key: cord-1031813-mtne3vvg authors: Wang, Qian; Anang, Saumya; Iketani, Sho; Guo, Yicheng; Liu, Lihong; Katsamba, Phinikoula S.; Shapiro, Lawrence; Ho, David D.; Sodroski, Joseph G. title: Functional properties of the spike glycoprotein of the emerging SARS-CoV-2 variant B.1.1.529 date: 2022-05-20 journal: Cell Rep DOI: 10.1016/j.celrep.2022.110924 sha: 257b82ac1ec770b68a9212e515f6a8fcdfe5a7ca doc_id: 1031813 cord_uid: mtne3vvg The recently emerged B.1.1.529 (Omicron) SARS-CoV-2 variant has a highly divergent spike (S) glycoprotein. We compared the functional properties of B.1.1.529 BA.1 S with those of previous globally prevalent SARS-CoV-2 variants, D614G and B.1.617.2. Relative to these variants, B.1.1.529 S exhibits decreases in processing, syncytium formation, virion incorporation and ability to mediate infection of cells with high TMPRSS2 expression. B.1.1.529 and B.1.617.2 S glycoproteins bind ACE2 with higher affinity than D614G S. The unliganded B.1.1.529 S trimer is less stable at low temperatures than the other SARS-CoV-2 spikes, a property related to its more “open” spike conformation. Upon ACE2 binding, the B.1.1.529 S trimer sheds S1 at 37 degrees but not at 0 degrees C. B.1.1.529 pseudoviruses are relatively resistant to neutralization by sera from patients with COVID-19 and vaccinees. These properties of the B.1.1.529 spike glycoprotein likely influence the transmission, cytopathic effects and immune evasion of this emerging variant. The continuing coronavirus disease 2019 pandemic has stimulated the implementation of a number of countermeasures, including vaccines, therapeutic antibodies and antiviral agents. Vaccines that elicit neutralizing antibodies against the spike (S) glycoprotein of the etiologic agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), have effectively reduced the probability of infection and death from this virus (Baden et al., 2021; Dai and Gao, 2021; Polack et al., 2020; Sadoff et al., 2021) . However, the emergence of a SARS-CoV-2 variant in Botswana and South Africa in mid-November 2021, coupled with its rapid spread throughout the world, has raised concerns (Grabowski et al., 2022; Pulliam et al., 2022; Scott et al., 2021) . The high rate of transmission has caused the World Health Organization to classify this variant as a Variant of Concern (VOC), and it has been termed Omicron (formally, B.1.1.529), now found in over 70 countries (WHO, 2021) . B.1.1.529 was found to have an unprecedented level of divergence, with more than 55 mutations compared to the ancestral Wuhan-Hu-1 strain. Of particular importance to antibody-mediated protection, more than 30 of these mutations alter the sequence of viral spike (S) glycoprotein (Scott et al., 2021) . Several of these changes in S, such as K417N and N501Y, have been previously demonstrated to confer resistance to antibody neutralization in other SARS-CoV-2 variants (Wang et al., 2021a; Wang et al., 2021b) . Residue Glu 484, which is altered to Lys in the B.1.351 and P.1 variants, is an Ala in B.1.1.529, resulting in a similar loss of sensitivity to a subset of antibodies . These features suggest that the B.1.1.529 virus may be highly adapted to resist antibodies directed against the ancestral SARS-CoV-2. J o u r n a l P r e -p r o o f expression, pseudovirus infection was mediated equivalently by the three S glycoproteins. Like other viruses descended from D614G, B.1.1.529 exhibits a higher affinity of its spike for the ACE2 receptor. The B.1.1.529 spike resists soluble ACE2induced shedding of the S1 exterior glycoprotein at lower temperatures. After extended incubation at 0˚C, the B.1.1.529 spike glycoprotein spontaneously sheds the S1 subunit and loses its ability to support virus entry. The spontaneous shedding of S1 and the cold inactivation of the virus spike are related to the conformation of the receptor-binding domains (RBDs) on the spike glycoprotein. We confirm that the B.1.1.529 S glycoprotein is less sensitive to neutralization by sera from patients convalescing from COVID-19 and recipients of two doses of the mRNA-1273 vaccine (Moderna) . The binding of the sera to recombinant S trimers indicates that many antibodies in the convalescent patient sera bind conserved epitopes on the B.1.1.529 spike that are not available as neutralization targets. Serum binding to the D614G and B.1.1.529 RBDs correlated better with the virus neutralization activity of the sera. Collectively, our study reveals biological properties of the divergent B.1.1.529 spike that may contribute to its rapid transmission, altered pathogenesis and immune escape. J o u r n a l P r e -p r o o f The significant number of changes in the B.1.1.529 BA.1 spike glycoprotein ( Figure 1A ) led us to compare its properties with those of spike glycoproteins from two other globally prevalent SARS-CoV-2 strains, D614G and B.1.617.2. We studied the expression and processing of the D614G, B.1.617.2 and B.1.1.529 S glycoproteins. We generated vesicular stomatitis virus (VSV) and lentivirus (HIV-1)-based pseudovirus particles as previously described, and examined the incorporated S glycoproteins by Western Blot (Schmidt et al., 2020; Wang et al., 2021c) . In both pseudoviral systems, relative to the D614G spike, the B.1.617.2 S glycoprotein was proteolytically processed more efficiently, whereas the B.1.1.529 S glycoprotein exhibited significantly less cleavage (Figure 1B and C) . For all these SARS-CoV-2 S glycoproteins, the cleaved (S1 and S2) glycoproteins were preferentially incorporated into pseudovirus particles. However, the decrease in B.1.1.529 S glycoprotein cleavage in the expressing cells was associated with reduced incorporation of spike glycoproteins into pseudovirus particles ( Figure 1B) . Next, we examined the glycosylation of these three SARS-CoV-2 S glycoproteins in the two pseudovirus systems. Cell lysates and pseudovirus particles were treated with either PNGase F or Endo Hf, and then visualized by Western blotting ( Figure 1D ). The S glycosylation patterns of the VSV and lentivirus pseudotypes were similar. As previously observed for SARS-CoV-2 spike glycoproteins (Nguyen et al., 2020; Wang et al., 2021c; Zhang et al., 2021) , the vast majority of the uncleaved S glycoprotein in cell lysates contained only high-mannose and hybrid glycans, whereas the S1 and S2 glycoproteins on pseudovirus particles were highly modified by complex carbohydrates. Following PNGase F digestion, the S2 glycoproteins incorporated into J o u r n a l P r e -p r o o f pseudovirus particles were more homogeneous than those in cell lysates; posttranslational modifications of S2 other than N-or O-linked glycosylation account for this difference (Zhang et al., 2021) . The untreated and the Endo Hf-treated S1 from B.1.617.2 migrated slightly faster than those of D614G and B.1.1.529, which may be due to a missing N-linked glycosylation site resulting from the T19R change in the Nterminal domain. We also evaluated the effects of coexpression of the SARS-CoV-2 membrane (M), envelope (E) and nucleocapsid (N) proteins on D614G, B.1.617.2 and B.1.1.529 S glycoprotein processing. We examined cell lysates and particulate fractions concentrated from the supernatants of 293T cells transfected with plasmids expressing the variant S glycoproteins along with plasmids expressing the M, E and N proteins, alone or in combination. Coexpression of the M, E and N proteins, alone or in combination, did not significantly affect S glycoprotein processing ( Figure 1E ). To determine if the reduced cleavage and virion incorporation of the B. Vero-E6, and Vero-E6-ACE2-TMPRSS2 target cells. No significant differences were observed in the infectivity of lentiviruses or VSV pseudotyped by the three SARS-CoV-2 S