Generation of the Rosa26-LSL-Nsp12 conditional knock-in (cKI) mouse
pLVX-EF1a-SARS-CoV-2-nsp12-2xstrep-IRES-Puro plasmid (Addgene #141378) was purchased from Addgene27. To generate Rosa26 cKI mice conditionally expressing SARS-CoV-2 Nsp12, the SARS-CoV-2 Nsp12 cDNA followed by ires (internal ribosome entry site) and GFP (green fluorescent protein) was inserted into the mouse Gt(ROSA)26Sor locus containing a loxP-flanked STOP cassette (LSL) and the neomycin resistance gene (Extended Data Fig.1). The generation of Rosa26-LSL-Nsp12 cKI mice was performed at the BioSafety Research Center Inc. (Iwata, Japan).
To construct the targetting vector, a 3.3 kb upstream DNA fragment and a 4.3 kb downstream fragment relative to the unique XbaI site in intron 1 of the Gt(Rosa)26Sor locus were amplified by PCR from genomic DNA of RENKA ES cells47 and used as the homology arms for the targetting vector. These amplified genomic fragments were subcloned into a plasmid containing the MC1_DTA cassette (polyoma enhancer/herpes simplex virus thymidine kinase promoter-driven diphtheriae toxin A gene) as a negative selection marker, a splicing acceptor sequence, and an LSL cassette containing CAT (chloramphenicol acetyltransferase) cDNA as a stuffer sequence48. A FRT-flanked PGK neo cassette (phosphoglycerate kinase I promoter-driven neomycin resistance gene) was inserted downstream of the CAT sequence as a positive selection marker.
To construct the expression unit, the SARS-CoV-2 Nsp12 gene coding sequence plus ires-GFP were amplified by PCR and subcloned into the targetting vector downstream of the second loxP site of the stop cassette along with a bGH (bovine growth factor) poly A signal. The resulting targetting vector contained: MC1_DTA, 3.3 kb 5’ homologous arm, splicing acceptor, first loxP site, CAT cDNA, poly A, frt flanked PGK neo cassette, second loxP site, Nsp12-ires-GFP, bGH polyA and 4.3 kb 3’ homology arm.
The above Nsp12 expression vector was linearized and introduced into RENKA ES cells (C57BL/6N) by electroporation. After selection using Geneticin, resistant clones were isolated and their DNA screened for homologous recombination by PCR using the following primer sets: rosa_F2731: 5’-CCA TGC TGG AAG GAT TGG AAC TAT GC-3’ and SA-R3: 5’-AGG ATA AGT ATG ACA TCA TCA AGG-3’. PCR-positive ES clones were expanded, and DNA isolated from each clone was further analysed by PCR amplification using the following primer sets: rosa_F2731 and SA-R3 for 5’ amplification; and neo100: 5’-AGG TGA GAT GAC AGG AGA TC-3’ and rosa_R10578: 5’-AAG CTT ACC ATC AAC CTT ATA GTA CAC-3’ for 3’ amplification (Extended Data Fig.2a,b). Homologous recombination of these clones was also confirmed by genomic Southern blot hybridization using the neomycin resistance gene and the GFP gene as probes (Extended Data Fig.3a,b).
Homologous recombinant ES cell clones were aggregated with ICR 8-cell embryos to generate chimeric mice. F1 heterozygous mice showing germline transmission were obtained by crossing chimeric mice with a high contribution of the RENKA background to C57BL/6N mice. The targetted allele was identified by PCR using the following primer set: rosa_F2731 and SA-R3.
Genotyping of Rosa26-LSL-Nsp12 cKI mice
Heterozygous Rosa26-LSL-Nsp12fl/- mice were mated to each other to obtain homozygous Rosa26-LSL-Nsp12fl/fl mice, which were genotyped using the following primer set: rosa-F5982: 5’-TTT GGA GGC AGG AAG CAC TTG CTC TCC-3’, rosa-R6262: 5’-ACA CAC CAG GTT AGC CTT TAA GCC CTG-3’, GFP-F649: 5’-GAT CAC ATG GTC CTG CTG GAG TTC-3’. For PCR analysis, genomic DNA was subjected to 35 cycles of 30 sec at 94°C, 30 sec at 63°C, and 30 sec at 72°C to amplify the 441 bp product from the Rosa26-LSL-Nsp12 allele and the 281 bp product from the wild-type allele (Extended Data Fig. 4). KOD FX (TOYOBO, Osaka, Japan) was used for PCR.
