Cell lines and culture conditions. HEK293T (human embryonic kidney 293T), HeLa, and A549 cells were cultured in DMEM medium (Gibco) with 10% (vol/vol) fetal bovine serum (Gibco) and 1% glutamine (Gibco). Calu-3 cells were cultured in MEM (Gibco) with 1% NEAA (Gibco), 1% sodium pyruvate (Gibco) and 10% (vol/vol) fetal bovine serum (Gibco). Primary nasal epithelial cells (hNEPCs) were isolated from fresh nasal specimens by means of enzymatic digestion with Dispase II (Sigma) at 48 °C overnight. Biopsy specimens of nasal mucosa were obtained from patients with chronic rhinosinusitis (CRS) who underwent functional endoscopic surgery at the First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China. None of the patients had other systemic diseases and did not receive glucocorticoids, antibiotics, or both within 3 months before the study. Approval to conduct this study was obtained from the Institutional Review Board of the First Affiliated Hospital, Sun Yat-sen University. All patients provided written informed consent. A modified culture method based on the previous described36. To induce starvation, cells were washed with phosphate-buffered saline (PBS) and incubated in EBSS (Gibco). All cells were incubated at 37 °C incubator with 5% CO2.
Antibodies and reagents. Horseradish peroxidase anti-Flag (M2; A8592) and anti-β-actin (A1978) were purchased from Sigma. Horseradish peroxidase anti-hemagglutinin (HA; 12013819001) and anti-c-myc (Myc; 11814150001) were purchased from Roche Applied Science. Anti-ACE2 (21115-1-AP), anti-PIAS4 (14242-1-AP), anti-Beclin-1 (11306-1-AP), anti-ATG5 (10181-2-AP), anti-p62/SQSTM1 (18420-1-AP) and anti-TOLLIP (11315-1-AP) were acquired from Proteintech. K48-linkage specific polyubiquitin antibody (4289) and anti-ACE2 (4355) were purchased from Cell Signaling Technology. Anti-SENP3 (sc-133149), anti-SUMO1 (sc-5308), and anti-SUMO2/3/4 (sc-393144) were from Santa Cruz Biotechnology. Anti-CoV2-N (40588-T62) was acquired from Sino Biological. ML-792 (HY-108702) and 2-D08 (HY-114166) were from MedChemExpress (MCE). MG132 (C-2211), 3-methyladenine (M9281), bafilomycin A1 (H2714), and N-Ethylmaleimide (NEM) (E3876) were purchased from Sigma.
Plasmids and siRNA Transfection. Constructs coding for ACE2 were cloned in the pcDNA3.1 vector for transient expression and into the FG-EH-DEST (provided by Xiaofeng Qin laboratory) for retroviral expression. HEK293T transfection was performed using Lipofectamine 2000 (Invitrogen) according to procedures recommended by the manufacturer. Chemically synthesized 21-nucleotide siRNA duplexes were obtained from Sangon Biotech and transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. siRNA sequences were:
Scramble (Scr) siRNA: 5′-GUGAGCGUCUAUAUACCAUTT-3′;
PIAS4 siRNA #1: 5′-GGAGUAAGAGUGGACUGAATT-3′;
PIAS4 siRNA #2: 5′-GCUCUACGGAAAGUACUUATT-3′;
TOLLIP siRNA #1: 5′-CACACAAUGGCGCCAAGAAUCTT-3′;
TOLLIP siRNA #2: 5′-CGACUGAACAUCACGGUGGUATT-3′;
SENP3 siRNA #1: 5′-ACGUGGACAUCUUCAAUAATT-3′;
SENP3 siRNA #2: 5′-CAAUAAGGAGCUACUGCUATT-3′.
Generation of stable expression cell lines. For ACE2 ectopic expression, lentiviral particles were produced by transfecting HEK293T cells with FG-EH-DEST-ACE2-Puro, VSGV and Δ8.9. The medium was changed the following day and the viral containing supernatant was collected 48 hr after transfection, filtered through a 0.45 mm filter. A549 cells were infected by incubation with lentivirus-containing supernatant for 48 hr together with Polybrene (8 μg ml−1). Transduced cells were purified by puromycin selection. For SENP KO cells, target sequences were cloned into pLentiCRISPRv2 by cutted with BsmBI. The target sequences used were as follows:
SENP1 target: 5′-TCGGTCCAAATGTCCTTGCC-3′;
SENP2 target: 5′-GCACCGACCGGTCGCAGAAA-3′;
SENP3 target: 5′-GTGGCGTCGCACCCCCTTGC-3′;
SENP5 target: 5′-GGACCATCAAGAGACCCGTA-3′;
SENP6 target: 5′-GTAAGGTTAAGTCGGCTCCA-3′；
SENP7 target: 5′-GAGTCTAGTATAAGAATACA-3′;
SENP8 target: 5′-GGTCCATCTTGTACTGACGA-3′.