glycoproteins for 293T-ACE2 or Vero-E6 cells (Figure 2A and 2B) SARS-CoV-2 can achieve entry into cells via two routes, either at the cell surface following TMPRSS2 proteolytic activation or from the endosome following proteolytic activation by the endosomal proteases Cathepsin B or L (Peacock et al., 2021 Figure 2E . The levels of the cleaved S1 and S2 glycoproteins in the cell lysates and on the cell surface were comparable for the D614G and B.1.617.2 variants. Although the B.1.1.529 S glycoprotein was expressed well, the level of processed S1 and S2 glycoproteins in cell lysates and on the cell surface was relatively low. To evaluate the efficiency of cell-cell fusion mediated by the D614G, B.1.617.2 and B.1.1.529 S glycoproteins, the COS-1 cells expressing these S glycoproteins and α-gal were cocultivated with 293T cells expressing ACE2 and ω-gal for 4 hours at 37°C. The formation of syncytia between the S-expressing cells and ACE2-expressing cells results in the activation of -galactosidase. Cell-cell fusion mediated by the B.1.1.529 S glycoprotein was mildly reduced compared with the levels observed for the D614G and B.1.617.2 S glycoproteins ( Figure 2F ). This result is consistent with the expectation that the process of syncytium formation is mediated by cleaved S1/S2 glycoprotein trimers on the surface of the expressing cell (Nguyen et al., 2020) . The decreased processing of the B.1.1.529 S glycoprotein results in lower levels of mature S1/S2 trimers in expressing cells, compared with the levels J o u r n a l P r e -p r o o f of the D614G and B.1.617.2 S glycoproteins, likely contributing to the observed reduction in cell-cell fusion. ACE2 binding and soluble ACE2-induced S1 shedding of variant spikes. We investigated the interaction of the D614G, B. 293T-ACE2 cells ( Figure 3A ). As expected for the lower level of ACE2 expression in Vero-E6 cells compared with that in 293T-ACE2 cells (Wang et al., 2021c) , the differences in huACE2-Fc sensitivity between D614G and the other viruses were more pronounced in the former cells. No significant differences were observed between the B.1.617.2 and B.1.1.529 pseudotypes in these assays. Using a surface plasmon resonance assay, we found that the KD value for huACE2-Fc binding to the B.1.1.529 spike (1.07 nM) was around 3-fold lower than that of the D614G spike (3.01 nM) ( Figure 3B ). The higher affinity of huACE2-Fc for the B.1.1.529 spike glycoprotein likely contributes to the enhanced huACE2-Fc inhibition of B.1.1.529 pseudovirus infection relative to that of D614G pseudoviruses ( Figure 3A) . We compared ACE2-induced shedding of S1 from the spike trimers of the D614G, B.1.617.2 and B.1.1.529 pseudoviruses. As differences in soluble ACE2induced shedding of S1 among some SARS-CoV-2 strains were revealed at 0˚C (Wang et al., 2021c) , we performed these experiments at 0˚C and 37˚C. In the absence of huACE2-Fc, the D614G, B.1.617.2 and B.1.1.529 spikes on the pseudovirus particles were stable for at least one hour at both temperatures ( Figure 3C ). Each variant pseudovirus was incubated with increasing concentrations of huACE2-Fc at J o u r n a l P r e -p r o o f either 0°C or 37°C for 1 h and then S1 shedding was quantified by Western blotting ( Figure 3D ). While S1 shedding was similar for all three pseudoviruses at 37˚C, the B.1.1.529 spike was significantly more stable at 0°C, with minimal S1 shedding compared to those of the D614G or B.1.617.2 spikes ( Figure 3E ). The observed differences in S1 shedding were not explained by differences in huACE2-Fc binding by the variant S glycoproteins. These results indicate that the ACE2-bound B.1.1.529 S trimer resists the disruptive effects of incubation at 0˚C better than the D614G and B.1.617.2 S trimers. Extended periods of incubation at near freezing temperatures can reveal differences in the stability of the spike