Methods

Surfaceome CRISPR Screen Identifies OLFML3 as a Rhinovirus-inducible IFN Antagonist

Design and construction of genome-wide and surfaceome CRISPR libraries

sgRNAs were designed to target to protein coding regions (NCBI CCDS data, released on 8-Sep-2016) 44 and optimized by two steps. First, off-target scores were calculated according to an established algorithm 30. Second, on-target scores were calculated using Rule Set 2 29. sgRNAs were ranked by on-target scores and the top 12 sgRNAs with off-target scores of less than 20 were selected for each gene. If less than 12 sgRNAs were obtained, the cutoff of off-target scores was increased sequentially to 40, 60, 80 and 100 until 12 sgRNAs were obtained. The genome-wide CRISPR library was divided into three sub-libraries A, B and C according to sgRNA rank. Non-targeting sgRNAs were included in each library.

Pooled sgRNA oligonucleotides were synthesized as 76-mers by Custom Array (Bothell, WA, USA) and were amplified by PCR with NEBNext High Fidelity PCR Master Mix (New England BioLabs, NEB, Ipswich, MA, USA) using customized primers (Additional file 2: Table S1). The PCR products were purified using MinElute PCR purification kit (Qiagen, Dusseldorf, Germany). Lentiviral vector LentiCRISPR-v2 was digested with Esp3I (Thermo Fisher Scientific, Waltham, MA, USA) at 37 °C for 3 h and gel-purified using Gel Extraction kit (Omega, Norcross, GA, USA). Purified digestion products were ligated to Esp3I-treated LentiCRISPR-v2 using Gibson assembly kit (NEB) following manufacturer’s instructions. The ligation product was purified by isopropanol precipitation and then transformed into electrocompetent Escherichia coli (Lucigen, Middleton, WI, USA). Transformed cells were plated on to 15 cm Luria-Bertani (LB) agar plates supplemented with 50 μg/mL ampicillin (Sangon Biotech, Shanghai, China). Approximately 1-3 × 107 colonies were collected for each library to ensure 500-fold coverage. Plasmid DNA was extracted as pooled libraries using NucleoBond Xtra Maxi EF kit (Macherey-Nagel, Duere, Germany) and stored at −20 ℃.

Cell culture

H1-Hela cells were obtained from the American Type Culture Collection (ATCC). HEK293T cells were obtained from the Cell Bank of Shanghai Institutes for Biological Science (SIBS) and were validated by VivaCell Biosciences (Shanghai, China). H1-Hela and HEK293T were grown in Dulbecco’s modified Eagle’s medium (DMEM, Thermo) supplemented with 10% fetal bovine serum (FBS, Thermo) and 1% penicillin-streptomycin (Thermo) and maintained at 37 ℃ in a fully humidified incubator containing 5% CO2. All cells were confirmed by PCR to be free of mycoplasma contamination.

LV production and transduction

To produce LVs with high titers, HEK293T cells were seeded on to 6-well plates with 2 × 106 cells per well for single sgRNA, or 10 cm petri dishes with 107 cells for library construction. At 24 h after seeding, HEK293T cells at a confluence of 70%-90% were transfected with LV packaging plasmid pMD2.G, envelope plasmid psPAX and transfer plasmid pLentiCRISPR-v2 that carries single or pooled sgRNAs using Lipofectamine 3000 (Thermo). At 6 h after transfection, the medium was replaced with fresh medium. The medium supernatant containing LVs was harvested at 48 h post transfection by centrifugation at 2,000 rpm for 10 min, filtrated through a 0.45-μm filter and stored at −80 ℃.

H1-Hela cells were transduced with LVs at an MOI of 0.3 using spinfection. Briefly, H1-Hela cells were washed with phosphate buffered saline (PBS) and incubated with LVs in serum-free DMEM under centrifugation at 2,000 rpm for 2 h. Upon completion of spinfection, LV-containing medium was removed and cells were incubated in DMEM supplemented with 10% FBS and 2 μg/mL puromycin (Thermo) for 2 to 3 days to purge empty cells containing no LVs or sgRNAs. For each library, cells of more than 500-fold coverage of the library size were collected.

RV production and infection

The full-length cDNA clones of RV-A16 (pR16.11, Cat. No. VRMC-8) and RV-B14 (pWR3.26, Cat. No. VRMC-7) were obtained from ATCC. To produce infectious viral RNA, RV-A16 and RV-B14 plasmids were linearized by SacI (NEB) digestion and then in vitro transcribed using HiScribe T7 Transcription Kit (NEB). The RNA transcripts were extracted using Trizol (Thermo) and chloroform (Titan, Shanghai, China), followed by isopropanol precipitation. Viral RNA was transfected into H1-HeLa cells using Lipofectamine 3000 (Thermo) to generate infectious RV-A16 or RV-B14 particles. At 48 h post transfection, the supernatant containing RVs were collected for further infection on H1-Hela cells to produce RVs with higher titers. The aliquots of purified RVs were stored at −80 ℃. Virus titers were determined by the 50% tissue culture infectious dose (TCID50) assay.

