Cell lines and primary keratinocytes

The human immortalized, but non-transformed HaCaT keratinocyte cell line [25] was kindly provided by Dr. Petra Boukamp, Leibniz Institute for Environmental Research, Düsseldorf, Germany.

CaCo2 cells were purchased from Sigma-Aldrich, St. Louis, MO. HeLa cells were obtained from ATCC, Manassas, VA. Huh7 cells were kindly provided by Dr. Volker Thiel, University of Berne, Switzerland. All cell lines were cultured in DMEM/10% fetal bovine serum (FBS) for propagation, and in DMEM/5% FBS for viral infection studies. Absence of mycoplasma was confirmed by PCR using the PCR Mycoplasma Test Kit I/C (PromoKine, Heidelberg, Germany) on a monthly basis.

Human primary keratinocytes (HPK) were kindly provided by Dr. Hans-Dietmar Beer, University Hospital Zurich.

Establishment and culture of mouse intestinal organoids

Mouse intestinal organoids were kindly provided by Drs. Annika Hausmann and Wolf-Dietrich Hardt (ETH Zurich). They were isolated and cultured as previously described [26]. Briefly, the organoids were embedded in Matrigel (Chemie Brunschwig, Basel, Switzerland) domes, covered with Complete Intesticult medium (Stemcell Technologies, Vancouver, Canada) and cultured at 37 °C/5% CO2. The medium was partially replaced every 3–4 days, and the organoids were sub-cultured upon mechanical shearing in Gentle Dissociation Reagent.

Virus strains

HSV-1 was produced as described [7]. HSV-1 recombinant strain C12 expressing green fluorescent protein (GFP) under control of the major cytomegalovirus (CMV) promoter [27] was kindly provided by Dr. Stacey Efstathiou (University of Cambridge, UK). Encephalomyocarditis virus (EMCV) was kindly provided by Drs. Roman Spörri and Annette Oxenius (ETH Zurich, Switzerland). HCoV229E/GFP Gemini was kindly provided by Prof. Volker Thiel (University of Berne, Switzerland). In this virus, the open reading frame 4 had been replaced by the GFP cDNA [28]. Influenza Wisconsin (IAV WSN) strain was kindly provided by Dr. Silke Stertz (University of Zurich, Switzerland).

Lactate dehydrogenase (LDH) activity quantification

LDH activity in the supernatant of treated cells was measured using the CyQUANT™ LDH Cytotoxicity Assay (C20300, Invitrogen, Waltham, MA) according to the manufacturer`s protocol. As per the protocol, addition of 10 µL water to the cells was used as negative control and addition of 10 µL lysis buffer provided with the kit was used as a positive control. The values for those were set to 0% and 100%, respectively, and used to calculate cytotoxicity levels in the test samples according to the manufacturer`s instructions.

Tyrosine kinase inhibitors

The following inhibitors were used (all from Selleckchem, Houston, TX): FGFR1/2/3 inhibitors AZD4547 (S2801), BGJ398 (NVP-BGJ398; S2183), Erdafitinib (JNJ-42756493, S8401), Debio 1347 (CH5183284, S7665), FGFR4 inhibitor Roblitinib (Roblitinib-FGF401, S8548,) and pan-Src kinase inhibitor AZD0530 (S1006). All inhibitors were dissolved in dimethyl sulfoxide (DMSO), and DMSO (vehicle) was used as control in all experiments. FGFR inhibitors were generally used at a concentration of 3.6 μm (BJG398) or 5 to 10 µM (all other inhibitors), unless indicated otherwise in the text and figure legends.

Lyn A kinase activity assay

Lyn A kinase was dissolved according to the manufacturer`s protocol for the Lyn A Kinase Enzyme System (Promega, Madison, WI; VA7476) to a final concentration of 10 ng per reaction. The enzymatic activity of Lyn A was measured in the presence of vehicle or inhibitors using the ADP-Glo™ Assay (Promega, V6930) following the manufacturer`s protocol. For each measurement, a standard curve with 100%, 60%, 20%, 5%, 3% and 0% conversion rate was made, using the provided ADP and ATP. The final concentrations in the reaction mix were 10 µM ATP and 0.2 µg/µl poly (Glu4, Tyr1) substrate. The reaction was performed for 1 h at room temperature (RT), then ADPGlo reagent was added for 30 min at RT, and the kinase reaction buffer was added for 40 min, followed by luminescence detection.

