Cell lines
Human embryonic kidney (HEK) 293T cells provided by Prof. Hai-Yan Ren, human lung cancer A549 cells purchased from National Collection of Authenticated Cell Cultures, and mouse leukemic monocyte/macrophage cell line Raw264.7 provided by Jia-Yi Xu were grown at 37°C in 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS). HEK293T cells were used for co-IP assays, Gluc activity assays and minigene splicing assays because of their very high transfection efficiency. A549 cells were used for the fluorescence microscopy and SARS-CoV-2 spike-mediated pseudovirus entry assays, since this kind of cells adhered well to the surface of coverslips placed in 6-well plates and at the bottom of 96-well white plates.
Plasmid construction
Total RNA was extracted from HEK293T cells with Trizol reagent (Invitrogen) and was then used for the synthesis of single-stranded cDNA by reverse transcription. Different kinds of nucleic-acid fragments were amplified with the corresponding primers (Table S1). The plasmids used for co-IP, fluorescence microscopy, protein expression, gene editing, Gluc activity assays and minigene splicing assays were constructed. All constructs used in this study were confirmed by DNA sequencing. The operations are as follows:
The plasmids pET21b-NSP12, pET21b-NSP7 and pET32a-NSP8 separately expressing SARS-CoV-2 (QSE99647.1) NSP12, NSP7 and NSP8 were provided by Prof. Li-Chuan Gu. The NSP12 and NSP7 gene were cloned into a modified pET21b vector with the C-terminus possessing a 6×His-tag. The NSP8 gene was cloned into the modified pET32a vector with the N-terminus possessing a trx-His6-tag and PreScission Protease site.
To generate the plasmid for expressing NSP12 and co-IP assays, the fragments NSP12, NSP12-HA, NSP12N representing N-terminal domain (1-356 aa) and NSP12C representing C-terminal domain (366–932 aa) were amplified with primers NSP12-F/-R, NSP12-F/NSP12-HA-R, NSP12-F/NSP12N-HA-R and NSP12C-F/NSP12-HA-R, respectively. The ORFs of NSP7, NSP8, SLU7, PPIL3 genes were amplified with primers NSP7-F/NSP7-HA-R, NSP8-F/NSP8-HA-R, SLU7-F/SLU7-3Flag-R, PPIL3-F/PPIL3-3Flag-R and PPIL3-F/PPIL3-HA-R, respectively. All fragments obtained above were separately cloned into pcDNA3.1(+) to construct the corresponding plasmids. The ORFs of NSP12, AKAP8, TCF12, CRTC3, RBM41 and BCKDK genes were amplified with primers NSP12-F/-R2, AKAP8-F/R, TCF12-F/R, CRTC3-F/R, RBM41-F/R and BCKDK-F/R, and cloned into a modified pcDNA3.1(+) vector with the C-terminus possessing a 3×Flag-tag, respectively. The codon-optimized SARS-CoV (AAP33696.1) NSP12 and MERS-CoV (NC_019843) NSP12 separately fused with HA-tag at the C-terminus were synthetized by GENEWIZ and cloned into the vector pcDNA3.1(+).
To construct plasmids for fluorescence microscopy, the ORFs of NSP12, NSP7, NSP8 and PPIL3 were amplified with primers NSP12-F2/R2, NSP7-F2/R2, NSP8-F2/R2 and PPIL3-F2/R2 and then cloned into pEGFP-N3, respectively. The ORF of the SLU7, PPIL3, AKAP8, TCF12, CRTC3, RBM41 or BCKDK gene was amplified with primers SLU7-F2/R2, PPIL3-F3/R3, AKAP8-F2/R2 TCF12-F2/R2, CRTC3-F2/R2, RBM41-F2/R2 or BCKDK-F2/R2 and separately cloned into pDsRed2-C1.
To construct plasmids for the prokaryotic expression of PPIL3 or SLU7, the ORF of PPIL3 gene was amplified with primers PPIL3-F4/R4 and cloned into pGEX-6P-1 to produce pGEX-PPIL3. The ORF of SLU7 gene was amplified with primers SLU7-F3/R3 and cloned into pET15b to produce pET15b-SLU7.
