Evolutionary remodelling of N-terminal domain loops fine-tunes SARS-CoV-2 spike. (2022)
Figure 2

Figure 2. NTD deletions are necessary for efficient entry by Alpha and Omicron spike. Lentiviral pseudovirus (PV), encoding a luciferase reporter gene, were used to evaluate spike-mediated entry by Wu-Hu-1 (Wuhan-Hu-1 reference strain) and Alpha.

A. Cellular expression and PV incorporation of spike were assessed by Western blotting using an anti-S2 mAb. Lentiviral capsid components were detected using anti-p24/55.

B. Representative raw luciferase activity upon PV infection of the stated cell lines, annotated values represent fold enhancement of Alpha entry compared to Wu-Hu-1, n=4 technical repeats.

C. Alpha infection of HeLa ACE2 and HEK 293T cells relative to Wu-Hu-1 control. Data points represent the mean of independent experiments, n=8 biological repeats.

D. Surface expression of ACE2 (goat anti-ACE2) in HeLa ACE2 and HEK 293T cells was assessed by flow cytometry, negative control represents cells incubated with secondary antibody only.

E. ACE2 RNA transcripts were assessed by qPCR, abundance is expressed relative to negative control (no-RT). Representative data, n=2 technical repeats.

F. Western blot detection of ACE2 (rabbit anti-ACE2) in cell lysates of the stated cells. ACE2 was detected in HEK 293T samples after loading 3X more lysate and increasing signal exposure time (labelled in boldface).

G. Representative raw luciferase activity after PV infection of HEK 293T cells preincubated with 20µg/ml goat anti-ACE2, n=4 technical repeats.

H. Alpha and Middle Eastern respiratory syndrome-CoV PV infection of HEK 293T cells treated with a serial dilution of anti-ACE2, data points are expressed relative to untreated control cells and represent the mean of n=3 biological repeats.

I. Raw luciferase values after Wu-Hu-1 and Alpha PV infection of HEK 293T cells transfected to over-express ACE2 or TMPRSS2, annotated values represent fold enhancement compared to Wu-Hu-1, n=8 technical repeats.

J. Over-expression confirmed by Western blotting, actin and CD81 were used as loading controls for ACE2 and TMPRSS2 respectively.

K. Entry of PV bearing the stated spike proteins into HeLa ACE2 and HEK 293T. Infection is expressed relative to Wu-Hu-1, data points represent mean values from n=8 (HeLa ACE2) and 4 (HEK 293T) biological repeats.

L. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting. The displayed samples are taken from the same image and have been cropped to remove irrelevant lanes/bands.

M. Entry of PV bearing the stated spike proteins into HeLa ACE2 and HEK 293T. Infection is expressed relative to Wu-Hu-1, data points represent mean values from n=5 biological repeats.

N. Cellular expression and PV incorporation of the stated spike proteins were assessed by Western blotting, note the change in sample order relative to panel M.

Data information: In all plots, error bars indicate standard error of the mean, statistical analysis (T-test and ANOVA) performed in GraphPad Prism. Fitted curves were determined to be statistically different using an F-test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, ns = not significant. Comparisons performed against control Wu-Hu-1 apart from in (K and M) where statistical analyses are performed relative to respective parental spike i.e. Wu-Hu-1 Δ69/70 Δ144 vs Wu-Hu-1 and Alpha +H69 +V70 +Y144 vs Alpha.