Fig 4.

Enhancement of VLP release by the inhibition of lysosomal acidification. (A) Effect of bafilomycin A1 (BafA1) on VLP release. Thirty-six hours after transfection, the VLPs in culture supernatants from the HEK293T cells expressing the indicated SARS-CoV-2 structural proteins and treated with or without 50 nM of BafA1 were purified using serial centrifugation. HiBiT activities in the cells and supernatant fractions were measured. The secretion rate was calculated by dividing the culture supernatant HiBiT value by the total HiBiT value of the cell culture. After calculating the secretion rate, the fold-change secretion was determined based on that of cells expressing E + M + N + S. *P < 0.05; ns, not significant (Tukey’s t-test). (B) Expression of the SARS-CoV-2 ORF3A protein. SARS-CoV-2 ORF3A-expressing vectors were transfected into HEK293T cells. Thirty-six hours after transfection, the expression of ORF3A protein (upper panel) or alpha-tubulin (lower panel) was confirmed using western blotting and specific antibodies. (C) Effect of ORF3A protein expression on VLP release. Thirty-six hours after transfection, the VLPs in the culture supernatants from the HEK293T cells that expressed the indicated SARS-CoV-2 structural proteins with or without ORF3A protein were purified using serial centrifugation. HiBiT activities in the cells and supernatant fractions were measured. The secretion rate was calculated by dividing the culture supernatant HiBiT value by the total HiBiT value of the cell culture. *P < 0.05; ns, not significant (Tukey’s t-test). After calculating the secretion rate, the fold-change secretion was determined based on that of cells expressing E + M + N + S. **P < 0.01; ***P < 0.001; ns, not significant (Tukey’s t-test).