Isolation of RNA and qRT-PCR

Total RNA was isolated from cultured cells using the IBI Scientific RNA isolation kit (IBI Scientific, Dubuque, IO; IB47303) following the manufacturer`s protocol. cDNA was synthesized using the iScript kit (Bio-Rad Laboratories, Berkeley, CA). Relative gene expression was determined using the LightCycler 480 SYBR Green system (Roche, Rotkreuz, Switzerland). Expression of the gene encoding ribosomal protein lateral stalk subunit P0 (RPLP0) was used for normalization of expression levels. The following primers were used:

RPLP0

5’-CCA CAT TGT CTG CTC CCA CA-3’ and 5’-GAA GAC AGG GCG ACC TGG AA-3’.

RSAD2

5’-GGA GGT GGT GCA GGG ATT AC-3’ and 5’-GGA AAA CCT TCC AGC GCA CA-3’.

ISG15

5’-CTT TGC CAG TAC AGG AGC T-3’ and 5’-GAC ACC TGG AAT TCG TTG C-3’.

ANXA2

5’-TGT GCA AGC TCA GCT TGG A- 3’and 5’-AGG TGT CTT CAA TAG GCC CAA- 3’.

STAT1

5’-AAA GGA AGC ACC AGA GCC AAT- 3’ and 5’-TCC GAG ACA CCT CGT CAA AC- 3.

IFIT1

5’-ATT TAC AGC AAC CAT GAG GAA AG- 3’ and 5’-GCT CCA GAC TAT CCT TGA CCT G- 3’.

NECT1

5’-CTA CCA CAT GGA CCG CTT CAA G- 3’ and 5’-CTT TGC AGG TGA GCT TCA CGT C- 3’.

IFNB1

5’-TGG GAG GAT TCT GCA TTA CC- 3’ and 5’-CAG CAT CTG CTG GTT GAA GA- 3’.

MXB

5’-TCT GTC ACT ATC AGT GTC CAT CTC TAC- 3’ and 5’-TCT TTG CTT TAT TAA ATT CCT CTT CAA- 3’.

RNA-Seq and data analysis

HaCaT cells were cultured in DMEM/10% FBS until they reached 90-100% confluency, and treated for 5 h with DMSO, 10 µM AZD4547 or 3.6 µM BGJ398 in DMEM/5% FBS. Total RNA was isolated as described above. RNA quality and concentration were checked using a NanoDrop spectrophotometer. RNA-seq and data analysis were performed as previously described [29]. Pathway analysis was performed based on the significantly regulated genes (p ≤ 0.05, false discovery rate (FDR) ≤ 0.1 and log2FC ≥ 1) using Ingenuity Pathway Analysis (IPA) software, Version 26,127,183 (Qiagen, Hilden, Germany) and the built-in right-tailed Fisher Exact Test with Benjamini Hochberg (BH) multiple testing correction.

Viral cell association assay

HaCaT cells were cultured in DMEM/10% FBS and seeded on cover slips. Cells were incubated with 4.7 × 105 FFU/ml WT HSV-1 (F) in RPMI medium with 20 mM HEPES and 0.2% bovine serum albumin (BSA) for 1 h on ice to allow for viral binding, but not entry. Cells were then washed twice with cold RPMI, and the medium was replaced with DMEM/5% FBS containing vehicle (DMSO) or inhibitors. Cells were incubated for 1 h at 37 °C to allow viral entry, fixed with 4% paraformaldehyde (PFA) and stained for HSV-1 with a rabbit anti-heavy chain polyclonal antibody against purified DNA-containing HSV-1 capsids (kind gift by R. Eisenberg and G. Cohen, University of Pennsylvania, Philadelphia, PA). Cells were counterstained with DAPI and Alexa Fluor 647 NHS ester (Thermo Fisher Scientific, Waltham, MA; A200006).

After mounting coverslips on slides, the samples were imaged with a Leica SP8 inverse FALCON confocal laser scanning microscope using a 63x magnification oil objective. Signal was quantified using CellProfiler version 4.2.1, and data sorting was performed with KNIME version 4.5.1. For quantification of virus signal, nuclei were segmented based on the DAPI channel, and the resulting nuclear mask was expanded or shrunk by 10 pixels. Virus signal was quantified over the expanded (at 0 min pi) or shrunk (at 60 min pi) nuclear area.

