tailed). 697 \n 698 \nFigure 4. HRV16 induced changes in gene expression during infection in 3D cultured 699 \nprimary bronchial epithelial tissues.  3D tissues were cultured at the air-liquid interface for 700 \n4 weeks, then mock infected or infected apically with 3x10 6 pfu HRV16 for 48 hours. RNA 701 \nsequencing was performed on three replicates for each condition. A. Volcano plot of 702 \ndifferentially expressed genes (DEGs) (scatte red dots, n=4034) between infected and mock-703 \ninfected samples (FDR<0.05). The x-axis is the log2 fold change (infected/controls) while the 704 \ny-axis is the -log(FDR) calculated value. Black dots indicate the DEGs with log2 fold change 705 \nwithin the range of -1 to 1. Red dots indicate DEGs with changes less than -1 or greater than 706 \n1. B. Violin plot comparing the distributi on of gene expression between mock-infected (Ctrs) 707 \nand infected (HRV16) tissues. C. Heatmap of the top 20 upregulated and downregulated 708 \ngenes in infected (HRV) and mock-infected (CTR) samples. Each row represents 1 of 3 709 \nreplicates per condition. The color scale ranges from deep purple (no expression) to yellow 710 \n(high level expression). D. Gene networks induced during HRV16 infection of HBECs in air-711 \nliquid interface culture. PPI network functional enrichment analysis (generated using 712 \nSTRING) using DEGs with log2-fold changes of greater than 2 or less than -2. Nodes 713 \nrepresent the interacting proteins with lines r epresenting direct links. A colour key shows the 714 \ndifferent networks. Up to four colours per node are shown. 715 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n34 \n \n 716 \nFigure 5. HRV16 infection in 3D cultured primary bronchial epithelial tissues leads to 717 \nchanges in the splicing of cilia-related genes. A. Pie chart of the relative proportions of 718 \nthe various splicing events identified in 1469 single alternative splicing events identified 719 \nusing SplAdder. B. Pathway analysis of the si gnificantly differentially spliced genes during 720 \nHRV16 infection in primary bronchial epithelial 3D cultures (p<0.05, student’s t-test, 2-tailed). 721 \n 722 \nFigure 6. Representative changes in cilia-related RNA splicing using Sashimi plots. A. 723 \nDiagram of the structure of a cilium showing the positions of the proteins encoded by the 724 \nRNAs shown in B-D (generated using biorender: https://biorender.com). B-D. Sashimi plots 725 \nof read coverage across genes encoding B. RSPH1, C. IFT74, and D. TMEM67. Dark blue 726 \nlines, read coverage from mock-infected air- liquid interface cultures. Green lines, read 727 \ncoverage from HRV16-infected air-liquid interface cultures. Blue lines and black arrows 728 \nbeneath the Sashimi plots indicate skipped exons. 729 \n 730 \nFigure 7. Cilia are reduced in number and in length due to HRV16 infection in 3D 731 \ncultured primary bronchial epithelial tissues.  A. H&E-stained 3D air-liquid interface 732 \nculture grown for 4 weeks of mock-infected HBECs. A magnification of the upper surface of 733 \nthe tissue is shown above the main image. B. H&E-stained 3D air-liquid interface culture of 734 \nHBECs grown for 4 weeks then infected with 3x10 6 pfu HRV16 for 48 hours. Scale 735 \nbars=50µm. A magnification of the upper surface of the tissue is shown above the main 736 \nimage. C. Graph of quantification of cilia numbers in mock-infected and HRV16-infected 3D 737 \ncultures. D. Average cilium length on mock-infected and HRV16-infected 3D cutlures. 738 \nCounts were taken from five sections from each of three replicate tissues at 16, 24 and 48 739 \nhours post-infection. The data show the mean and standard error of the mean. Significant 740 \nstatistical difference, *p=<0.05, **p,<0.005, ***p<0.0005 (students t-test, 2-tailed). 741 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n35 \n \n 742 \nFigure 8. HRV16 infection of 3D cultured primary bronchial epithelial tissues leads to 743 \ndecreased expression of the cilia proteins β -tubulin and TMEM67. Fluorescence 744 \nconfocal microscopy image of HBEC 3D air-liqui d interface cultures grown for 4 weeks using 745 \nan antibody to detect β -tubulin in A. mock-infected (Control) and C. HRV16-infected 746 \n(HRV16: 3x10 6 pfu) tissues at 48 hours post-infection. B & D. The same images but with 747 \nDAPI staining added to show the cell nuclei in the tissues. Fluorescence confocal 748 \nmicroscopy image of an HBEC 3D air-liquid interface culture grown for 4 weeks using an 749 \nantibody to detect TMEM67 in E. mock-infected (Control) and G. HRV16-infected (HRV16: 750 \n3x106 pfu) tissues at 48 hours post-infection. F & H. The same images but with DAPI staining 751 \nadded to show the cell nuclei in the tissues. Scale bars=20µm. I. Graph of the quantification 752 \nof staining intensity of the β -tubulin antibody over time in mock-infected (Control) and 753 \nHRV16-infected (HRV16) 3D HBEC cultures. J. Graph of the quantification of staining 754 \nintensity of the TMEM67 antibody over time in mock-infected (Control) and HRV16-infected 755 \n(HRV16) 3D HBEC cultures. The data show the mean and standard error of the mean from 5 756 \nsections from three separate air-liquid interf ace cultures. *p<0.01,  **p<0.001, ***p<0.0001 757 \n(student’s t-test, 2-tailed). 758 \n  759 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n36 \n \nReferences 760 \n 761 \n1. 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It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint \n\n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted August 26, 2025. ; https://doi.org/10.1101/2025.08.26.672366doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}