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

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

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

(XLSX)</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

Competition assay, additional targets.

Calu-3 cells were infected with a mixture of B.1 and VOC Alpha at indicated ratios (B.1:VOC Alpha/v1 ratio of 1:1, 9:1, and 1:9) with a total infectious dose of 10,000 PFU (corresponding to an MOI of 0.04). After serial passaging, viral RNA from the supernatant was isolated, sequenced, and the relative proportion of B.1- and VOC Alpha-corresponding sequences, discriminated by mutations in NSP3 (A), Spike amino acid positions 501 (B) and 681 (C) was plotted. Data show individual values of triplicates of 1 experiment. MOI, multiplicity of infection; PFU, plaque-forming units; p0-p5, passage 0–passage 5; VOC, variant of concern. See S1 Data. (TIF)</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

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

VOC Alpha spike protein shows decreased proteolytic processing.

(A) Spike processing in lysates of HEK293T cells expressing empty vector or SARS-CoV-2 spike-HA encoding individual or all VOC Alpha-corresponding mutations was quantified by immunoblotting (upper panel). At least 4 independent biological replicates (using independent lentivirus particle preparations) were performed. Shown is 1 representative immunoblot (bottom panel) out of 4. (B) Protein in lysed lentiviral particles pseudotyped with SARS-CoV-2 spike-HA was quantified by immunoblotting (upper panel). Shown is 1 representative immunoblot (bottom panel) out of 4. (C) Vero E6 cells were infected with SARS-CoV-2 (MOI 5). Cells and virus-containing supernatants were harvested at 48 hours postinfection and processed for detection of spike and nucleocapsid by immunoblotting. Processing of spike in cell lysates (left panel) and spike incorporation in concentrated virion preparations (middle panel) was quantified. One representative blot out of 2 is shown (right panel). Black and white arrowheads indicate the bands of the uncleaved spike-HA precursor and of the cleaved S2-HA subunit, respectively. Statistical significance was calculated by a 2-tailed, paired Student t test. kDa, kilodalton; MOI, multiplicity of infection; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; UI, uninfected; VOC, variant of concern. See S1 Data.</p

VOC Alpha and B.1 share efficient prevention of induction of innate immunity despite VOC Alpha-specific enhanced production of viral subgenomic RNA transcripts.

Calu-3 cells were infected with B.1 or VOC Alpha (MOI of 2) and cell lysates were generated either 24 or 48 hours postinfection, following total RNA extraction. Samples were generated in 4 individual experiments each conducted in quadruplicates. (A) Expression of cell-associated envelope was determined in Calu-3 at 24 and 48 hours postinfection by Q-RT-PCR. TBP was used for normalization. (B) Expression of cell-associated sgN in Calu-3 cells at 24 and 48 hours postinfection was determined by Q-RT-PCR. TBP was used for normalization. (C) RNA-seq analysis was conducted from total cell lysates that were obtained 24 hours postinfection to quantify sgRNA proportions in infected cells. Stacked bars depict the relative proportion of total sgRNA in Calu-3 cells mapping to each viral sgRNA from the RNA-seq dataset. (D) Canonical, as well as ORF9b and N* sgRNAs were quantified from the RNA-seq dataset. Data were normalized to the respective B.1- or VOC Alpha-specific genomic reads. (E) Expression of the indicated genes was determined by Q-RT-PCR. Shown is the mean fold change +/− SD. RVFV cl.13, which is devoid of its IFN antagonist NSs, was included as an induction control for the expression of IFNs and ISGs. IFN, interferon; MOI, multiplicity of infection; Q-RT-PCR, quantitative real-time PCR; RVFV cl.13, Rift Valley Fever Virus clone 13; sgN, subgenomic nucleocapsid; sgRNA, subgenomic RNA; TBP, TATA-binding protein; VOC, variant of concern. See S1 Data.</p