For RV infection, H1-Hela cells were seeded on to 96- or 12-well plates with a density of 2×104 or 1.5×104 cells per well, respectively. Unless noted otherwise, at 24 h after seeding, cells were infected with RVs at an MOI of 2 for 1.5 h, washed with PBS for three times and then cultured in DMEM (Thermo) supplemented with 10% fetal bovine serum (FBS, Thermo) and 1% penicillin-streptomycin (Thermo) for 24 h.

CRISPR screen using RV-B14

H1-Hela cells carrying genome-wide sub-library A or surfaceome library were seeded on to 15 cm petri dishes. Approximately 1.5 ×107 cells were seeded to ensure more than 200-fold coverage of sgRNA. These cells were infected with RV-14 at an MOI of 1 for 48 h. At the end point of RV-14 challenge, the cells were washed with PBS and attached cells were collected from the plates. Two biological replicates were performed for mock and test groups. Genomic DNA of the harvested cells was extracted using phenol: chloroform: isoamyl alcohol (v/v/v, 25:24:1) and then purified using ethanol precipitation.

Next-generation sequencing (NGS) analyses of sgRNA enrichment

Genome-integrated sgRNAs were amplified from extracted genomic DNA by PCR using the primers containing Illumina adaptor (Additional file 2: Table S1). The PCR product was gel-purified and then analyzed on Illumina HiSeq 3000 platform by Genewiz (Suzhou, Jiangsu, China). After removing the adaptors, the 20 bp sgRNA was mapped to the reference sgRNA libraries with one nucleotide mismatch allowed for each sgRNA. Gini index was calculated to analyze the distribution of sgRNAs. The raw read counts were subjected to MAGeCK analyses 45 to determine the enriched sgRNA and genes. Enrichment of sgRNAs and genes was analyzed using MAGeCK (v0.5.7) by comparing the read counts from the cells infected with RV-B14 with those from uninfected cells. A false discovery rate (FDR) of less than 0.01 was applied to identify significantly enriched sgRNAs or gene knockout.

Generation of CRISPR-Cas9 knockout cells

The transfer plasmid pLentiCRISPR-v2 carrying single sgRNA for targeted gene knockout was constructed as described above. Briefly, forward and reverse oligonucleotides encoding the 20 bp sgRNA (Additional file 2: Table S2) were annealed to generate double-stranded DNA with overhang that matched the sticky ends of Esp3I (Thermo)-treated pLeniCRISPR-v2 vector. Annealed sgRNA sequence was ligated into digested pLeniCRISPR-v2 and then transformed into DH5α E. coli (Tsingke, Beijing, China).

The LVs carrying single sgRNA were packaged and transduced on to cells as described above. To evaluate the knockout efficiency, the genomic DNA of edited cells were extracted using Quick Extraction kit (Lucigen). Target sites carrying gene-edited sequences were PCR amplified using gene-specific primers (Additional file 2: Table S3). The knockout efficiency of each sgRNA was determined using T7E1 analysis. Single clones were obtained by limited dilution and genotyped by Sanger sequencing to determine the mutations at each allele. Knockout of target proteins was verified by western blotting (WB).

Cell viability assay

H1-Hela cells were seeded into 96- or 24-well plates with a density of 5,000 or 50,000 cells per well respectively. At 24 h after seeding, cells were infected with RVs at an MOI of 1 for 1.5 h, washed with PBS for three times and cultured in fresh medium for 24 h unless noted otherwise. Cell counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) was applied to determine cell viability according to manufacturer’s instructions. The absorbance at 450 nm was determined by Enspire multimode plate reader (PerkinElmer, Waltham, MA, USA).

Immunofluorescence (IF) and fluorescence in situ hybridization (FISH)

IF staining of RV-B14 was performed using mouse anti-RV VP3 antibody (1:50, clone G47A, Thermo) and Alexa Fluor Plus 488 goat anti-mouse IgG (H+L) (1:1,000, A32723, Thermo). IF images were acquired and analyzed using Operatta high-content analysis system (PerkinElmer). At least 2,000 fluorescent cells were imaged and quantified for each replicate.

FISH was performed using an RNAscope Multiplex Fluorescent V2 Assay kit (Advanced Cell Diagnostics, Newark, USA) according to the manufacturer’s instructions. After fixation and pretreatment, RV-B14 RNA was detected using an RVB RNA probe (Advanced Cell Diagnostics, Cat. No. 447141) and TSA Plus Fluorescein (PerkinElmer, Cat. No. NEL741E001KT). FISH images were acquired and analyzed using a TissueFAXS 200 flow-type tissue quantitative analyser (TissueGnostics GmbH, Vienna, Austria). At least 5,000 cells in each replicate were included in analyses.