Three pairs of oligos SLU7-sgRNA-F/R, PPIL3-sgRNA-F/R and AKAP8-sgRNA-F/R were designed according to a previous report [24]. The fragments SLU7-sgRNA, PPIL3-sgRNA and AKAP8-sgRNA were obtained by annealing and separately cloned into plasmid lentiCRISPRv2. The plasmids pMD2-G and psPAX2 were purchased from Addgene (www.addgene.org).
To generate plasmid pCoV-Gluc, which produces the positive-strand of vRNA encoding Gluc, the fragment 5′UTR-Gluc-3′UTR representing the ORF of Gluc flanked by the 5′UTR (untranslated region) and 3′UTR of SARS-CoV-2 was synthesized and inserted into the BamHI and NotI sites of pRetroX-tight-Pur vector purchased from Miaoling Bio (www.miaolingbio.com).
The fragment IRF7exons5 − 6-2A representing exons5-6 of mouse IRF7 (NM_016850.3) containing its endogenous intron fused with 2A self-cleaving peptide was amplified with primers IRF7-F and IRF7-2A-R using Raw264.7 genome as template, and cloned into pEGFP-N3 to produce pEGFP-IRF7-2A. The fragment IRF7-2A was amplified with primers IRF7-F2/R2 using pEGFP-IRF7-2A as template, and cloned into the XhoI and NcoI sites of MSCV vector (Addgene 21654) to produce pMSCV-IRF7-2A. The SARS-CoV-2 NSP16 (YP_009725311.1) fused with HA-tag at the C-terminus was synthetized and cloned into the vector pcDNA3.1(+). The ORF of GAPDH gene was amplified with primers GAPDH-F/R and cloned into the vector pcDNA3.1(+).
Co-IP assays
Co-IP assays were performed as previously described with minor modifications [25]. Briefly, HEK293T cells seeded in 10 cm dishes were cotransfected with 7.5 µg of pcDNA3.1-NSP12-HA and 7.5 µg of pcDNA3.1-SLU7-3Flag or other indicated plasmids. As a control, 7.5 µg of empty vector pcDNA3.1(+) was transfected in parallel. At 24 h post-transfection (hpt), cells were lysed with 1 mL of a radio immunoprecipitation assay (RIPA) buffer (50 mM Tris pH 7.4, 150 mM NaCl, 1% NP-40, 0.25% sodium deoxycholate) containing protease inhibitor cocktail (Sigma) and 1 mM phenylmethylsulfonyl fluoride (PMSF) for 1 h. The cell lysates were centrifuged to remove cell debris and the lysate supernatants were collected and divided into two parts. Five hundred microliters of lysate supernatant was used to analyze the coexpression of NSP12 and other proteins by Western blot analysis using anti-HA and anti-Flag antibodies. Other 500 µL of lysate supernatant was incubated with red anti-HA affinity gel (Sigma) or anti-Flag affinity gel (Sigma) overnight at 4°C. The precipitates were collected by centrifugation, washed with ice-cold RIPA buffer, eluted with 40 µL of phosphate-buffered saline (PBS), and finally subjected to Western blot analysis.
Western blot analysis
Western blot analysis was performed as described previously [25]. Protein samples were resolved by SDS-PAGE, followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. The blots were probed with anti-HA rabbit monoclonal antibody (1: 1,000, Cell Signaling), anti-Flag rabbit monoclonal antibody (1: 1,000, Cell Signaling), anti-PPIL3 rabbit polyclonal antibody (1:1000, Sino Biological), anti-SLU7 rabbit polyclonal antibody (1:1000, Novus) or anti-AKAP8 rabbit polyclonal antibody (1:1000, Abclonal). Peroxidase-conjugated goat anti-rabbit IgG (H + L) antibody was used as the secondary antibody. The signals were detected with a chemiluminescent horseradish peroxidase (HRP) substrate (Millipore).
