HEK293T cells (a human embryonic kidney cell line; ATCC, CRL-1573), HEK293 cells (a human embryonic kidney cell line; ATCC, CRL-1573) and HOS-ACE2/TMPRSS2 cells (HOS cells stably expressing human ACE2 and TMPRSS2) (25) were cultured in high glucose Dulbecco’s Modified Eagle Medium (DMEM, high glucose) (Wako, Cat# 044-29765) supplemented with 10% fetal bovine serum (FBS) (NICHIREI, Cat# 175012) and 1% penicillin/streptomycin (P/S) (Wako, Cat# 168-23191). VeroE6/TMPRSS2 cells (VeroE6 cells stably expressing human TMPRSS2; JCRB Cell Bank, JCRB1819) (26) were maintained in DMEM (low glucose, Wako, Cat# 041-29775) containing 10% FBS, G418 (1 mg/ml; Wako, Cat#070-06803) and 1% P/S. Calu-3 cells (ATCC, HTB-55) were maintained in EMEM (Wako, Cat#055-08975) containing 20% FBS and 1% P/S. Calu-3/DSP_1-7 cells [Calu-3 cells stably expressing dual split protein_1-7 (DSP_1-7)] (27) were maintained in EMEM (Wako, Cat# 055-08975) containing 20% FBS and 1% P/S. All cells were maintained at 37°C with 5% CO₂.

SARS-CoV-2 Wuhan variant (strain SARS-CoV-2/Hu/DP/Kng/19-020 strain, Genbank accession no. LC528232) (28) was provided by Drs. Tomohiko Takasaki and Jun-Ichi Sakuragi (Kanagawa Prefectural Institute of Public Health). SARS-CoV-2 Alpha (strain QHN001, GISAID ID: EPI_ISL_804007) (28), Beta (strain TY8-612, GISAID ID: EPI_ISL_1123289) (29), Gamma (strain TY7-503, GISAID ID: EPI_ISL_877769) (28, 29), Delta (strain TY11-927, GISAID ID: EPI_ISL_2158617), Lambda (strain TY33-456, GISAID ID: EPI_ISL_4204973), Mu (strain TY26-717, GISAID ID: EPI_ISL_4470503), Omicron BA.1 (strain TY38-873, GISAID ID: EPI_ISL_7418017) (23), and Omicron BA.2 (strain TY40-385, GISAID ID: EPI_ISL_9595859) (22, 30) variants were obtained from National Institute of Infectious Diseases. D614G-bearing B.1.1 (strain TKYT41838, Genbank accession no. LC606020) and BA.5 (strain TKYS14631; GISAID ID: EPI_ISL_12812500) (20, 22) variants were provided by Tokyo Metropolitan Institute of Public Health.

Virus propagation was performed as previously described (28, 30, 31). Briefly, VeroE6/TMPRSS2 cells (5 × 10⁶ cells) were seeded in a T-75 flask the day before infection. Virus was diluted in virus dilution buffer [1M HEPES, DMEM (low glucose), Non-essential Amino acid (gibco, Cat# 11140-050), 1% P/S] and the dilution buffer containing virus was added to the flask after removing the initial medium. After 1 hour of incubation at 37°C, the supernatant was replaced with 15 ml of 2% FBS/DMEM (low glucose) and cell culture was continued to incubate at 37°C until visible cytopathic effect (CPE) was clearly observed. Then, cell culture supernatant was collected, centrifuged at 300 × g for 10 minutes and frozen at –80°C as working virus stock. The titer of the prepared working virus was determined as the 50% tissue culture infectious dose (TCID₅₀) (20, 30, 32). The day before infection, VeroE6/TMPRSS2 cells (10,000 cells) were seeded in a 96-well plate and infected with serially diluted working virus stocks. The infected cells were incubated at 37°C for 4 days and the appearance of CPEs in the infected cells was observed by a microscope. The value of TCID₅₀/ml was calculated by the Reed-Muench method (33).