CreERT2;Nsp12 conditional KI mouse
To examine the effects of lung-specific expression of the SARS-CoV-2 Nsp12 gene, Sftpc-CreERT2 transgenic mice (JAX database strain #0028054) were purchased from the Jackson Laboratory31. Sftpc-CreERT2 transgenic mice and Rosa26-LSL-Nsp12fl/- mice were interbred to generate lung-specific CreERT2;Nsp12 mice.
In vivo treatments of Nsp12 and CreERT2;Nsp12 mice
Treatment protocols are illustrated in Extended Data Fig. 6. To examine the in vivo effects of Nsp12 induction in lungs, lung-specific CreERT2;Nsp12 mice were intravenously injected on days 0, 1, 2, 3 and 7, 8, 9, 10 (see Extended Data Fig. 6a) with (Z)-4-hydroxytamoxifen (4-OHT) (5mg Kg-1, Lot #APN18056-5-1, Cat. #ab141943, Abcam, Cambridge, UK) in serum-free stem cell medium S-Clone SF-03 (-) that lacked BSA, cytokines and penicillin/streptomycin (Iwai North America Inc., Signal Hill, CA, USA). Nsp12 mice were intravenously injected on days 0, 1, 2, 3 and 7, 8, 9, 10 (see Extended Data Fig. 6b) with TAT-Cre recombinase (1,000 unit Kg-1, Lot #3906499 and #3941016, Cat. #SCR508, Merck Millipore, Burlington, MA, UAS) in SF-03 (-).
To examine the in vivo effects of ISR inhibitors (ISRis), Nsp12 mice or CreERT2;Nsp12 mice (simultaneously induced with 4-OHT or TAT-Cre as described above) were intravenously injected on day -1 and day 6 with ISRIB33 (Trans-isomer, 5mg Kg-1, Lot #S740001, Cat. #S7400, Selleckchem, Houston, TX, USA) in SF-03 (-) (see Extended Data Fig. 6c). Oral administration of A92 (ref.34) (GCN2-IN-1, 0.4mg Kg-1 Lot #30198, Cat. #HY-100877, MedChemExpress, Monmouth Junction, NJ, USA) in artificial gastric fluid solution (993ml ddH2O containing 2.0g NaCl, 7ml conc. HCl, and 3.2g pepsin) followed on days 0, 1, 2, 3 and 7, 8, 9, 10, 11.
To examine the in vivo effects of an RdRp inhibitor, CreERT2;Nsp12 mice (simultaneously induced with 4-OHT or TAT-Cre as described above) received oral administration of EIDD-2801 (ref.38-42) (10mg Kg-1, Lot #0000182503, Cat. #SML2873, Sigma-Aldrich, Merck, St. Louis, MO, USA) in artificial gastric fluid solution on days 0, 1, 2, 3 and 7, 8, 9, 10, 11 (see Extended Data Fig. 6d).
C57BL/6 mice were purchased as control mice from Crea Japan, Inc. (Tokyo, Japan). For all protocols, mice were humanely sacrificed by method on day 12 or at 12-22 weeks post-administration. All animal care and experiments were carried out at the animal facilities of the Research Institute for Radiation Biology and Medicine, and Natural Science Center for Basic Research and Development, Hiroshima University, in accordance with the guidelines for animal and recombinant DNA experiments of Hiroshima University (Authorized Protocol Numbers A20-111, A21-52, A22-95, 2020-60, 2020-199, 2021-61, 2021-97, and 2021-97-2).
Histology and immunofluorescence
For frozen lung and heart sections, tissues were fixed in Periodate Lysine Paraformaldehyde (PLP) solution (Lot #LEJ1749, Cat. #290-63201, Fujifilm Wako Chemical Corporation, Osaka, Japan) for 1 hr on ice. For lung fixations, bilateral lungs were injected with PLP solution through the trachea using a 26G needle and syringe. Paraffin-embedded sections (3 mm) were prepared using a Retratome REM-700 microtome (Yamato Kohki Industrial Co., Saitama, Japan). Frozen sections (10 mm) were prepared using a cryostat (7732, CM1950, Leica Microsystems GmbH, Wetzlar, Germany). For paraffin-embedded histological sections, mouse lung and heart tissues were fixed in 10% formalin natural buffer solution (Lot #ACN4253, Cat. #062-01661, Fujifilm Wako Chemical) for 8 hr on ice. Paraffin-embedded sections were subjected to haematoxylin and eosin (H&E) staining, whereas frozen sections were immunostained as described below. Histological images were acquired using optical microscopy (IX70, Olympus Corporation, Tokyo, Japan), CellSens Standard software (Ver 1.6 Build 9464, Olympus), and Photoshop software (Photoshop 2022.23.2.1. Adobe, San Jose, CA, USA).
