Materials and correspondence
Further information and requests for resources and reagents should be directed to and will be fulfilled by Mirko Trilling (Mirko.Trilling@uk-essen.de).
Cells, viruses, and infection
Vero E6 (ATCC CRL-1586) and Caco-2 (ATCC HTB-37) were grown in high-glucose Dulbecco’s minimal essential medium (DMEM [Gibco 41,966–029]) and Roswell Park Memorial Institute 1640 (RPMI-1640 [Gibco 21,875–034]), respectively, supplemented with 10% (v/v) FCS, penicillin, and streptomycin. Calu-3 (ATCC HTB-55) were cultivated in minimal essential medium (MEM [Gibco 31,095–029]) supplemented with 10% (v/v) FCS, 1 mM sodium pyruvate, penicillin, and streptomycin. All cells were kept at 37 °C in an atmosphere of 5% CO2. All cells were tested for mycoplasma contamination and only used when free of mycoplasma.
The SARS-CoV-2 strains B.1, B1.1.232, Alpha (B.1.1.7), Beta (B.1.351), Delta (B.1.617.2 and AY.6), and Omicron (BA.1) were isolated from patient samples obtained in May 2020, March to May 2021, and December 2021. For the isolation, permissive cells were incubated with virus-containing clinical nasopharyngeal swab samples until CPE was observed. All strains except Delta and Omicron were amplified in Vero E6 cells. The Delta isolates B.1.617.2 and AY.6 as well as the Omicron isolate BA.1 were isolated and amplified using Calu-3 cells. All SARS-CoV-2 isolates were analyzed by Next Generation Sequencing (data not shown) and classified with the help of GISAID and assigned into clades according to pangolin [68,69,70,71]. The authors made use of the outbreak.info . Viral titers were determined by 50% tissue culture infectious dose (TCID50) titration. The virus isolation has been approved by the ethics committee of the medical faculty of the University of Duisburg-Essen (20–9511-BO and 20–9512-BO). HSV-1-ΔgE-GFP was generated and described  by the laboratory of Prof. David C Johnson (Oregon Health & Science University, USA). With Prof. Johnson’s written permission, we received the virus from Prof. Hartmut Hengel (University of Freiburg, Germany).
Generation of aqueous infusions of herbs
The herbal infusions were prepared by boiling up 15 g of fresh herbal leaves in 100 ml of water and subsequent simmering at 60 °C for 2 h. The infusions were stored overnight at 4 °C before the leaves were removed and the aqueous solutions were sterile-filtered (200 µM filter, Whatman/GE Healthcare). Afterwards, the herbal infusions were stored in aliquots at − 80 °C. For the infusions based on dried herbs, 3 g of material per 100 ml was utilized. The 10-min infusion was prepared by boiling up dried sage leaves in water (30 g per liter) and subsequent incubation for 10 min before the herb was removed. Sterile-filtered aliquots were then stored at − 80 °C. Infusions of frozen and re-thawed herbal material were prepared in the same manner as those based on fresh herbs. The concentration of 150 g of herbal material per liter was calculated based on the fresh weight before freezing. The sources of supply for the herbal leaves and plants are as follows: coriander, sage and mint, farmer’s market (Essen, Germany); red and green perilla plants, online vendor Naturkraeutergarten (Kleinich, Germany); bi-color perilla plant, home-grown; dried red perilla, home-dried; dried green perilla, Keiko Shiso Finest Selection (Japan); dried sage and dried thyme, vom-Achterhof Bio-Salbei/Bio-Thymian (Uplengen, Germany), mint tea bags, Meßmer Pfefferminze (Germany).