Plasmids expressing the codon-optimized SARS-CoV-2 S proteins of Wuhan (34), B.1.1 (S gene bearing D614G mutation) (34), Alpha (21), Beta (21), Gamma (35), Delta (21), Lambda (35), Mu (36), BA.1 (23), BA.2 (24), and BA.5 (20) variants were prepared in previous studies. For creation of S proteins with 19 amino acids deletion at the C-terminal end, each S gene was amplified with primers; forward, 5’-NNN NNG GTA CCA TGT TTG TGT TCC TGG TGC T-3´ and reverse 5’-GTG GCG GCC GCT CTA GAT TCA ACA ACA GGA GCC ACA GGA A-3’. The resulting PCR fragment was digested with KpnI (New England Biolabs, Cat# R3142S) and NotI (New England Biolabs, Cat# R3189L) and inserted into the corresponding site of the pCAGGS vector (37). All constructs were confirmed by Sanger sequencing (AZENTA) and the sequence data were analyzed by Sequencher v5.4.6 software (Gene Codes Corporation).

A SARS-CoV-2 S-based fusion assay with dual split protein (DSP) encoding Renilla luciferase (RL) and green fluorescence protein (GFP) genes was performed as previously described (38). On day 1, effector cells (i.e., S-expressing) and target cells (Calu-3/DSP_1-7 cells) were prepared at a density of 0.6-0.8 × 10⁶ cells/well in a 6 well plate. On day 2, to prepare effector cells, HEK293 cells were cotransfected with the S expression plasmids (400 ng) and pDSP_8-11 (39, 40) (400ng) using TransIT-LT1 (Takara, Cat# MIR2306). On day 3 (24 hours post transfection), effector cells were detached by pipetting and 16,000 effector cells/well were reseeded into a 96 well black plate (PerKinElmer, Cat# 6005225), and target cells were reseeded at a density of 1 × 10⁶ cells/2 ml/well in a 6 well plate. On day 4 (48 hours post transfection), the target cells were incubated with Enduren live cell substrate (Pomega, Cat# E6481) for 3 hours and then detached, and 32,000 target cells/well were added to a 96 well plate with effector cells. RL activity was measured at the indicated time points using a Centro XS3 LB960 (Berthhold Technologies). To measure the surface expression level of the S protein, effector cells were stained with rabbit anti-SARS-CoV-2 S S1/S2 polyclonal antibody (Thermo Fisher Scientific, Cat#PA5-112048, 1:100 dilution). Normal rabbit IgG (SouthernBiotech, Cat# 0111-01, 1:100) was used as negative control, and APC-conjugated goat anti-rabbit IgG polyclonal antibody (Jackson immunoresearch, Cat# 111-136-144, 1:50) was used as a secondary antibody. The expression level of the surface S proteins was detected by FACS Canto II (BD Biosciences) and analyzed by FlowJo software v10.7.1 (BD Biosciences). RL activity was normalized to the mean fluorescence intensity (MFI) of surface S proteins, and the normalized values are shown as fusion activity.

For pseudovirus production, lentivirus (HIV-1)-based luciferase-expressing reporter viruses were pseudotyped with the SARS-CoV-2 S protein (20, 22, 34, 41–43). HEK293T cells (2 × 10⁶ cells) were cotransfected with 2 μg of psPAX2-IN/HiBiT (44), 2 μg of pWPI-Luc2 (44) and 1 μg of plasmids expressing SARS-CoV-2 S protein with the C-terminal deletion of 19 amino acids (Δ19CT) using TransIT-LT1 according to the manufacturer’s protocol. After 2 days, cell culture supernatants were collected and filtered, and the pseudoviruses were stored at –80°C until use. The amount of pseudovirus prepared was quantified using Nano Glo HiBiT lytic detection system (Promega, Cat# N3040) because HiBiT-based luciferase activity is perfectly correlated with the amount of p24 antigen levels (44). Then, the same amount of pseudoviruses normalized by the HiBiT value was inoculated into HOS-ACE2/TMPRSS2 cells (10,000 cells/well/50μl) in a 96 well plate. At 2 days postinfection, the infected cells were lysed with a Bright-Glo Luciferase Assay System (Promega, cat# E2620) and the luminescent signal was measured using a Centro XS3 LB960 plate reader (Berthhold Technologies).