Fluorescence microscopy
A549 or A549-SLU7KO, A549-PPIL3KO or A549-AKAP8KO cells seeded on the surface of coverslips placed in 6-well plates were alone transfected with one plasmid, such as pEGFP-NSP12 or cotransfected with two plasmids, such as pEGFP-NSP12 + pDsRed2-SLU7. At 24 hpt, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100, stained by Hoechst 33342 and observed under a Leica DMi8 fluorescence microscope (objective 64×) or a Leica Stellaris laser confocal microscope (objective 100×), as described previously [26].
siRNA
SLU7, PPIL3 or AKAP8 specific siRNA was designed and synthetized by Sangon Biotech (Shanghai) Co., Ltd. The sequences of siRNAs were listed in Table S1. HEK293T cells were transfected siRNA by jetPRIME® (Polyplus Transfection).
Gluc activity assay
To test the SARS-CoV-2 RdRp-dependent Gluc reporter system [27], 1.6×105 HEK293T were cotransfected with 10 ng of pCoV-Gluc, 0.5 µg of pcDNA3.1-NSP12-HA, 0.5 µg of pcDNA3.1-NSP7-HA and 0.5 µg of pcDNA3.1-NSP8-HA. The total DNA quantities were consistent by adding to the empty vector pcDNA3.1(+) in control group. At 24 hpt, the supernatant was collected for Gluc activity assay. The Gluc activity in supernatant was determined according to Secrete-Pair™ Gaussia Luciferase Assay Kit User Manual. For luminescence assay, 10 µL of supernatant was added to each well of a white and opaque 96-well plate, and 100 µL of Gluc working buffer (GL-S) was injected, and luminescence was measured using a BioTek Cytation 5 Cell Imaging Multi-Mode Reader.
To analyze the effect of SLU7, PPIL3 or AKAP8 overexpression on the polymerase activity of RdRp, 1.6×105 HEK293T were cotransfected with 10 ng of pCoV-Gluc, 0.5 µg of pcDNA3.1-NSP12-HA, 0.5 µg of pcDNA3.1-NSP7-HA, 0.5 µg of pcDNA3.1-NSP8-HA and 0.5 µg of pcDNA3.1-SLU7-3Flag or other indicated plasmid. At 24 hpt, the supernatant was collected for Gluc activity assay.
To analyze the effect of SLU7, PPIL3 or AKAP8 knockdown on the polymerase activity of RdRp, 1.2×105 HEK293T transfected with 3 µL of 55 nM siNC, siSLU7-606, siPPIL3-325 or siAKAP8-1665 for 24 h were cotransfected with 10 ng of pCoV-Gluc, 0.5 µg of pcDNA3.1-NSP12-HA, 0.5 µg of pcDNA3.1-NSP7-HA, 0.5 µg of pcDNA3.1-NSP8-HA. At 24 hpt, the supernatant was collected for analysis as above.
Protein expression and purification
The protein expression and purification were performed as previously described with minor modifications [28, 29]. The plasmid pET21b-NSP12 or pET32a-NSP8, was separately transformed into E. coli BL21 (DE3). The plasmid pET21b-NSP7 was transformed into E. coli Rosetta-gami2 (DE3). These proteins were then expressed after induction with 0.2 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) at 16°C for 16 h. Harvested cell pellets were resuspended in buffer A (25 mM Tris-HCl pH 8.0, 250 mM NaCl, 4 mM MgCl2, 10% Glycerol). The cell lysate was applied to a nickel-affinity column (Ni-NTA; GE Healthcare) for purification. NSP12 and NSP7 were eluted with buffer B (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 4 mM MgCl2, 250 mM Imidazole). NSP8 was subjected to on-column tag cleavage by PPase (PreScission Protease) and then eluted by the buffer A. Next, the three proteins were purified by ion exchange chromatography (Source Q, GE Healthcare) and size exclusion chromatography (GE Healthcare) with buffer C (25 mM Tris-HCl pH 8.0, 200 mM NaCl, 4 mM MgCl2).
For RdRp complex assembly, protein samples were mixed with the molar ratio of NSP7:NSP8:NSP12 = 2:2:1 at 4°C overnight. The incubated RdRp complex was then concentrated with a 100 kDa molecular weight cut-off centrifugal filter unit (Millipore Corporation) and further purified by size exclusion chromatography in buffer C with 5 mM DTT.
