FIG 2.

Hypervariable NTD loops control S protein stability and membrane fusion potential. (a) Plasmids encoding SARS-CoV-2 spike (S), envelope (E), membrane (M), and amino-terminal HiBiT-tagged nucleoprotein (HiBiT-N) were expressed in HEK293T cells, supernatants (sup) were harvested after 2 days, and HiBiT-VLPs were purified by size exclusion chromatography. VLPs were compared as isogenic pairs: SARS-1 versus SARS-1/2 (panels b to d), SARS-2 versus SARS-2/1 (panels e to g), and D614G-2 versus D614G-2/1 (panels h to j). Each pair was evaluated by Western blot (left), cell entry (middle), and cell-free fusion (right) assays. Western blot assays detected uncleaved S (S-unc), S1, S2, and HiBiT-N. Cell entry assays detected HiBiT-VLP entry into ACE2-LgBiT/hTMPRSS2 target cells. The cell entry data are presented relative to the cell entry of control inoculations of spikeless (No S) VLPs. Cell-free fusion data are presented as HiBiT-VLP:ACE2-LgBiT EV fusion levels relative to data under control conditions with spikeless VLPs. For the cell entry and cell-free fusion data, the error bars present standard deviations (SD) from three technical replicates (n = 3), with data being representative of three biological repeats.