HIV-1-based BlaM-Vpr assay (45, 46) was modified to perform SARS-CoV-2 S-based BlaM-Vpr assay. To produce SARS-CoV-2 S pseudovirus with β-lactamase-Vpr (BlaM-Vpr), HEK293T cells (2 × 10⁶ cells) were cotransfected with 1.6 μg of psPAX2-IN/HiBiT, 1.6 μg of pWPI-Luc2, 0.8 μg of plasmids expressing SARS-CoV-2 S protein with Δ19CT and 0.8 μg of BlaM-Vpr expression plasmid, pMM310 (HIV reagent program, Cat# ARP-11444) using TransIT-LT1 according to the manufacturer’s protocol. At 2 days posttransfection, cell culture supernatants were collected, filtered, and mixed with 40% polyethylene glycol #6000 (Nacalai Tesque Cat# 10200-25), followed by incubation at 4°C for 16 hours. After spinning down at 1,700 × g for 40 minutes at 4°C, the viral pellets were resuspended in 10% FBS/DMEM (high glucose) and the amount of pseudovirus prepared was quantified using the HiBiT assay. Note that the 24 well-collagen plate used in the following step should be kept at 4°C to inhibit starting fusion. The same amount of pseudoviruses normalized by HiBiT value was inoculated into HOS-ACE2/TMPRSS2 cells (2 × 10⁵ cells/well/1 ml) in a 24 well-collagen plate on ice and spinoculation was performed at 1,500 × g for 1 hour at 4°C to allow virus-cell attachment. After 1 hour, the cell culture supernatant including pseudoviruses was replaced with warmed fresh DMEM (high glucose), followed by incubation for 3 hours at 37°C. Then, each well was washed with phosphate-buffered saline (PBS) and β-lactamase loading solution (Invitrogen, Cat# K1030) [CCF4 substrate, solution B, solution C and anion transport inhibitor (Invitrogen, Cat# K1156) in Opti-MEM] was loaded. The plate was covered with aluminum foil to avoid light exposure and kept for 2 hours at room temperature. After 2 hours, cells were harvested, washed with PBS twice, and fixed with 2% paraformaldehyde phosphate (Nacalai Tesque, Cat# 09154-85). The fluorescence intensities at 520 nm (Pacific Blue; uncleaved CCF4) and 447 nm (AmCyan; cleaved CCF4) were detected by FACS Canto II (BD Biosciences) and analyzed by FlowJo software v10.7.1 (BD Biosciences). The ratio of cleaved CCF4 signal to the sum of uncleaved and cleaved CCF4 signals was calculated and shown as entry efficiency.

The day before infection, 1 × 10⁴ VeroE6/TMPRSS2 and Calu-3 cells were seeded into a 96-well plate. Then, VeroE6/TMPRSS2 (100 TCID₅₀) and Calu-3 (5000 TCID₅₀) cells were inoculated with SARS-CoV-2 and incubated at 37°C for 1 hour. After washing, 180 μl of fresh cell culture medium was added. 15 μl of cell culture supernatant was harvested at the indicated timepoints and used for RT-qPCR to quantify the viral RNA copy number (see “RT-qPCR” section).