For the quantification of viral protein amounts in infected cells, an icELISA was applied. A detailed icELISA protocol is provided in . Briefly, cells were infected with SARS-CoV-2 and fixed after 20 or 30 h of infection with 4% (w/v) paraformaldehyde/PBS. Cells were permeabilized with 1% (v/v) Triton-X-100/PBS and blocked with 3% (v/v) FCS/PBS. The primary antibody was added and incubated for 2 h at room temperature (RT) or overnight at 4 °C. Peroxidase-labelled secondary antibody was incubated for 1–2 h. Washing steps were performed with 0.05% (v/v) Tween-20/PBS. Tetramethylbenzidin (TMB) substrate was added to visualize the enzyme reaction. The reaction was stopped with 0.5 M HCl before the absorbance was determined using a microplate multireader and MicroWin software (Mithras2 LB 943; Berthold Technologies). The resulting data were analyzed using Excel and GraphPad Prism software. The α-S mAb (kindly provided by Peter Miethe, fzmb, Bad Langensalza, Germany), α-N mAb (ABIN6952435), and POD-coupled secondary antibodies (Dianova) were used.
Dose–response curves of antiviral activity
To enable comparison among different icELISA measurements and experiments, we included on every plate a virus calibration curve. Residual infectivity after treatment was calculated using the formula computed from the calibration curve (see Additional file 2: Fig. S1 as an example). Dose–response curves were compiled based on the relative change in infectivity compared to the untreated control.
Cells were infected with SARS-CoV-2 and fixed after 20 or 30 h of infection using 4% (w/v) paraformaldehyde/PBS for > 2 h before they were discharged from the BSL-3 laboratory. Cells were permeabilized with 1% (v/v) Triton-X-100/PBS and blocked with 3% (v/v) FCS/PBS. SARS-CoV-2 infection was visualized by use of α-S mAb (kindly provided by Peter Miethe, fzmb, Bad Langensalza, Germany) and Cy2-conjugated goat anti-mouse IgG (Dianova). Nuclei were counterstained with 4′6-diamidino-2-phenylindole (DAPI; Sigma). Fluorescence was visualized using a THUNDER Imager 3D Cell Culture (Leica). Image analysis and processing were performed with LAS X Premium imaging software (Leica).
Quantitative reverse-transcription PCR (qRT-PCR)
SARS-CoV-2 progeny was analyzed by quantification of viral RNA extracted from the culture supernatants of infected cells using the INSTANT Virus RNA/DNA Kit (Analytik Jena, Germany). Viral RNA was quantified by diagnostic qRT-PCR targeting the SARS-CoV-2 genes S and E (RealStar ® SARS-CoV-2 RT-PCR kit, Altona, Hamburg, Germany). In the case of Omicron, undiluted RNA preparations were used as template for qRT-PCR, for all other variants, a 1:50 dilution was used.
Interferons, inhibitors, substances, and size exclusion
Human IFNα2 was purchased from PBL Assay Science (#11101) and IFNβ from Peprotech (#300-02BC). Remdesivir and ruxolitinib were obtained from Cayman Chemicals (#30354) and Cell Guidance Systems (#SM87-10), respectively. The substances cinnamic acid, hydroxy-cinnamic acid, and dihydroxy-cinammic acid/caffeic acid were purchased from Sigma (#8002350250, #8002370050, #8220290010). Perilla aldehyde and perillyl alcohol were also obtained from Sigma (#W355704 and #218391). Size exclusion and protein fractionation of components of herbal infusions were conducted by use of Amicon 100 K, 30 K, 10 K, and 3 K filters (Sigma #UFC510024, #UFC501096, #UFC503096, #Z740183-96EA). The fraction of proteins > 1 kDa was obtained by dialysis using the Mini Dialysis Kit 1 kDa (Sigma #GE80-6483–94). All kits were applied according to the manufacturer’s instructions.
Sample preparation for MS
For protein extract preparation, 80–90% confluent cell monolayers of a T25 flask of Vero E6 or Caco-2 cells were harvested using a cell scraper. Medium and cells were transferred to a 15-ml tube and centrifuged for 3 min at 350 g. Pellets were washed twice with 10 ml PBS, resuspended in 400 µl of lysis buffer (50 mM Tris, 150 mM NaCl, 1% [w/v] SDS, pH 7.8 supplemented with one cOmplete mini EDTA-free protease inhibitor tablet as well as one PhosSTOP tablet [both Roche] per 10 ml volume of lysis buffer), and transferred into 1.5-ml tubes. Lysates were incubated for 30 min at 4 °C before they were subjected to virus inactivation at 70 °C for 10 min followed by 10 min at 95 °C. After ultrasonic treatment for 1 min, cell lysates were stored at − 80 °C until further analysis. Six microliters benzonase (27 units/µl, Merck) and 1 µl MgCl2 (2.5 mM final concentration) were added per sample, followed by incubation at 37 °C for 30 min. Protein concentration was determined by a bicinchoninic acid assay (Pierce), and 100 µg protein lysate per replicate (n = 5 for all analyzed conditions) was processed further as described below.