The plasmid pGEX-PPIL3 was transformed into E. coli BL21 (DE3). PPIL3 was then expressed after induction with 0.1 mM of IPTG. Harvested cell pellets were resuspended in buffer D (25 mM Tris-HCl 7.5, 250 mM NaCl, 3 mM DTT). The cell lysate was then loaded onto glutathione-coated Sepharose resin (GE Healthcare) for purification. GST-PPIL3 was subjected to on-column tag cleavage by PPase and then eluted by buffer D. PPIL3 was purified by size exclusion chromatography with buffer E (25 mM Tris-HCl 7.5, 100 mM NaCl, 3 mM DTT).
The plasmid pET15b-SLU7 was transformed into E. coli BL21 (DE3). SLU7 was then expressed after induction with 0.2 mM of IPTG. Harvested cell pellets were resuspended in buffer F (25 mM Tris-HCl pH 7.5, 250 mM NaCl). The cell lysate was applied to a nickel-affinity column and eluted with elution buffer G (25 mM Tris-HCl pH 7.5, 50 mM NaCl, 300 mM Imidazole). SLU7 was purified by ion exchange chromatography and size exclusion chromatography with buffer H (25 mM Tris-HCl pH 7.5, 100 mM NaCl).
In vitro RNA primer extension assay using RdRp complex
RNA template-product duplex was designed according to the published SARS-CoV-2 RNA extension assays [11]. FAM labeled 13 nt oligonucleotide (FAM-5'-GCUAUGUGAGAUU-3') and the 23 nt complementary oligonucleotide (3'-CGAUACACUCUAAUUUCAAUUGA-5') were synthesized for polymerase activity assay. The oligonucleotides were mixed in equal molar ratio in DEPC (diethy pyrocarbonate) water, annealed by heating it to 95°C for 10 min and gradually cooling to room temperature to make the RNA duplex.
To optimize the quantity of RdRp and nucleoside triphosphate (NTP), 2, 3 or 4 µM RdRp and 0.1, 0.5 or 1 mM NTP were added to the reaction buffer (R buffer) containing 200 nM 13 + 23 nt RNA duplex, 20 mM Tris-HCl pH 8.0, 10 mM KCl,10 mM MgCl2, 0.01% Triton-X100, 1 mM DTT. The volume was adjusted to 20 µL by added DEPC-treated ddH2O. DEPC-treated water was used for all buffer preparation. The system was reacted at 37 ℃ for 30 min. Eventually, glycerol was added to a concentration of 6.5%. Ten microliters of RNA product for each reaction was resolved on 20% denaturing polyacrylamide-urea gels and imaged with a ChemiDoc™ MP imaging system (BIO-RAD).
To test the effect of SLU7 or PPIL3 on RdRp polymerase in vitro, 4 ~ 8 µL of purified SLU7 (3.75 mg/mL, about 11 ~ 22 µM) or 2 ~ 8 µL of PPIL3 (5.6 mg/mL, about 30 ~ 120 µM) and 2 µM RdRp were added to the reaction buffer (R buffer). The volume was adjusted to 20 µL by added DEPC-treated ddH2O. The system was reacted for 30 min at 37 ℃, and then 0.5 mM NTP was added to the reaction system for 30 min at 37 ℃. The detection of RNA product was as described above.
CRISPR/Cas9 gene editing
CRISPR/Cas9 gene editing was performed as previously described with minor modifications [24]. A549 cells lacking SLU7, PPIL3 or AKAP8 were generated as follows. Approximately 8×105 HEK293T cells in six-well plates were cotransfected with 0.51 µg of pMD2-G, 0.78 µg of psPAX2 and 1.20 µg of lentiCRISPRv2-SLU7 sgRNA or other indicated plasmid. At 48 hpt and 72 hpt, the supernatants were collected and mixed. A549 cells in six-well plates were inoculated with 500 µL of harvested supernatant for 24 h and then selected with DMEM supplemented with 10% FBS containing 2 µg/mL puromycin until clones formed. All clones were sorted into 96-well plates by flow cytometer (FACSAria SORP) and each well contained only one cell. Individual clones formed in 96-well plates were further transferred into 6-well plates for culture and then identified by DNA sequencing and Western blot analysis. For DNA sequencing, the genomes of cells in the 6-well plates were extracted and amplified by PCR using corresponding primers. The PCR products were sequenced using corresponding primers.