RT-qPCR was performed as previously described (32, 34, 43, 47–50). Briefly, 5 μl of culture supernatant was mixed with 5 μl of 2 × RNA lysis buffer [2% Triton X-100 (Nacalai Tesque, Cat#12969-25), 50 mM KCl, 100mM Tris-HCl (pH 7.4), 40% glycerol, 0.8 U/μl recombinant RNase inhibitor (Takara, Cat# 2313A)] and incubated at room temperature for 10 minutes. 90 μl of RNase free water was added and then 2.5 μl of diluted sample was used for real-time RT-PCR according to the manufacturer’s protocol with One step TB green PrimeScript PLUS RT-PCR Kit (Takara, Cat# RR096A) and primers for Nucleocapsid (N) gene; Forward N, 5’-AGC CTC TTC TCG TTC CTC ATC-3’ and Reverse N, 5’-CCG CCA TTG CCA GCC ATT C-3’. The viral RNA copy number was standardized using a SARS-CoV-2 direct detection RT-qPCR kit (Takara, Cat# RC300A). Fluorescent signals from resulting PCR products were acquired using a Thermal Cycler Dice Real Time System III (Takara).

Plaque assay was performed as previously described (21, 31, 48, 51). One day before infection, 1 × 10⁵ VeroE6/TMPRSS2 cells were seeded into 24 well plate and infected with SARS-CoV-2 (50,000, 5000, 500, and 50 TCID₅₀ respectively) at 37°C for 1 hour. 3% FBS and 1.5% carboxymethyl cellulose (Wako, Cat# 039-1335) containing mounting solution was overlaid, followed by incubation at 37°C. At 3 days postinfection, the cell culture medium was removed, and the cells were washed with PBS three times and fixed with 4% paraformaldehyde phosphate (Nacalai Tesque, Cat# 09154-85). The fixed cells were washed with tap water, dried, and stained with 0.1% methylene blue (Nacalai Tesque, Cat# 22412-14) in water for 30 minutes. The stained cells were washed with tap water and dried. The size of plaques was measured using Fiji software v2.2.0 (Image J).

As previously described, sample preparation for western blotting was performed with minor modifications (38, 50, 52–54). The cell culture supernatants containing pseudoviruses produced from the HEK293T cells (see “Pseudovirus assay” section and “BlaM-Vpr assay” above) and the HEK293 cells transfected with the S expression plasmids (see “SARS-CoV-2 S-based fusion assay” section above) were used for western blotting. For viral lysate preparation, at 2 days posttransfection, cell culture supernatants were collected, filtered, and subjected to ultracentrifugation using 20% sucrose (22,000 × g, 4˚C, 2 hours). Then, virions were dissolved in phosphate-buffered saline (PBS). To quantify HIV-1 p24 antigen in the pseudovirus, HiBiT-based luciferase activity in viral supernatants was determined with a Nano Glo HiBiT lytic detection system and translated into p24 antigen levels. After normalization with HiBiT value, the samples were diluted with 2 × SDS sample buffer [100 mM Tris-HCl (pH6.8), 4% SDS, 12% β- mercaptoethanol, 20% glycerol, 0.05% bromophenol blue] and boiled for 5–10 minutes at 100°C. For cell lysate preparation, the transfected cells were detached, washed twice with PBS, and lysed in lysis buffer [25mM HEPES (pH7.2), 20% glycerol, 125 mM NaCl, 1% Nonidet P40 substitute (Nacalai Tesque, Cat# 18558-54), protease inhibitor cocktail (Nacalai Tesque, Cat# 03969-21)]. Quantification of total protein in the cell lysates was done by protein assay dye (Bio-Rad, Cat# 5000006) according to manufacturer’s instruction. Then, cell lysates were diluted with 2 × SDS sample buffer and boiled for 5–10 minutes. After cooling down, viral (pseudovirus) and cell lysates were mixed with diluted sample buffer (proteinsimple, Cat# 99351). Then, 5 × Fluorescent Master mix (proteinsimple, Cat# PS-ST01EZ-8) was added at a ratio of 4:1. Simple Western System was used for protein analysis. For protein detection, the following antibodies were used: rabbit anti-SARS-CoV-2 S polyclonal antibody (Novus Biologicals, Cat# NB100-56578, viral lysate; 1:40, cell lysate; 1:200). mouse anti-HIV-1 p24 monoclonal antibody (HIV Reagent Program, ARP-3537, 1:500), mouse anti-α tubulin monoclonal antibody (Sigma-Aldrich, Cat# T5168, 1:300), anti-rabbit secondary antibody (proteinsimple, Cat# 042-206), and anti-mouse secondary antibody (proteinsimple, Cat# 042-205). Bands were visualized and analyzed using Compass for Simple Western v6.1.0 (proteinsimple).