Positive pressure filter-aided sample preparation (FASP) in 96-well format
Cysteines were reduced using 10 mM dithiothreitol (Roche) at 56 °C for 30 min and alkylated in the presence of 25 mM iodoacetamide (Sigma) for 30 min at RT in the dark. Samples were diluted at least 1:4 in 8 M urea (Sigma-Aldrich) dissolved in 100 mM Tris–HCl, pH 8.5 (Applichem), and transferred to a 30-kDa AcroPrep Omega filter membrane plate (PALL, New York, USA, purchased via VWR, Hannover, Germany, REF 8035/518–0028) in a blocked randomized order, which was also kept for subsequent LC–MS measurements. The filter plate was placed on top of a 2.2-ml MegaBlock collection plate (Sarstedt, Nümbrecht, Germany) and the liquid of the protein solution was forced through the filter using a Resolvex A200 (Tecan, Männedorf, Switzerland) connected to nitrogen gas (N2, 5.5 bar, purity 4.8 or higher, Linde, Düsseldorf, Germany) using a relative pressure of 20% of the low profile setting. Subsequently, the dispensing function of the A200 was used to wash the filter twice with 200 µl 8 M urea buffer in Tris–HCl pH 8.5 and twice with 200 µl 50 mM ammonium bicarbonate (ABC, Fluka). After each washing step, the liquid was forced through the filter using the same pressure profile as for loading. Afterwards, the plate was centrifuged for 2 min at RT and 1000 g to remove residual drops under the membrane. For digestion, 100 µl digestion buffer were added comprising 100 mM urea, 50 mM ABC, and 2 mM CaCl2 (Merck) including sequencing-grade trypsin (Promega, sequencing-grade modified trypsin) in a concentration to meet a 1:3 (w/w) enzyme-to-sample ratio. After incubation for 16 h at 37 °C, the digested protein fraction was forced through the filter and collected in a 500-µl LoBind plate (Eppendorf, Hamburg, Germany). The filter was further washed with 100 µl 50 mM ABC followed by 100 µl H2O, both wash steps were also collected in the LoBind plate. Then, tryptic digestion was stopped by reducing the pH < 2 through the addition of trifluoroacetic acid (TFA; Biosolve, Valkenswaard, Netherlands) to a final concentration of 1% (v/v). Aliquots were transferred to 700 µl glass-vial plates (Waters, Eschborn, Germany) for injection on a monolithic column HPLC (for tryptic digestion quality control) . Based on the results, 4–5 replicates per condition were further analyzed by LC–MS.
LC–MS in data-dependent acquisition-mode (DDA)
LC–MS was conducted using an UltiMate 3000 RSLCnano ProFlow UPLC system operated by Chromeleon Client 6.80 and online-coupled to a Q Exactive HF MS operated by Tune application 2.8 SP1 and Thermo Scientific Xcalibur 3.0.63 (both Thermo Scientific, Dreieich, Germany, including HPLC columns). Employed solvents were LC–MS grade or higher (Biosolve, Valkenswaard, Netherlands). In total, 825 ng of tryptic peptides per LC–MS injection was analyzed. Samples were loaded on a trapping column (Acclaim PepMap C18, 0.1 × 20 mm, 5 µm, 100 Å) for 3 min in 0.1% TFA at a flow rate of 30 µl/min. Then, the trapping column was switched in line with the analytical column (Acclaim PepMap C18; 0.075 × 500 mm, 2 µm, 100 Å) and peptides were separated at a flow rate of 250 nl/min using a 102-min linear gradient of buffer B (84% v/v acetonitrile [ACN], 0.1% v/v formic acid [FA]) in buffer A (0.1% v/v FA) ranging from 3 to 25% B, followed by a 10-min linear gradient ranging from 25 to 35% B, washing steps and reconditioning of the analytical column to 3% B. Both columns were kept at 60 °C temperature.
