Figure 4.

The route of SARS-CoV-2 S-mediated virus entry in cell lines is not altered by ΔH69/V70 spike

(A) Schematic illustrating spike in producer cells with CMK targeting and blocking furin cleavage (left panel). In target cells, camostat inhibits TMPRSS2 and, therefore, cell fusion at the plasma membrane, and E64D blocks cathepsins and targets endocytic viral entry (right panel).

(B) Western blots show that CMK inhibits spike S1/S2 cleavage in producer cells transfected with the S ΔH69/V70 plasmid, and spikes with altered S1/S2 cleavage are incorporated into the virions. Antibodies against HIV-1 p24 and spike S2 were used with anti-GAPDH as a loading control.

(C) The viruses produced from transfected HEK293T cells in the presence of CMK were used to transduce target cells. The luciferase reading is used as a surrogate for the spike infectivity bearing with various S2/FL ratios. The data shown are technical triplicates or quadruplicates, and statical analysis was done using an unpaired t test.

(D) Comparison of the infectivity of spike with the PBCS deleted (ΔPBCS) with and without ΔH69/V70. The effect of ΔH69/V70 is independent of the PBCS.

(E) ΔH69/V70 does not alter the virus entry route. S pseudotyped lentiviruses bearing WT S, ΔH69/V70 S, or VSV-G were used to transduce 293T-ACE2 or 293T-ACE2/TMPRSS2 cells in the presence of E64D or camostat at different drug concentrations. The cells were then harvested after 2 days and assayed for luciferase expression, which was then normalized against the non-drug control (set as 100%). The data shown are technical duplicates.

The data are representative of at least two independent experiments.