Isolation of primary lung cells
Primary murine lung cells were isolated from bilateral lungs of Nsp12 or CreERT2;Nsp12 mice. For tissue digestion, lungs and heart were incubated with Dri Tumor & Tissue Dissociation Reagent (Lot #1166901, Cat. #661563, BD Biosciences, Franklin Lakes, NJ, USA) for 30 min at 37°C (pipetting 100 times, incubation for 15 min at 37°C, repeated for two cycles). The resulting homogenate was passed through a 40 mm filter (BD Biosciences) and red blood cells were removed by treatment with 0.17M NH4Cl solution for 2 min at room temperature. The cell suspension was washed twice with 5% FBS/2mM EDTA/PBS solution and once with 5% FBS/PBS. To remove haematopoietic cells and endothelial cells, the suspension was incubated with biotin-conjugated anti-mouse CD31 Ab (clone #390, Lot #B316988, Cat. #102404, Biolegend, San Diego, CA, USA), biotin-conjugated anti-mouse CD45 Ab (clone #30-F100, Lot #B360907, Cat. #103104, Biolegend), and biotin-conjugated anti-mouse CD16/32 Ab (clone #93, Lot #B355428, Cat. #101303, Biolegend) for 15 min on ice, and then with 100ml BD iMagTM Streptavidin Particle Plus (Lot #2217592, Cat. #557812, BD Biosciences) for 30 min on ice. Primary lung epithelial cells and heart cells were isolated by negative selection using the iMagTM system.
Primary murine lung cells isolated from Nsp12 or CreERT2;Nsp12 mice were cultured in a 3% O2 incubator at 37°C containing 200ml PneumaCultTM EX Plus medium (Lot #1000116700, Cat. #05040, Stemcell Technologies, Vancouver, British Columbia, Canada) supplemented with 5mM SB203580 (p38MAPK inhibitor, Lot #ARV-106, Cat. #S-3400, LC Laboratories, Woburn, MA, USA), 1mM CHIR-99021 (GSK-3b inhibitor, Lot #X104110, Cat. #10-1279, FOCUS Biomolecules, Plymouth Meeting, PA, USA), 10mM Y-27632 2HCl (Rock inhibitor, Lot #S104920, Cat. #S1049, Selleckchem), 5mM DMH1 (BMP inhibitor, Lot #S104920, Cat. #S1049, Selleckchem), 5mM A83-01 (ALK5, ALK4, ALK7 inhibitor, Batch #9B/238070, Cat. #2939, Tocris Bioscience, Avonmouth, Bristol, UK), 5ng ml-1 mouse Wnt3a, (Lot #HTR10115031, Cat. #1324-WN-002, R&D Systems, Minneapolis, MN, USA), 50ng ml-1 mouse R-Spondin 3 (Lot #TWP0815091, Cat. #4120-RS-025, R&D Systems), 20ng ml-1 EGF (Lot #08H27390, Cat. #02633, Stemcell Technologies), bFGF, 10ng ml-1 (Lot #08H27183, Cat. #026334, Stemcell Technologies), and 50ng ml-1 Noggin (Lot #SKR2092, Cat. #146-08991, FUJIFILM Wako Chemical), as described previously with modifications49.
In vitro treatment of primary murine lung epithelial cells
For activation of Cre recombinase, primary lung epithelial cells isolated from CreERT2;Nsp12 mice were treated with 1mM 4-OHT (Lot #APN18056-5-1, Cat. #ab141943, Abcam) for 2 days in a 3% O2 incubator at 37°C in 96-well, ultralow cluster, flat bottom plates (Lot #01711007, Costar 3471, Corning). Primary lung epithelial cells isolated from Nsp12 mice were treated with 50 unit ml-1 of TAT-Cre recombinase (Lot #3906499 and #3941016, Cat. #SCR508, Merck Millipore) for 2 days in a 3% O2 incubator at 37°C in 96-well, ultralow cluster, flat bottom plates (Lot #01711007, Costar 3471, Corning). For treatment in vitro with an ISR inhibitor, primary lung epithelial cells isolated from either mutant strain were pre-treated with 1mM ISRIB (Trans-isomer, Lot #S740001, Cat. #S7400, Selleckchem) or A92 (GCN2-IN-1, Lot #30198, Cat. #HY-100877, Med Chem Express) for 1 day in a 3% O2 incubator at 37°C in 96-well, ultralow cluster, flat bottom plates (Lot #01711007, Costar 3471, Corning). ISR inhibitor-treated cells were then incubated in the presence of 4-OHT (Lot, APN18056-5-1, Cat. #ab141943, Abcam) or TAT-Cre recombinase (Lot #3906499 and #3941016, Cat. #SCR508, Merck Millipore) for 2 days. Treated cells were twice washed with PBS and incubated with TrypLETM Express enzyme (Cat. #12604013, Invitrogen, Thermo Fisher Scientific) at 37°C for 10 min. To purify forward scatter (FSC)-high primary lung epithelial cells and deplete FSC-low haematopoietic cells, cells were passed through a FACS Aria III cell sorter (S/N, P64828201002, BD Biosciences) equipped with BD FACSDiva software ver. 6.1.3 (BD Biosciences). To assay mitochondrial activity of primary lung epithelial cells, cells were plated on microscope slides freshly precoated with poly-L-lysine (Lot # RNBB0228, Cat. #P4832, Sigma-Aldrich, Merck)50 and treated with 100nM MitoTrackerTM DeepRed FM (Lot #2325947, Cat. #M22426, Invitrogen, Thermo Fisher Scientific) for 1hr in a 3% O2 incubator at 37°C.