The UPLC system was coupled to the Q Exactive HF MS via a NSI Source (Thermo). Coated emitters (Silica Tip, 20 μm inner diameter, 10 μm tip inner diameter, New Objectives, Woburn, MA) and a static voltage of 1.8 kV were applied for electrospray ionization, and ion transfer tube temperature was set to 250 °C. The MS was operated in data-dependent acquisition (DDA) mode at positive polarity; all spectra were acquired in profile mode with survey scans acquired at a resolution of 60,000 followed by 15 MS/MS scans at a resolution of 15,000 (top15). Precursor ions were selected for MS/MS by intensity, isolated in a 1.6 m/z window and subjected to fragmentation by higher-energy collision-induced dissociation using a normalized collision energy (NCE) of 27. Automatic gain control target values were set to 106 and 5 × 104 and the maximum ion injection was set to 120 ms and 50 ms for MS and MS/MS, respectively. Precursor masses were excluded from re-fragmentation for 20 s (dynamic exclusion).
Protein identification and relative quantification with Proteome Discoverer
DDA files were processed with Proteome Discoverer 2.4 (Thermo Scientific) using Spectrum Files RC and Sequest HT nodes as database search algorithm and Percolator  in conjunction with Peptide validator and Protein FDR validator nodes for adjusting the false discovery rate to 1% on PSM, peptide, and protein levels. Database search was conducted against Uniprot homo sapiens database for Caco-2 (UP000005640, 20,376 entries, retrieved in November 2019) or Chlorocebus sabaeus database for Vero E6 (UP000029965, 19,230 entries, retrieved in January 2021) in conjunction with the SARS-CoV-2 database (UP000464024, 14 entries, retrieved in January 2021) supplemented with reported putative novel ORFs , common contaminants, and an additional fasta file containing the amino acid sequence of indexed retention time peptides added as an internal standard  by the Sequest HT search engine with the following parameters: error tolerances of 10 ppm and 0.02 Da for precursor and fragment, trypsin (full) as enzyme with a maximum of two missed cleavage sites, oxidation of Met as variable modification (+ 15.995 Da) and carbamidomethylation of Cys (+ 57.021 Da) as fixed modification. Full settings are provided as additional material via ProteomeXchange. Quantification was performed using the Minora feature detector node in conjunction with the Feature mapper node with a maximum retention time shift of 10 min and mass tolerance of 10 ppm for chromatographic alignment and minimum signal to noise threshold of 10 for feature linking and mapping. Precursor Ions Quantifier node accepted only unique peptides for quantification while considering protein groups for peptide uniqueness with disabled scaling and normalization to total peptide amount. Sample abundances of the connected peptide groups were summed to calculate protein abundances. Common contaminant proteins and proteins with less than two unique peptides per protein group were filtered out. Normalized protein abundances were used for further data analysis.
Analysis of MS data
For all analyses, only proteins with a maximum of one missing replicate value per respective condition were considered. Lightweight normalizing and testing tool (LNTT)  was used for the creation of volcano plots in Fig. 5 and Additional file 11: Fig. S10. Proteome Discoverer normalized (total protein normalization) data were filtered (at least two unique peptides per protein group required) and proteins with a coefficient of variation < 20.0% over all measurements (also across treatments) were removed. Independent two-sided two-sample Welch’s t-test was performed for remaining proteins. Computed p values were adjusted using the Benjamini and Hochberg multiple test correction (https://0-www-jstor-org.brum.beds.ac.uk/stable/2346101) and results were plotted by LNTT. Gene names for labeling were retrieved by accession numbers using Uniprot API. For heatmap analysis, only proteins quantified across all conditions and time points were considered for Vero E6 and Caco-2 samples. Displayed order of the proteins was sorted accordingly to the mean value for the respective 6 h p.i.
Statistical significance was determined using one-way ANOVA as described in the figure legends. A p value of < 0.05 was considered statistically significant. *, p value < 0.05. **, p value < 0.01. ***, p value < 0.001. Calculation of p values for analysis of MS data are described above. IC50 values were calculated using GraphPad Prism by nonlinear regression.