Staining of viral particles in 96-well plates or on cover slips

After fixation, cells were treated with 25 mM NH4Cl for 10 min, washed with PBS, permeabilized with Triton X-100 for 5 min, and washed again. Blocking was done with 5% goat serum for 30 min at room temperature (RT), followed by overnight incubation with the primary antibody at 4 °C. The next day, after washing with PBS, cells were stained with the secondary antibody (anti-rabbit Alexa-488) for 1 h at RT. When staining for viral cell association experiments, cells were also stained with Alexa-Fluor 647 NHS ester (Thermo Fisher Scientific, Waltham, MA; A200006) during the last washing step to visualize the cell body. The following antibodies were used: mouse anti-nucleoprotein HB65 of the Influenza strain Wisconsin (kindly provided by Dr. Yohei Yamauchi, ETH Zurich, Switzerland) and a rabbit anti-heavy chain polyclonal antibody against purified DNA-containing HSV-1 capsids (kindly provided by Drs. Robert Eisenberg and Gary Cohen, University of Pennsylvania, Philadelphia, PA) [27].

Isolation of genomic and viral DNA from HSV-1 infected cells and from cell supernatant

Genomic and viral DNA were obtained from infected cells as previously described [30]. Viral DNA from the supernatant was isolated using the same procedure, but instead of a cell lysate, a mix of 50 µl supernatant and 100 µl lysis buffer was used in the first step. Samples were used for quantitative PCR to measure HSV-1 viral load. Primers for amplification of the human β-actin (ACTB) DNA (5’-TAC TCC TGC TTG CTG ATC CAC-3’ and 5’TGT GTG GGG AGC TGT CAC AT-3’) and the viral glycoprotein D (GlycD) DNA (5’ ACGTCCGGAAACAACCCTAC-3’ and 5’- CCCAGGTTATCCTCGCTGAC-3’) were used. For quantification of viral load in the supernatant, only GlycD DNA was measured, because no genomic cellular DNA was detectable in the supernatant.

EMCV viral burden analysis

Mouse intestinal organoids were harvested using cold phosphate-buffered saline (PBS) containing 0.1% BSA. The harvested organoids were centrifuged at 300 g twice for 5 min and twice for 2 min to gradually remove the matrigel and to obtain a pellet that includes only the organoids. Once the purified pellet was obtained, RNA was isolated from the organoids as described above. To obtain enough material, organoids from 2 to 3 wells were pooled. Relative viral load was assessed by qRT-PCR using the following primers:

EMCV-3D: 5`GACGCTTGAAGACGTTGTCTTCTTA-3`, 5`-CCCTACCTCACGGAATGGGGCAAAG-3` human ACTB; 5`-TACTCCTGCTTGCTGATCCAC-3`, 5`-TGTGTGGGGAGCTGTCACAT-3`.

Infection experiments

For all experiments, cells (HaCaT, CaCo2, Huh7, HeLa cells) were cultured in DMEM/10% FBS until they reached 90–100% confluency. HPKs were cultured in Keratinocyte-SFM medium (Gibco, Carlsbad, CA) with supplements for keratinocytes (Gibco; epidermal growth factor (EGF) #10450-013, bovine pituitary extract # 13028-014) and 10% FBS. For infection with HSV-1-GFP, cells were pre-treated overnight (ON) or for 1 h with the respective concentrations of inhibitor, while control cells were treated similarly, but with DMSO (vehicle) only. No pre-treatment with the inhibitors was performed for HSV-1 infection, unless stated otherwise. Pre-treatment of the cells and infection were done in DMEM/5% FBS. Infection with HSV-1-GFP was done at the described concentrations for 1 h at 37 °C unless stated otherwise in the figure legend, then the virus was washed away with sterile PBS, and cells were incubated in the presence or absence of inhibitors at 37 °C. Cells were fixed with 4% PFA at different time points post infection. For experiments where viral burden was assessed by extraction of gDNA followed by qPCR, cells were infected with HSV-1, but the virus was not removed until the time of cell lysis. Prior to lysis, cells were washed twice with PBS. An exception were experiments designed to measure the amount of viral DNA in the supernatant in which the virus was removed 1 h after infection, and cells were incubated in DMEM/5% FBS with DMSO or inhibitors.

For HCoV-229E/GFP infection, cells were pre-treated, then infected, and further incubated in the presence of the virus unless stated otherwise. Infected cells were incubated at 33.5 °C.

For Influenza Virus infection, cells were cultured in DMEM with 1% non-essential amino acids and 1% penicillin-streptomycin (both from Sigma-Aldrich).

