Uncropped and unadjusted blots.

(TIF)</p

Analysis of <i>ACE2</i>, <i>TMPRSS2</i>, and <i>FURIN</i> expression in human alveolar-like organoids.

Expression of ACE2, TMPRSS2, FURIN and GAPDH and β-ACTIN as housekeeping genes measured by qPCR on bulk RNA of human alveolar-like organoids. Shown are Ct values of four different donors (P4-P7) and two technical replicates.</p

Replication kinetics of SARS-CoV-2-infected human alveolar-like organoids.

Human alveolar-like organoids were infected with SARS-CoV-2 (MOI = 1) and viral replication was assessed by plaque assay (A) and via viral E gene-based RT-qPCR (B). Data of seven (plaque assay) and four (RT-qPCR) biological replicates are shown as individual data points. The mean is visualized by a horizontal black line.</p

Delayed cytopathic onset of VOC Alpha SARS-CoV-2 infection.

Vero E6 cells were infected with B.1, VOC Alpha/v1, and VOC Alpha/v2 (MOI 0.001). Onset of CPE was monitored by live cell imaging until 70 hours postinfection. CPE, cytopathogenic effect; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; VOC, variant of concern. (MP4)</p

Primers used for reconstruction of recombinant SARS-CoV-2.

(XLSX)</p

Relative sgRNA level normalized to total RNA reads and infection efficiency in B.1- and VOC Alpha-infected Calu-3 cells.

(A) RNA-seq analysis was conducted from total cell lysates that were obtained 24 hours postinfection to quantify sgRNA proportions in SARS-CoV-2-infected cells (MOI of 2). Canonical, as well as ORF9b and N* sgRNAs were quantified from the RNA-seq dataset. Data were normalized to total RNA reads. (B, C) Number of SARS-CoV-2 nucleocapsid (N)-positive Calu-3 cells was determined by flow cytometry. Calu-3 were left either UI or were infected with B.1 and VOC Alpha (MOI of 2) for 24 hours, permeabilized and immunostained with rabbit-anti-SARS-CoV-2 nucleocapsid antibody, followed by goat anti-rabbit Alexa 488 secondary antibody. (B) Percentage of SARS-CoV-2 N-positive cells. (C) Gating strategy of living-, single-, and N-positive cells is depicted for UI, B.1-, and VOC Alpha-infected cells. MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, subgenomic RNA; UI, uninfected; VOC, variant of concern. See S1 Data. (TIF)</p

VOC Alpha and B.1 efficiently dampen induction of innate immunity in hBAECs.

hBAECs were infected with B.1 or VOC Alpha (MOI of 0.5) and cell lysates were generated at the indicated time points followed by total RNA extraction. The experiment was performed with cells derived from 1–5 adult donors and that were infected in duplicates. (A) Cell-associated expression of envelope in hBAECs during the early phase of infection determined by Q-RT-PCR. TBP was used for normalization. (B) Cell-associated expression of sgN in hBAECs during an early phase of infection determined by Q-RT-PCR. (C–I) Expression of the indicated genes was determined by Q-RT-PCR. Shown is the mean fold change +/− SD. (J) Relative change (to preinfection) of cytokines and chemokines concentration in the basal medium of infected hBAECs (MOI 0.5). Concentration of cytokines and chemokines was determined by MagPix Luminex technology. Paired t tests were conducted between B.1 and VOC Alpha-infected groups and scored negative. AEC, airway epithelial cells; hBAEC, human bronchial airway epithelial cell; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein; VOC, variant of concern. See S1 Data.</p

VOC Alpha spike is not superior in mediating entry compared to B.1 spike.

(A) Calu-3 cells were transduced for 72 hours with increasing amounts of lentiviral particles (0.1 μl, 1 μl, and 10 μl) pseudotyped with either B.1 or VOC Alpha spike proteins. Pseudotype entry was analyzed luminometrically in cell lysates. (B) Calu-3 cells were pretreated with 25 μM MDL28170 (Cathepsin L inhibitor), 25 μM pitstop II (clathrin inhibitor), 100 μM Camostat (TMPRSS2 inhibitor), or 15 μM CMK (furin inhibitor), infected and entry efficiency was determined by sgN Q-RT-PCR. (C) Calu-3 cells were infected with Calu-3-derived virus stocks. Entry efficiency was determined by sgN-specific Q-RT-PCR from cell lysates at 4 hours postinfection. Cam, Camostat mesylate; CatL, Cathepsin L; DMSO, Dimethylsulfoxid; PS: PitStop; Q-RT-PCR, quantitative real-time PCR; sgN, subgenomic nucleocapsid; TMPRSS2, transmembrane protease serine subtype 2; VOC, variant of concern. See S1 Data. (TIF)</p

Primers used for quantitative RT-PCR.

(XLSX)</p

Similar abundance of sgN RNAs, genome replication, and low but similar expression of IFNs, proinflammatory cytokines, and ISGs in B.1 and VOC Alpha-infected H1299 cells.

NCI-H1299 cells were infected with B.1 or VOC Alpha (MOI of 2), and viral replication, viral transcription, and expression of innate immune genes were determined by Q-RT-PCR from cell lysates at 24 and 48 hours postinfection. (A) Expression of cell-associated envelope. (B) Expression of cell-associated sgN RNA. TBP was used for normalization. (C) Expression of the indicated genes was determined by specific Q-RT-PCR. TBP was used for normalization. Shown is the mean fold change +/− SD of 3 biologically independent experiments that were each conducted in quadruples. RVFV cl.13, which is devoid of its IFN antagonist NSs, was included for the expression of IFNs, ISGs, and pro-inflammatory cytokines. GE, genome equivalents; IFN, interferon; ISG, IFN-stimulated gene; Q-RT-PCR, quantitative real-time PCR; RVFV cl.13, Rift Valley Fever Virus clone 13; sgN, subgenomic nucleocapsid; TBP, TATA-binding protein. See S1 Data. (TIF)</p