SARS-CoV-2 spike-mediated pseudovirus entry assay
To analyze the effect of host protein knockout on SARS-CoV-2 pseudovirus entry, 5×104 A549, A549-SLU7KO, A549-PPIL3KO or A549-AKAP8KO cells were seeded in 96-well white plates and grown overnight. The pseudovirus infection and measurement of entry efficiency were according to previous report [24]. The culture medium was replaced with fresh medium containing 8 µg/mL polybrene for 1 h and then the cells were inoculated with 4E + 6 RLU (relative light units) VSV-SARS-2-S-luc pseudovirus. At 16 h post infection (hpi), the culture medium was replaced with fresh medium. Entry efficiency was quantified at 48 hpi by measuring the activity of Renilla luciferase in cell lysates using the ONE-Glo™ Luciferase Assay according to the manufacturer’s instructions (PekinElmer Envision). The infection experiments were performed under biosafety level 2 (BSL2) laboratory conditions.
RNA-Seq processing and analysis
To investigate whether SLU7 or PPIL3 knockout affects the transcriptions of some genes involved in SARS-CoV-2 entry, the transcriptomes of A549, A549-SLU7KO and A549-PPIL3KO cells were sequenced and analyzed. To analyze the effect of NSP12 on the global cellular AS, the transcriptome of HEK293T cells transfected with pcDNA3.1 or pcDNA3.1-NSP12 for 24 h was sequenced and analyzed. RNAs were sequenced using the standard Illumina protocol (LC-Bio Technology CO., Ltd.). Reads were mapped to the genome using HISAT2. Genes differential expression analysis was performed by DESeq2 software between two different groups (and by edgeR between two samples). The genes with the parameter of false discovery rate (FDR) below 0.05 and absolute fold change ≥ 2 were considered differentially expressed genes (DEGs). DEGs were then subjected to enrichment analysis of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways. rMATS (version 4.1.1) was used to identify AS events and analyze differential alternative splicing events between samples. We identified AS events with a false discovery rate (FDR) < 0.05 in a comparison as significant AS events. The classification of AS is as follows: skipped exon (SE), retained intron (RI), mutually exclusive exon (MXE), alternative 5’ splice site (A5SS) and alternative 3’ splice site (A3SS).
Minigene splicing assays
Minigene splicing assays were performed as previously described with minor modifications [21]. To test the effect of NSP12, its N-terminal domain (1-356 aa) and C-terminal domain (366–932 aa), SLU7, PPIL3, AKAP8, NSP12a or NSP12b on cellular AS, 1.6×105 HEK293T were cotransfected with 0.5 µg of pMSCV-IRF7-2A and 0.5 µg of pcDNA3.1-NSP12-HA or other corresponding plasmids. GAPDH was as a negative control. SARS-CoV-2 NSP16 was as a positive control. At 48 hpt, cells were collected and measured by flow cytometry (Cytoflex) and analyzed using Flowjo analysis software. Three independent biological replicates per condition were prepared.
To analyze the effect of SLU7, PPIL3 or AKAP8 overexpression on the ability of NSP12, NSP12a or NSP12b in inhibiting cellular AS, 1.6×105 HEK293T were cotransfected with 0.5 µg of pMSCV-IRF7-2A, 0.5 µg of plasmid expressing one of the three virus proteins, 0.5 µg of plasmid expressing one of the three host proteins. At 48 hpt, cells were measured as above. To analyze the effect of SLU7, PPIL3 or AKAP8 knockdown on the ability of NSP12, NSP12a or NSP12b in inhibiting cellular AS, 1.2×105 HEK293T transfected with corresponding siRNA for 24 h were cotransfected with 0.5 µg of pMSCV-IRF7-2A and 0.5 µg of plasmid expressing one of the three virus proteins. At 24 hpt, cells were measured as above.