Quantification of viral infection

After fixation with 4% PFA in PBS, cells were permeabilized, and nuclei were stained with 1 µg/ml DAPI in 0.5% Triton X-100 in PBS for 5 min at RT. Samples were washed with PBS and imaged in a high-throughput microscope (IXM-XL or IXMc; Molecular Devices, San Jose, CA) in widefield mode. A 4x objective was used for plaque assays and a 20x objective to observe single-round infection. For the quantification of infection with CellProfiler [31], nuclei were segmented according to the DAPI signal, and the GFP signal over the nuclear mask was measured. For plaque assays, the number and size of the plaques were determined based on the GFP signal using the Plaque2.0 software [32]. Uninfected cells were used to distinguish between background fluorescent signal and signal resulting from viral plaques. No plaques were detectable in these cells.

Live imaging of viral infection

For live cell imaging, cells were seeded in a flat bottom black 96-well plate (Greiner Bio-One, Kremsmünster, Austria) and incubated at 37 °C/5% CO2. On the next day, cells were treated with inhibitors and infected as described in the figure legends. After incubation with virus, the inoculum was removed and replaced with imaging media consisting of phenol red-free DMEM (Thermo Fisher Scientific) supplemented with 1% penicillin-streptomycin (Sigma-Aldrich), 1% L-glutamine (Sigma-Aldrich), 1% non-essential amino acids (Sigma-Aldrich), and 1 mM sodium pyruvate (final concentration), 250 ng/ml Hoechst 33,342 (Sigma-Aldrich) and 5% FBS. Live imaging was performed using an IXM-C fluorescence microscope (Molecular Devices, San Jose, CA) using a 4x objective. The plate was monitored every 15–30 min for a total time of 60 to 70 h.

Analysis of kinome data

Data from a kinome analysis of AZD4547-treated HaCaT cells [33] were analyzed with the BioNavigator v6.3.67 software and public databases (PhosphoNet, published for in vitro or in vivo experiments, or Kinexus), as described previously [34].

Western blot analysis

Cells were harvested in Laemmli 4x lysis buffer (16 g SDS, 48 ml Tris pH 6.8, 70 ml ddH2O, 80 ml glycerol), 1:1 diluted with ddH2O prior use. For analysis of pLyn (Y397) levels, cells were lysed in NP-40 lysis buffer (150 mM NaCl, 1% NP-40, 50 mM Tris pH 8.0) with protease inhibitors (Roche, 04 693 159 001) and phosphatase inhibitors (Roche, 04 906 837 001), placed on a rotator at 4 °C for 30 min, and centrifuged for 15 min at 13,800 g. The supernatant was transferred to new Eppendorf tubes, and lysates were stored at -80 °C. Protein concentrations were measured using BCA Protein assay (Thermo Fisher Scientific). Protein samples were then run on an SDS–PAGE and transferred to nitrocellulose membranes. After blocking of unspecific binding sites with 5% BSA (Chemie Brunschwig AG, Basel, Switzerland; PANP06-1391100), membranes were incubated with the primary antibody overnight at 4 °C (see below), followed by a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 h at RT (anti-rabbit or anti-mouse IgG, Sigma-Aldrich). Signals were developed using ClarityTM Western ECL Substrate (BioRad, Hercules, CA).

The following primary antibodies were used: pLyn Tyr397 (Invitrogen; MA5-38270; 1:1000 diluted), total Lyn (Invitrogen; MA5-14924; 1:1000 diluted), Annexin A2 (Invitrogen; PA5-27085; 1:1000 diluted); pAnnexin A2 Tyr24 (Invitrogen; PA5-105372; 1:1000 diluted), vinculin (Sigma-Aldrich; v4505; 1:500 diluted), phospho-p42/44 ERK1(2 (Cell Signaling, Danvers, MA; 9101; 1:1000 diluted), total ERK1/2 (Cell Signaling; 9102; 1:1000 diluted), phospho-p38 (Cell Signaling; 9211 S; 1:1000 diluted), total p38 (Cell Signaling; 9212 S; 1:1000 diluted), GAPDH (HyTest, Turku, Finland; 5G4; 1:1000 diluted), α-tubulin (Sigma Aldrich, T5168, 1:5000 diluted), histone H3 (Abcam, Cambridge, UK; ab1791; 1:2000 diluted), total EGFR (Cell Signaling; 373746: 1:1000 diluted), pEGFR-Tyr1068 (Cell Signaling; 3777; 1:1000 diluted), and HSV-1 glycoprotein D (Abcam, ab18638, diluted 1:5000).

