SARS-CoV-2 spike engagement of ACE2 primes S2′ site cleavage and fusion initiation

Significance The SARS-CoV-2 spike protein is responsible for host receptor recognition, membrane fusion, and viral infection. Understanding the cellular and inhibiting the molecular mechanisms of spike-driven viral entry is a research priority in curbing the ongoing pandemic and preventing future coronavirus outbreaks. Here, we highlight that the generation of SARS-CoV-2 S2′ fragments, a proteolytic event occurring within the S2 subunit, is a molecular switch coupled to membrane fusion. Downstream of host receptor recognition, spike-driven syncytia formation requires the presence of an S2′ cleavage site at arginine 815 but not 685. Hence, the proteolytic processing of spike at the S2′ site upon its engagement of host ACE2 may serve as a potential antiviral target against the current SARS-CoV-2 and related coronavirus strains.


Silver staining and mass spectrometry
For purification of the S2' protein fragment, 1x10 6 HEK293T cells transiently expressing S and ACE2 were co-cultured at 1:1 ratio in a T25 flasks. After incubation for 16 hours at 37 o C, syncytia lysates were washed once in cold PBS before lysed in NP-40 lysis buffer supplemented with 1x EDTA-free protease inhibitor cocktail. Post-nuclear syncytia lysates were incubated with 1.5 μg rabbit anti-S2 antibody for 1 hour at 4 o C, before immunoprecipitated with Protein A/G magnetic beads. Pull-down proteins were then washed, eluted and boiled in the 2 x Laemmli loading buffer.
Total lysates (Input) and immunoprecipitates (IP) were separated by reducing SDS-PAGE on a 7.5% Tris-glycine gel before rinsed in fixation buffer (50% methanol 5% sodium acetate) for 20 min. The gel was sensitized in 0.02% sodium thiosulfate before reacted with 0.1% silver nitrate for 20 min. The gel was eventually developed in 2% sodium carbonate containing 0.045% formalin before rinsed and captured digitally; the S2' band was cut and subjected to in-gel digestion by 12.5 ng/μL trypsin in 25 mM NH 4 HCO 3 .
Mass spectrometry (MS) analysis was performed by Shanghai Applied Protein Technology Co.
Ltd. Briefly, a quadrupole Orbitrap mass spectrometer (Q Exactive) mass spectrometer coupled to Easy nLC (ThermoFisher) was operated in the positive ion mode. MS survey scans (300-1800 m/z) were obtained using the higher collision-energy dissociation (HCD) method in a datadependent acquisition mode. Normalized collision energy was 30 eV and the underfill ratio was set as 0.1%. The instrument was run with peptide recognition mode enabled. MS spectra were searched using MASCOT v2.2 (Matrix Science) against SARS-CoV-2 spike using the Uniprot reference P0DTC2; decoy hits were used to control the false discovery rate.
Immunofluorescence and confocal microscopy HEK293T cells were seeded overnight onto sterilized poly-D-lysine (100 ug/mL) (Sigma) treated 10 mm coverslips in 24-well plates at a density of 1.5 x 10 5 cells per well. After transfection with spike mutants, cells were washed with cold PBS once before fixed with 4% (w/v) paraformaldehyde (PFA) for 20 min. Fixed cells were blocked in 1% (w/v) goat serum in PBS without membrane permeabilization. Anti-SARS-CoV-2 S1 monoclonal antibody in 0.1% (w/v) goat serum in PBS was incubated for 1 hour at room temperature. Coverslips were then washed 3 times with PBS before incubation with goat anti-mouse AlexaFluor555 secondary antibodies (ThermoFisher) for 1 hour at room temperature; secondary-antibody-only controls were performed. Coverslips were washed 3 times before being mounted in neutral mounting medium (Xinyu). Fluorescent images covering various areas on the coverslips were captured at 14-bit depth in monochrome using a 60x oil immersion objective on the Olympus FV-1200 inverted 4 Figure

Figure S6, Spike arginine 815 is essential for syncytia formation and viral infection. (A)
Immunoblots showing the full-length S, S2, S2', ACE2 and tubulin detected from HEK293T cells  from cell-cell fusion lysates, treated without or with 10 μg/mL trypsin for 3 and 6 hours using the system described in Figure 1G. Data are shown as individual points with mean ± SEM from five independent experiments. (D) Luciferase activity (RLU) detected from S-WT, S-R685A or S-R815A spike variants induced cell-cell fusion lysates in the absence or presence of 10 μg/mL trypsin for 6 hours, employing the system described in Figure 1G. Data are shown as individual points with mean ± SEM from five independent experiments. P values were obtained by one-way ANOVA with Sidak's post hoc test and are indicated on figures.