A one-way ANOVA and Dunnett’s test were performed to compare the S1/S2 cleavage efficiency, plaque size, pseudoviral infectivity, and pseudovirus entry efficiency. Pearson’s correlation analysis was conducted to ascertain the relationship between two variables tested in this study. The P value was calculated by a two-tailed test. The magnitude of the linear relationship between two variables is classified into several groups according to prior studies (55, 56). GraphPad Prism software v8.4.3 (GraphPad Software) was used for these statistical tests. In the time-course experiments, we conducted a two-way ANOVA, incorporating both experimental conditions (namely, S protein and SARS-CoV-2 variants) and timepoints as indicator variables. These analyses were used to assess differences across all timepoints between the various experimental conditions. The time point zero was excluded from this analysis. The P value was calculated by a two-sided t test. Subsequently, familywise error rates (FWERs) were calculated using the Holm method. These analyses were performed in R v4.1.2 (https://www.r-project.org/).

SARS-CoV-2 S protein mediates the virus-infected cell membrane fusion (10, 11). To quantitatively monitor the fusion kinetics between effector cells expressing S protein from eleven SARS-CoV-2 variants (including the previous VOCs and VOIs) and target cell membranes, we performed S protein-mediated membrane fusion assay in Calu-3/DSP_1-7 cells ( Figure 1A ) (20, 22–24, 30, 32, 38, 42, 43, 50). In this assay, dual split protein (DSP), DSP_1–7 and DSP_8–11 was utilized (39, 40) and the reconstituted Renilla luciferase signal was measured over 24 hours ( Figure 1A ). Although the Alpha, Beta, Gamma, Delta Lambda, Mu, and BA.5 S protein expression levels were lower than that of the B.1.1 S protein (harboring the D614G mutation) on the transfected HEK293 cell surface, the surface S protein levels of the remaining variants were comparable ( Figure 1B ). Compared to B.1.1 S protein, the Wuhan S protein exhibited lower fusogenicity, while the Alpha, Beta, Gamma, Delta, Lambda, and Mu S proteins was significantly more fusogenic ( Figure 1C ). Notably, the Delta S protein exhibited profound fusogenicity (21, 23), being the highest of all tested S proteins ( Figure 1C ). The BA.1 and BA.2 S proteins exhibited lower fusogenicity compared to the B.1.1 S protein while the fusogenicity of the BA.5 S protein was not significantly different ( Figure 1C ). These data indicate that SARS-CoV-2 S protein fusogenicity is different before and after Omicron variant emergence.

