Extended Data Fig. 6: Cellular source of persistent SARS-CoV-2 viral RNA and sustained viral replication in lungs (matched to figure 2).

a. Quantification of genomic (gRNA) and subgenomic (sgRNA) viral RNA (E-gene) in whole homogenized lung tissue at 4, 14 and 28 d.p.i. 4 d.p.i.: n = 7, 14 d.p.i. n = 5, 28 d.p.i. n = 4 biologically independent mice examined over 3 independent experiments. Means with all datapoints and SD. b. Quantification of genomic (gRNA) and subgenomic (sgRNA) viral RNA (E-gene) in whole homogenized lung tissue at 14 d.p.i. in mice treated with combined therapy of Remdesivir and anti-IFNAR2. CTRL: n = 4, anti-IFNAR2+RDV: n = 4 biologically independent mice examined over 2 independent experiments. N.D.= not detected. c. Quantification of genomic (gRNA) and subgenomic (sgRNA) viral RNA (E-gene) in whole homogenized lung tissue at 28 d.p.i. in mice treated with Remdesivir, anti-IFNAR2 or combined therapy of Remdesivir and anti-IFNAR2. N = 3 biologically independent mice representative of 2 independent experiments. N.D.= not detected. d. Representative gating strategy for sorting human immune cells (human CD45⁺) or mouse epithelial cells (mouse EPCAM⁺) from lungs of mice infected with SARS-CoV-2 and quantification of viral RNA (E and N genes) in these sorted cells. N gene: 4 d.p.i. n = 3, 14 d.p.i. n = 6(epithelial), n = 5 (immune), 28 d.p.i. n = 4 (epithelial) n = 3 (immune) biologically independent mice analysed over 3 independent experiments. E gene: 4 d.p.i. n = 3, 14 d.p.i. n = 7 (epithelial), n = 6 (immune), 28 d.p.i. n = 4 (epithelial) n = 3 (immune) biologically independent mice analysed over 3 independent experiments. e. mNG signal in epithelial (EPCAM⁺) cells from lungs and BAL of mice infected with reporter SARS-CoV-2-mNG or control wild type SARS-CoV-2/WA1. mNG is expressed in infected cells following viral replication. Representative of n = 4 biologically independent mice examined over 2 independent experiments. f. Representative histograms of mNG expression in human or mouse lung macrophages isolated from BAL of infected MISTRG6-hACE2 mice at 4 d.p.i. Representative of n = 3 biologically independent mice examined over 2 independent experiments. g. Frequencies of mNG+ cells within human lung immune cells (hCD45⁺) of SARS-CoV-2-mNG infected MISTRG6-ACE2 mice at 4 d.p.i. and 14 d.p.i. 4 d.p.i. n = 4, 14 d.p.i. n = 6 biologically independent mice examined over at least 2 experiments. Unpaired, two-tailed t-test. P value = 0.066. h. Viral titres measured as PFU using Vero ACE2⁺TMPRSS2⁺ cells that over express human ACE2 from lung homogenates of MISTRG6 mice transduced with AAV-hACE2 (+AAV) or not (−AAV) and infected with SARS-CoV-2. MISTRG6-hACE2 (+AAV): 2 d.p.i. n = 2, 4 d.p.i. n = 5, 7 d.p.i. n = 2, 14 d.p.i. n = 6 MISTRG6(−AAV): 2 d.p.i. n = 4, 4 d.p.i. n = 10 and 7, 14 d.p.i. n = 2, biologically independent mice representative of at least 2 independent experiments. Viral titres using standard Vero E6 cells do not have any detectable titres (previously reported¹⁹) in MISTRG6 mice without AAV-hACE2. Some of the MISTRG6-hACE2 data presented here have been previously reported as part of the characterization of the model¹⁹. i. Frequencies of mNG⁺ cells within human macrophages (human CD68⁺) isolated from lungs of infected MISTRG6 mice transduced with AAV-hACE2 (AAV+) or not (AAV−). MISTRG6 mice with and without AAV-hACE2 were reconstituted with human progenitor cells from the same donor. AAV+ n = 6, AAV− n = 5 biologically independent mice examined over 3 independent experiments. j. Representative gating strategy for sorting mNG⁺ and mNG⁻ human immune cells, mNG⁺ and mNG⁻ mouse epithelial cells and mouse immune cells. Lung cells from SARS-CoV2-mNG infected MISTRG6-hACE2 mice were stained with antibodies against human CD45, mouse CD45, and mouse EPCAM. Sorted cells were used for viral RNA quantification (Fig. 2) and characterization of the inflammasome pathway (Fig. 3).

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