Real-time quantitative PCR (RT-qPCR)

To determine viral loads, the medium supernatant or cell lysate containing RVs were harvested at 24 h after infection with RVs at an MOI of 2 unless noted otherwise. The total RNA from supernatant or cell lysate was purified using Trizol (Thermo) and chloroform (Titan), followed by purification using isopropanol precipitation. Purified viral RNA (vRNA) was reverse transcribed into cDNA using PrimeScript RT reagent Kit with gDNA Eraser (Takara Bio Inc., Shiga, Japan). The number of RV genome copy in the medium supernatant was determined using RT-qPCR with general or serotype-specific Taqman probe and primers (Additional file 2: Table S4) on Applied Biosystems Q6 Real-Time PCR cycler. The absolute viral titers were calculated based on a standard curve of RV genome with known TCID50 and the R square of curve-fitting was guaranteed to be more than 0.99. The mRNA levels of RVs and IFN-stimulating genes (ISGs) in cell lysate were determined using RT-qPCR with SYBR green dye (Thermo) and specific primers (Additional file 2: Table S5) on Applied Biosystems Q6 Real-Time PCR cycler. All SYBR Green primers were validated with dissociation curves. The expression of vRNA and host genes in cell lysate is normalized to ribosomal gene RPLP0 (36b4).

Gene knockdown using siRNA

H1-Hela cells were seeded on to 6-well plates with a density of 5×105 cells per well. At 24 h after seeding, cells were transfected with 100 pmol SOCS3 siRNA (Genepharma, Shanghai, China) (Additional file 2: Table S6) using 7.5 μL Lipofectamine 2000 (Thermo) for 6 h, washed with PBS and then cultured in fresh DMEM (Thermo) supplemented with 10% fetal bovine serum (FBS, Thermo). At 48 h post transfection, cells were infected with RV-B14 at an MOI of 2 for 1.5 h, washed with PBS for three times and then cultured in fresh medium for 24 h. The medium supernatant or cell lysate containing RVs were harvested and lysed for total RNA extraction, and then the mRNA levels of RVs, SOCS3, STAT1/2 and IFN-stimulating genes (ISGs) in cell lysate were determined using RT-qPCR as described above.

Virus attachment and entry assays

H1-Hela cells were seeded in 12-well plate with a density of 200,000 cells per well and incubated overnight. For virus attachment assay, cells were incubated with RV-B14 or RV-A16 at an MOI of 20 in cold medium on ice for 60 min, washed by PBS for three times and then the total RNA was extracted using Trizol (Thermo). For virus entry assay, cells were incubated with RV-B14 or RV-A16 at an MOI of 20 in cold medium on ice for 60 min, washed by PBS for three times and then treated with pre-warmed medium for 40 min at 37 ℃. Then cells were washed with PBS for three times and treated with 0.25% trypsin for 2 min (Thermo, Cat. No. 25200072) to remove surface-bound viral particles. The internalized viral RNA was extracted using Trizol (Thermo) and viral loads were determined using RT-qPCR and FISH.

Rescue experiments by overexpression

ICAM1, RAB5C and OLFML3 genes were codon-optimized for expression in human cells and synthesized by Genewiz. The 20 bp sgRNA-targeting sites and PAM sequences were mutated with silent mutations. Myc and FLAG tags were added to the C-terminus of these genes for WB detection. These genes were cloned into the EcoRI and XhoI sites of pCAGG plasmid that carries a separate mScarlet fluorescent protein as a transfection reporter.

WB analysis

For WB analysis, cells were lysed with RIPA buffer (Beyotime Biotechnology, Beijing, China) on ice for 10 min. The total protein concentration in cell lysate was determined using the BCA Protein Assay Kit (Thermo). Cell lysate was mixed with SDS-PAGE loading buffer (Takara) containing 200 mM DTT, incubated at 95 °C for 10 min and resolved on NuPAGE 4-12% Bis-Tris gels (Thermo). Protein samples were transferred onto nitrocellulose membranes using an iBlot gel transfer system (Thermo). The following primary and secondary antibodies were used in WB including anti-ICAM1 rabbit antibody (Cell Signaling Technology, Cat. No. 4915S, Danvers, USA), anti-RAB5C rabbit antibody (Thermo, Cat. No. PA551932), anti-OLFML3 rabbit antibody (Thermo, Cat. No. PA531581), HRP-conjugated anti-rabbit IgG (CST, Cat. No. 7074S). Anti-β actin antibody conjugated with HRP (Abcam, Cat. No. ab49900, Cambridge, UK) was used as an internal control.