Lyn knock-down in HaCaT keratinocytes

HaCaT cells were used at around 50–60% confluency. They were incubated ON with a mixture of Opti-MEM medium ® (Gibco), scrambled siRNA (Negative Control #1 siRNA, Thermo Fisher Scientific, 4390843) or a mix of two Lyn siRNAs (5’-GCG ACA UGA UUA AAC AUU AUU TT-3’ and 5’-GUG AUG UUA UUA AGC ACU AUU TT-3’) and Lipofectamine™ RNAiMAX (Invitrogen; 56532). The following morning, the medium was replaced by DMEM/10% FBS. For infection experiments, cells were infected 48–60 h post transfection.

Statistics

Statistical analysis was performed using the PRISM software, version 9 for Mac OS X or Windows (GraphPad Software Inc., La Jolla, CA). For comparison of two groups, an unpaired Student`s t test was performed; for comparison of more than two groups, Bonferroni multiple comparisons test was used.

Supplemental information

Supplemental Information: Fig. S1-S4 and legends, Table S1.

Videos S1 and 2: Live imaging of HSV-1-GFP infection of HaCaT keratinocytes, with or without AZD4547 treatment, related to Fig. 1.

Fig. 1

FGFR inhibitors suppress HSV-1 infection in different cell types. A: Area covered by viral plaques (plaque area) at 24 h post infection (hpi) of human primary foreskin keratinocytes (HPK) from four donors, infected with HSV-1-GFP (3 × 102 focus-forming units (FFU)/ml) for 1 h in the presence of 5 or 10 µM AZD4547 (AZD) or BGJ398 (BGJ; 3.6 µM) or vehicle (DMSO). B: Live cell imaging of HaCaT keratinocytes, which had been pre-treated overnight (ON) with AZD4547 (10 µM) or vehicle and infected with HSV-1-GFP (1.5 × 103 FFU/ml) for 1 h. Representative image shows the endpoint at 70 hpi, supplementary videos S1 and S2 show infection progression between 17 and 70 hpi. Magnification bar: 500 μm. C: Plaque number and area at 24 hpi of HaCaT keratinocytes, which had been pre-treated ON with AZD4547 (10 µM) or vehicle, and infected for 1 h with HSV-1-GFP (1.5 × 103 FFU/ml). Infection was quantified at 24 hpi. D: Plaque number and area as well as cell number in the whole well at 24 hpi of HaCaT keratinocytes, which had been pre-treated for 1 h with AZD4547, BGJ398, LY28744, Erdafitinib (Erda), Debio-1347 (Debio)) (5 µM each) or vehicle and infected with HSV-1-GFP (1.5 × 103 FFU/ml) for 1 h. E: qPCR for viral GlycD relative to host β-actin (ACTB) using DNA from HaCaT keratinocytes, which had been infected with HSV-1 (1.8 × 105 plaque-forming units (PFU)/ml) overnight in the presence of AZD4547, BGJ398, or Roblitinib (10 µM each) or vehicle. The expression of GlycD was normalized to host β-actin. AU: arbitraty units. F: Plaque number and area at 24 hpi of HaCaT keratinocytes, which had been treated for 1 h with vehicle (ctrl) or different concentrations of AZD4547 and infected with HSV-1-GFP (1.5 × 103 FFU/ml) for 1 h. G: qPCR for GlycD relative to ACTB (i) or Western blot (ii) for GlycD and vinculin (loading control) with quantification of the GylcD/vinculin ratio (iii) using DNA or protein lysates from CaCo-2 cells, which had been infected with HSV-1 (0.45 to 1.8 × 105 PFU/ml) ON in the presence of AZD4547 (10 µM), BGJ398 (3.6 µM) or vehicle (DMSO). H: qPCR for GlycD relative to ACTB using DNA from mouse embryonic fibroblasts (MEFs), which had been infected with HSV-1 (1.8 × 105 PFU/ml) ON in the presence of AZD4547 (10 µM), BGJ398 (3.6 µM) or vehicle (DMSO). Experiments in (B-G) were performed in the presence of DMEM/5% FBS, which contains FGFs. Bar graphs show mean +/- SD. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. A, D-H: One-way ANOVA, C: Unpaired t-test. N (biological replicates) = 2–3 from 2 experiments (A), N = 14 from 5 experiments (C), N = 14–15 from 3 experiments (D), N = 8–9 from 3 experiments (E), N = 8 from 2 experiments (F), N = 6 (qPCR) from 2 experiments or N = 3 (Western blot) from 1 experiment (G) and N = 8–9 from 3 experiments (H)