Fluorescence immunostaining
For fluorescence immunostaining of primary lung epithelial cells, cells were fixed with 4% PFA for 1 hr after in vitro culture as described above. Fixed cells were permeabilised with 0.25% Triton-X100 for 15 min, washed, and blocked by incubation in 2% BSA in TBS for 1 hr, as described50. For fluorescence immunostaining of frozen sections, sections were washed and blocked by incubation in 2% BSA in TBS for 3 hr. Blocked cells or frozen sections were incubated overnight at 4°C with rabbit anti-phospho-eIF2a (Ser51) (D9G8) Ab (dilution 1:25, Lot #8, Cat. #3398S, Cell Signaling Technologies, Danvers, MA, USA), rabbit anti-SARS-CoV-2 RNA-dependent RNA Polymerase NSP12 Ab (dilution 1:100, Lot #1, Cat. #67988S, Cell Signaling Technologies), mouse anti-SARS-CoV RdRp R2 Ab (Clone 4E6, dilution 1:100, Lot #101229, Cat. #ARG11093, Arigo biolaboratories)36, rabbit anti-Strep-tag II Ab (dilution 1:100, Lot #1029430-4, Cat. #ab76949, Abcam)37, mouse anti-ATP5H Ab (Clone 7F9BG1, dilution 1:100, Lot #YG3988954, Cat. #459000, Thermo Fisher Scientific, Waltham, MA, USA), and/or rabbit anti-HIF1a Ab (dilution 1:100, Lot #4, Cat. #3716, Cell Signaling Technologies). Primary mAbs were visualised by incubating the cells with AlexaFluor 488-conjugated donkey anti-rabbit IgG (dilution 1:200, Lot # 1387792, Cat. #A21206, Molecular Probes®, Thermo Fisher Scientific) and AlexaFluor 555-conjugated donkey anti-mouse IgG (dilution 1:200, Lot # 1476616, Cat. #A31570, Molecular Probes®, Thermo Fisher Scientific). Nuclei were stained with 5mM of the DNA marker DAPI (Cat. #340-07971, Dojindo Laboratories, Kumamoto, Japan). Stained slides were mounted using Prolong Diamond® (Thermo Fisher Scientific). Fluorescent images were acquired by confocal microscopy using either an FV10i instrument (Olympus Corporation, Tokyo, Japan) or a Stellaris 5 instrument (Leica Microsystems GmbH), and Photoshop software (Photoshop 2022.23.2.1. Adobe).
Transmission electron microscopy (TEM)
For TME analysis of primary cardiomyocytes from mouse heart, 1-2 mm of tissue pieces were fixed in 2% formaldehyde (P6148, Sigma-Aldrich), 2% glutaraldehyde (071-02031, Fuji Film Wako Chemicals Corporation) in 0.1M cacodylate buffer (pH. 7.4) solution (039-18165, Fuji Film Wako Chemicals) for 2 days at 4°C. Tissue samples were washed four times 15 min in 0.1M cacodylate buffer, post-fixed with 2% osmiumtetroxid in 0.1 M cacodylate buffer, and washed again four times 15 min in 0.1M cacodylate buffer. The samples were dehydrated in ethanol (30, 50, 70, 90, and 100%) and embedded in the epoxy resin (TAAB EPON 812, TAAB Laboratories Equipment Ltd., Berkshire, UK). The fixed samples were cut in 80 nm sections on the ultramicrotome (UC7, Leica Microsystems GmbH) on a grid (Cu 150 mesh, EM fine grid, Nisshin EM, Tokyo, Japan). Ultrathin sections were stained with 2.0% uranylacetate aqueous solution 10 min, and lead staining solution for 5 min at room temperature. TEM (JEOL, JEM-1400, Tokyo, Japan) with a tungsten filament was used at 80kV at room temperature.
Statistical analyses and reproducibility
All experiments were repeated at least three times.