The SARS-CoV-2 S protein S1/S2 cleavage is pivotal, affecting viral fusogenicity (57). For example, the level of Delta S protein S1/S2 cleavage is higher than that of the B.1.1 and BA.1 S proteins, being associated with higher fusogenic potential in the Delta variant (21, 23, 38, 47). To investigate whether the relationship between S protein S1/S2 cleavage and its fusogenicity could apply to S proteins from the other SARS-CoV-2 variants, we subjected HEK293 cells expressing each S protein to western blotting and quantified the full-length S and cleaved S2 band intensities ( Figures 2A, B ). Full-length S and S2 band intensity variations indicated that each S protein exhibits a different susceptibility to cellular protease-induced S1/S2 cleavage. Consistent with previous results (21, 23, 38), the Delta and Lambda S proteins displayed the highest S1/S2 cleavage efficiencies ( Figures 2A, B ). In addition, the Wuhan, BA.1, and BA.2 S protein S1/S2 cleavage was significantly lower than that of the B.1.1 S protein ( Figures 2A, B ). Finally, we investigated how fusogenicity and S1/S2 cleavage efficiency correlate among the eleven SARS-CoV-2 variants ( Figure 2C ). Remarkably, the correlation coefficient (r = 0.7730) indicated a strong positive correlation between fusogenicity and S1/S2 cleavage efficiency of all eleven SARS-CoV-2 S protein variants ( Figure 2C , Supplementary Table S1 ). In summary, these results demonstrated that SARS-CoV-2 S protein fusogenicity is strongly associated with S protein S1 and S2 subunit cleavage.

The Delta variant with higher fusogenic S protein reportedly forms larger plaques than the B.1.1 variant in infected VeroE6/TMPRSS2 cells (21). However, the BA.1 variant encoding the lower fusogenic S gene displays smaller plaques compared to the B.1.1 variant (23). These results suggest that S protein fusogenicity is potentially associated with viral infection, indicated the size of the plaques formed by clinical isolates. To address this question, we performed plaque assays using the eleven clinical isolates including the previous VOCs and VOIs and compared the plaque diameters formed by each clinical isolate in VeroE6/TMPRSS2 cells ( Figures 3A, B ). We observed that the Alpha, Beta, Gamma, Delta, Lambda, and Mu variants formed significantly larger plaques than B.1.1 ( Figures 3A, B ). In contrast, the BA.1, BA.2, and BA.5 variant-related plaque sizes were smaller compared to that of the B.1.1 variant ( Figures 3A, B ). Interestingly, the SARS-CoV-2 infection-induced plaque size in VeroE6/TMPRSS2 very strongly and positively correlated with S protein fusogenicity and S1/S2 cleavage efficiency ( Figure 3C , Supplementary Table S1 ; r = 0.8356 and 0.8222, respectively). These results indicate that SARS-CoV-2 S protein fusogenicity strongly influences the SARS-CoV-2 infection plaque size.

Next, we examined the correlation of S protein-mediated cell-free viral infection with S protein fusogenicity. We used HEK293T cells to produce HIV-1 virions pseudotyped with SARS-CoV-2 S protein carrying the C-terminal deletion of 19 amino acids (Δ19CT), normalized by HiBiT-based luciferase activity, and measured viral infectivity as described previously (20–24, 30, 32, 34, 47, 51). Our results revealed that the BA.5 S pseudoviral infectivity measured using the HOS-ACE2/TMPRSS2 system were significantly higher than that of the B.1.1 S pseudovirus while that of the Wuhan, Alpha, Beta, Lambda, Mu, BA.1 and BA.2 S pseudoviruses significantly decreased in HOS-ACE2/TMPRSS2 cells compared to B.1.1 ( Figure 4A ). We observed no statistically significant difference between the Gamma and Delta S pseudoviral infectivities and that of the B.1.1 S pseudovirus ( Figure 4A ). Notably, we noticed that the infectivity degrees of the eleven SARS-CoV-2 variant-derived S protein-mediated cell-free pseudoviruses did not correlate with the fusogenicity mediated by these S proteins ( Figure 4B , Supplementary Table S1 ; r = 0.0574). This discrepancy suggests that each S protein incorporation level into the pseudoviruses might vary, thereby leading to incorrect viral infectivity validation. To test this hypothesis, we analyzed the S protein incorporation levels in the viral particles using western blotting ( Figures 4C, D ). The S protein band intensity quantification revealed that compared to the B.1.1 S pseudovirus, Wuhan, Alpha, Mu, BA.1, BA.2, and BA.5 variant-derived S proteins incorporated less in the pseudovirus particles ( Figures 4C, D ). These data indicate variable S protein incorporation levels, prompting that viral infectivity per incorporated S protein should be evaluated. Although most SARS-CoV-2 S pseudoviral infectivity divided by S protein incorporation levels became comparable or lower than that of the B.1.1 S pseudovirus, only those of the BA.2 and BA.5 S pseudoviruses were significantly higher ( Figure 4E ). In addition, the r value between the cell-free pseudoviral infectivity per S protein and fusogenicity mediated by these S proteins changed from 0.0574 to –0.3849 and indicated moderate negative correlation ( Figures 4B, F , Supplementary Table S1 ). In summary, these data suggest that S protein fusogenicity might have little effect on pseudovirus infection in target cells.