Quantitative RT-PCR. Total RNA was extracted from cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. For RT-PCR analysis, cDNA was generated with HiScript® II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, R223-01) and was analyzed by quantitative real-time PCR using the 2 × RealStar Green Power Mixture (GenStar). All data were normalized to RPL13A expression. The primer pair sequences were as follows: RPL13A Fwd, 5′-GCCATCGTGGCTAAACAGGTA-3′ and RPL13A Rev, 5′-GTTGGTGTTCATCCGCTTGC-3′; PIAS4 Fwd, 5′- TCAGATGCTCCTGGGTTTCGTG-3′ and PIAS4 Rev, 5′- TTCTTCTTGGCGTAGCGGGTC-3′; ACE2 Fwd, 5′- CTTTCCGTCTGAATGACAACA-3′ and ACE2 Rev, 5′- CACTATCACTCCCATCACAACTC-3′; SARS-CoV-2-NSP10 Fwd, 5′-CCCTGTGGGTTTTACACTTAA-3′ and SARS-CoV-2-NSP10 Rev, 5′-ACGATTGTGCATCAGCTGA-3′; SARS-CoV-2-N Fwd, 5′-GGGGAACTTCTCCTGCTAGAAT-3′ and SARS-CoV-2-N Rev, 5′-CAGACATTTTGCTCTCAAGCTG-3′; TNFA Fwd, 5′-CCTCTCTCTAATCAGCCCTCTG-3′ and TNFA Rev, 5′-GAGGACCTGGGAGTAGATGAG-3′; IFNL1 Fwd, 5′-GTGACTTTGGTGCTAGGCTTG-3′ and IFNL1 Rev, 5′-GCCTCAGGTCCCAATTCCC-3′; CXCL10 Fwd, 5′-GTGGCATTCAAGGAGTAGCTC-3′ and CXCL10 Rev, 5′-GCCTTCGATTCTTGGATTCAG-3′.
Immunoprecipitation and immunoblot analysis. For immunoprecipitation, whole-cell extracts were prepared after transfection or stimulation with appropriate ligands, followed by incubation overnight with the appropriate antibodies plus Protein A/G beads (Pierce). For immunoprecipitation with anti-Flag, α-FLAG M2 beads (Sigma) were used. Beads were then washed five times with low-salt lysis buffer (50 mM HEPES, 150 mM NaCl, 1mM EDTA, 10% glycerol, 1.5 mM MgCl2, and 1% Triton X-100), and immunoprecipitates were eluted with 2 × SDS Loading Buffer and resolved by SDS-PAGE. Proteins were transferred to PVDF membranes (Bio-Rad) and further incubated with the appropriate antibodies. Immobilon Western Chemiluminescent HRP Substrate (Millipore) was used for protein detection.
Mass spectrometry analysis. Total lysates were prepared from 293T cells transfected with Flag-ACE2 and followed by immunoprecipitation with α-FLAG M2 beads. Immunoprecipitated proteins were separated by SDS-PAGE, and then stained with Coomassie Blue. The entire lane was excised, digested with trypsin and analyzed with LC-MS/MS. LC-MS/MS identification of peptide mixtures was performed at Applied Protein Technology (aptbiotech, Inc. Shanghai, China). Briefly, peptides were chromatographed through the Easy-nLC 1000 system (Thermo Fisher, California, USA). Peptide samples were loaded by Thermo Scientific Acclaim PepMap100 (100 μm*2 cm, nanoViper C18) and separated by Thermo scientific EASY column (10 cm, ID75 μm, 3 μm, C18-A2) at 300 nL min−1 for 60 min using a three-step acetonitrile (0.1% formic acid in 84% acetonitrile) gradient: 0–35% over the first 50 min and 35–100% for 50–55 min and 100% for 55–60 min. The tandem mass spectrometry was performed by Q Exactive mass spectrometer (Thermo Fisher, California, USA). The MS1 survey scan (300–1800 m/z) was at a resolution of 70,000 at 200 m/z with automatic gain control (AGC) target of 1e6 and a maximum injection time of 50 ms. Dynamic exclusion was 60.0 s. Each full scan takes 20 MS2 scans. MS2 activation type was HCD model. Isolation window was 2 m/z. MS2 scan was at a resolution of 17,500 at 200 m/z with normalized collision energy 30 eV. Underfill was 0.1%. RAW files generated by spectrometer was subjected to Biopharma Finder1.0 software for protein identification.