RNA-Seq analysis of mock and OLFML3-/- cells

Non-targeting sgRNA-treated mock cells and OLFML3-/- cells were seeded on to 10 cm plates at a density of 1×106 cells per plate. At 24 h after seeding, the cells were infected with RV-B14 at an MOI of 1 or with PBS as mock infection. After 24 h of RV challenge, the remaining cells were washed with PBS and collected by Trizol (Thermo) treatment. Three biological triplicates were prepared for each group. The whole-transcriptome RNA sequencing was performed by Genewiz.

RNA-Seq short reads were aligned to the human genome (GRCh37) using Hisat2 (v2.0.1) 46. Gene expression was counted as the number of short reads fully or partially aligned to the annotated gene model using HTSeq (v0.6.1) 47. Genotype (non-targeting sgRNA-transduced mock cells and OLFML3-/- cells) and treatment conditions (mock and RV-B14 infection) were the factor variables in our RNA-Seq data. DESeq2 (v1.26.0) 48 was used to examine the difference between the response of mock and knockout cells to RV infection, which was captured by the interaction term. RV infection-induced gene upregulation and downregulation were calculated and the differentially upregulated or downregulated genes in mock and knockout cells were defined as DEGs. Significant DEGs were filtrated with an adjusted P value of less than 0.05 and a fold change value of more than 2.

GO enrichment analysis was performed using cluster Profiler (v3.14.3) by comparing DEGs to a list of all human genes 49. Adjusted P value of less than 0.001 or 0.05 was set as the filter for biological process and molecular function terms respectively.

Isolation of clinical RV strain

Nasopharyngeal swab samples were collected from the hospitalized patients bearing respiratory infection symptoms such as fever, cough, pharyngalgia and others. Nasopharyngeal swab samples were maintained in viral-transport medium and all samples were conserved on −80°C until analyses. The swab samples were diagnosed for respiratory viruses and the remaining samples were used for isolation of RVs. All procedures were complied with the Measures for the Ethical Review of Biomedical Research Involving Human Subjects issued by the National Health and Family Planning Commission of the People’s Republic of China. The Ruijin Hospital Ethics Committee, Shanghai Jiaotong University School of Medicine, approved the sample collection protocol with a permit number of Ruijin Hospital Ethics Committee 2018-48.

For isolation of RVs, the specimens were centrifuged at 2,000 rpm for 10 min and the supernatant was collected for RT-qPCR analysis using universal RV primers (Supplementary Table 4). The samples with high RV loads were selected for subsequent RV isolation in H1-Hela cells. H1-Hela cells were seeded on to 12-well plates at a cell density of 100,000 cells per well. Specimen supernatant (700 μL) was mixed with 300 μL of fresh medium and incubated with cells under centrifugation at 2,000 rpm for 2 h. Upon completion of spinfection, the supernatant was replaced with fresh medium and cells were incubated for another 2 days. Thereafter, medium supernatant and attached cells were collected and subjected to quick freeze-thaw cycles for three times to release viral particles. The medium supernatant or cell lysate from above was added to cells on 12-well plates. At 24 h after incubation, the supernatant was replaced with fresh medium and cells were incubated for 2 to 3 days before reaching 100% confluence. Isolation of RV strain was confirmed by CPEs and sequencing results.

Statistical analyses

All data are the results from at least three biological replicates and are shown as mean ± SD unless noted otherwise. Statistical analyses and graphing were performed with GraphPad Prism 7.0. The P values were determined using two-tailed unpaired Student’s t-test unless otherwise noted.

Article TitleSurfaceome CRISPR Screen Identifies OLFML3 as a Rhinovirus-inducible IFN Antagonist

Abstract

Background Rhinoviruses (RVs) cause more than half of common cold and, in some cases, more severe diseases. Functional genomics analyses of RVs using siRNA or genome-wide CRISPR screen uncovered a limited set of host factors, few of which has proven clinical relevance.

Results Herein, we systematically compared genome-wide CRISPR screen and surface protein-focused CRISPR screen, referred to as surfaceome CRISPR screen, for their efficiencies in identifying RV host factors. It was found that surfaceome screen outperformed genome-wide screen in the success rate of hit identification. Importantly, using surfaceome screen we have identified olfactomedin like 3 (OLFML3) as a novel host factor of RV serotypes A and B including a clinical isolate. We found that OLFML3 was a RV-inducible suppressor of the innate immune response and that OLFML3 antagonized type I interferon (IFN) signaling in a SOCS3-dependent manner.

Conclusion Our study has suggested that RV-induced OLFML3expression is an important mechanism for RV to hijack the immune system and underscored surfaceome CRISPR screen in identifying viral host factors.


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