The aforementioned pseudovirus assays indicated a weak correlation between S protein fusogenicity and SARS-CoV-2 S pseudoviral infectivity even after normalizing the S protein incorporation levels into the viral particles ( Figure 4F ). These data suggest that HIV-1 infection mechanisms, such as reverse transcription and integration, might affect the pseudovirus assay results. To address this hypothesis, we performed an assay using β-lactamase-Vpr (BlaM-Vpr) chimeric protein and S protein with Δ19CT enabling us to quantify cell-free virus entry efficiency into HOS-ACE2/TMPRSS2 cells ( Figure 5A ). The BlaM-Vpr chimeric protein is incorporated into pseudoviral particles and cleaves a CCF4 fluorescent dye substrate upon entry into a target cell, leading to change in the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm) ( Figure 5A ) (45, 46). The results indicated that all S pseudoviruses exhibited comparable or lower entry efficiencies compared to the B.1.1 S pseudovirus ( Figure 5B ). Similar to the pseudovirus assays ( Figures 5B, C ), we normalized the fluorescence signals indicating entry efficiency into the target cells by the S protein incorporation levels due to the variable S protein incorporation levels into the viral particles ( Figures 5C–E ). Interestingly, although the entry efficiency determined by BlaM-Vpr assays and normalized by the viral S protein level strongly correlated with the S pseudovirus infectivities per S protein incorporation levels ( Figure 5F , Supplementary Table S1 ; r = 0.7880), the relationship with S protein fusogenicity was very weak ( Figure 5G , Supplementary Table S1 ; r = 0.0368). These data support the results suggesting that the S protein fusogenic potential could have little effect on pseudovirus entry into the HOS-ACE2/TMPRSS2 cells.

To investigate the clinical isolate replication kinetics, we inoculated the eleven SARS-CoV-2 variants into VeroE6/TMPRSS2 and Calu-3 cells and measured the viral RNA levels in the cell culture supernatants using RT-qPCR ( Figures 6A, B ). Compared to the B.1.1 variant, the Lambda, BA.1, and BA.5 variants displayed slower replication kinetics in VeroE6/TMPRSS2 cells while the other variants were comparable ( Figure 6A ). In Calu-3 cells, the Alpha, Gamma, Delta, Mu, and BA.5 variant replication kinetics indicated significantly higher and that of the BA.2 variant lower values than that of the B.1.1 variant ( Figure 6B ). The other variants, such as Wuhan, Beta, Lambda, and BA.1, displayed no statistically significant difference ( Figure 6B ). Finally, we aimed at addressing a potential correlation between viral RNA production in VeroE6/TMPRSS2 or Calu-3 cells 48 hours postinfection and S protein fusogenicity ( Figures 6C, D ). However, we obtained low r values, indicating a weak viral RNA production correlation with S protein fusogenicity ( Figures 6C, D , Supplementary Table S1 ; r = 0.1233 in VeroE6/TMPRSS2 cells and r = 0.3927 in Calu-3 cells, respectively). These data suggest that S protein fusogenicity might only slightly affect viral RNA production during viral replication in VeroE6/TMPRSS2 and Calu-3 cells.