Diltiazem inhibits the internalization of SARS-CoV-2.

(A) Cells were incubated with HRB25 (M.O.I. = 10) for 1 h at 4°C, then washed with acid buffer/trypsin to remove bound virus and lysed for qPCR to detect SARS-CoV-2. (B and C) Diltiazem-treated cells were incubated with HRB25 (M.O.I. = 1 or 10) at 4°C for 1 h and washed with PBS, and then shifted to 37°C for 1 h; they were then washed with PBS (B) or acid buffer/trypsin (C). The washed cells were lysed for qPCR to detect SARS-CoV-2 binding to cells (B) or internalized into cells (C). (D) HEK293T cells transiently expressing ACE2 and TMPRSS2 were treated with DMSO, E64D, camostat mesylate, or diltiazem, and then infected with HRB25 (M.O.I. = 5). The viral RNA level in the cell lysate was measured by qPCR at 6 h post-infection. (E) Schematic representation of the SARS-CoV-2 S protein-mediated cell-cell fusion assay. (F) Representative images of DMSO-, camostat mesylate-, or diltiazem-treated cell-cell fusion. Scale bar: 4 × field, 1000 μm; 10 × field, 400 μm; and 20 × field, 200 μm. The data shown are the means ± SDs of three independent experiments or replicates. The two-tailed unpaired Student’s t-test was used for the statistical analysis. ns, not significant, **p < 0.01, ***p < 0.001.