SUMOylation assay and prediction of SUMOylation sites. For SUMOylation assays, 2 × 106 293T cells were plated in 10 cm dishes and transfected with a total of 7.5 μg of DNA the following day. 48 hr post-transfection cells were lysed in low-salt lysis buffer supplemented with deSUMOylation inhibitor NEM (20 mM) and complete protease inhibitors. Cell lysates were sonicated for 20 s then clarified at 4 °C at 12,000 g for 5 min. 20 μl of α-FLAG M2 beads was rotated with cleared protein lysates at 4°C for overnight. Furthermore, 4 × washes in low-salt lysis buffer, supplemented with NEM (20 mM) and complete protease inhibitors, were performed, and samples eluted by boiling in 15 μl 2 × SDS loading dye. For the prediction of putative SUMOylation sites in ACE2, GPS-SUMO, SUMOplot analysis (Abgent) software, JASSA and Ron Hay’s SUMO consensus motif search tool were used as previously described37.
Fluorescence microscopy. Cells were cultured on Glass Bottom culture dishes (Nest Scientific) and directly observed as previously described16. For examination by immunofluorescence microscopy, cells were fixed with 4% paraformaldehyde for 10 min, and then permeabilized in methyl alcohol for 30 min at −20 °C. After washing with PBS for 3 times, cells were blocked in 5% goat serum for 1 hr, and then incubated with primary antibodies diluted in 5% goat serum overnight. The cells were washed and followed by a fluorescently labeled secondary antibody (CF488A rabbit anti-HA IgG, Biotium; Rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, Invitrogen; Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, Invitrogen; Goat anti-mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 568, Invitrogen; Goat anti-rabbit IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 568, Invitrogen; Goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 633, Invitrogen). Confocal images were examined using a microscope (TCS SP8 STED 3X; Leica) equipped with 100 × 1.40 NA oil objectives. The images were processed for gamma adjustments using Leica AS Lite or ImageJ software (National Institutes of Health). The data plotted on the line intensity plots were produced by using the Plot Profile function of Image J on a single plane z-stack of confocal microscopy pictures.
Virus infection. Cells were either mock-infected or infected with SARS-CoV-2 (Accession number: MT123290), as previously described38, 39. Virus was allowed to adsorb at 37 °C for 1 hr with gentle rocking every 15 min before incubation at 37 °C for indicated time points. Cells were infected at different MOI and time points as indicated.
SARS-CoV-2 S pseudotyped virus. Pseudotyped particles were obtained from package of pHIV-Luciferase, psPAX2, and SARS-CoV-2 S protein expressing pcDNA3.1 vector (all these plasmids are gifts from Yi-Ping Li laboratory). The pseudovirions contain SARS-CoV-2 spike protein and the firefly luciferase gene. Therefore, the spike-mediated cell entry can be measured via luciferase reporter activity.
Immunofluorescence staining. Paraffin-embedded sections of lung biopsies were processed as reported previously40. Briefly, sections were deparaffinized, incubated with blocking buffer (PBS with 5% normal donkey or goat serum and 0.3% Triton X-100) at room temperature for 1 hr, and stained with primary antibodies overnight in a wet chamber at 4 °C in the dark. Sections were then washed with PBS, incubated with secondary antibodies (Goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488, Invitrogen and goat anti-mouse IgG (H+L) cross-adsorbed secondary antibody, Alexa Fluor 594, Invitrogen) for 1 hr at room temperature in the dark, and mounted with DAPI for nuclear staining. Immunofluorescent images were acquired with a Nikon Eclipse Ni-E microscope using a × 40 objective. Post-acquisition processing (brightness, opacity, contrast, and color balance) was applied to the entire image and accurately reflected the results of the original image. The protein expression levels were quantified in square micrometers by using ImageJ software.
RNA-seq analysis. Total RNA was isolated from cells using TRIzol reagent (Invitrogen), and sequencing was performed by Sangon Biotech. Sample quality was assessed using a Bioanalyzer (Agilent 2100 Bioanalyzer). RNA-seq libraries of polyadenylated RNA were prepared using mRNA-seq V2 Library Prep Kit and sequenced on MGISEQ-2000 platform. All clean data were mapped to the human genome GRCh38 using HISAT2 v2.1.0 with default parameters. Bam files were sorted by Samtools 1.9. Reads counts were summarized using the featureCounts program as part of the Subread package release 2.0.0 (http://subread.sourceforge.net/)41. To identify differentially expressed genes (DEGs) between groups, DESeq2 was used to normalize reads counts and p value < 0.05 and absolute logged fold-change ≥ 1 were determined as DEGs73 using Bioconductor clusterProfiler package v3.14.3 for the functional enrichment of DEGs42, 43. RNA-seq sequence density profiles were normalized using bamCoverage and visualized in IGV genome browser44.
Statistical analysis. Data from three or more experiments were displayed as mean ± SEM unless otherwise indicated, and data of immunofluorescent staining experiments are represented as mean ± SD of indicated image numbers. Student’s t-test was used for all statistical analyses with the GraphPad Prism 8 software. Differences between two groups were considered significant when P value was less than 0.05.