glycoprotein trimers of SARS-CoV-2 variants (Nguyen et al., 2020; Wang et al., 2021c) . We evaluated the effect of temperature on the infectivity of VSV pseudotyped by the D614G, B.1.617.2 and B.1.1.529 S glycoproteins. We incubated VSV pseudotypes at 0°C, 4°C, room temperature (RT), or 37°C for various periods of time before measuring their infectivity on Vero-E6 cells. No differences between the SARS-CoV-2 variants were observed at 4°C, RT, or 37°C; the infectivity of all these variants declined slightly faster at 37°C than at the other temperatures ( Figure 4A) . Notably, at 0°C, the infectivity of the B.1.1.529 pseudotype decayed faster than the infectivities of the D614G or B.1.617.2 pseudotypes. As the observed reduction in infectivity of the B.1.1.529 pseudovirus at 0°C may be due to the shedding of the S1 glycoprotein from the spike, we quantified S1 retention on the viral particles following various periods of incubation at different temperatures ( Figure 4B and C) . At 0˚C, a higher rate of spontaneous S1 shedding from the B.1.1.529 pseudoviruses compared with the D614G and B.1.617.2 J o u r n a l P r e -p r o o f pseudoviruses was observed, suggesting that spike disassembly may be part of the mechanism for the observed loss of infectivity at this temperature. The integrity of the B.1.617.2 and B.1.1.529 spikes decreased faster than that of D614G at 4°C; as the infectivity of these pseudoviruses differed only modestly at 4˚C, mechanisms of spike inactivation that do not result in S1 shedding are likely involved (Wang et al., 2021c) . In a previous study (Wang et al., 2021c) , we found that, similar to the B.1.1.529 variant S glycoprotein, the spike glycoprotein of the P.1 (Gamma) SARS-CoV-2 variant also exhibited an unusually high degree of S1 shedding after a two-day incubation at 0˚C. Interestingly, both P.1 and B.1.1.529 variant spike trimers were reported to reside exclusively in a conformational state with only one receptor-binding domain (RBD) in the "up" position (Cerutti et al., 2022; Wang et al., 2021a) . We found a correlation between the S1/S2 ratios of different SARS-CoV-2 variant spike trimers after 48 hours of incubation at 0˚C (Wang et al., 2021c) The B.1.1.529 variant has been reported to be significantly more resistant to antibody neutralization than previously characterized SARS-CoV-2 variants Planas et al., 2022) . We tested the binding of sera from patients convalescing from COVID-19 and vaccinees who had received two doses of mRNA-1273 (Moderna) to recombinant D614G and B.1.1.529 spike trimers (S2P) and RBDs ( Figure 5A , 5C, S2, and S3). Unexpectedly, we observed that while binding to the B.1.1.529 S2P was lower than that to the D614G S2P for all serum samples, the magnitude of the decrease differed significantly between the groups; convalescent patient sera J o u r n a l P r e -p r o o f exhibited only a 1.6-fold decrease in binding titer, whereas sera from Moderna vaccinees exhibited a 9.6-fold decrease, on average. We also observed a decrease in binding to the B.1.1.529 RBD compared with that to the D614G RBD for both convalescent patient sera (15.9-fold decrease) and vaccinee sera (44.1-fold decrease); however, the magnitude of the decrease was more similar between the two groups of sera than that seen for binding to the D614G and B.1.1.529 S2P trimers. The vaccinees elicit higher titers of antibodies against the SARS-CoV-2 spike and against the RBD region than the patients convalescing from COVID-19. Apparently, the S2P and RBD epitopes targeted by the vaccinee sera are less conserved between the D614G and B.1.1.529 S glycoproteins than the S2P epitopes recognized by the sera from patients convalescing from COVID-19. To assess the functional consequence of the observed differences in antibody binding to the S glycoprotein trimers, we tested the capability of these sera to neutralize VSV pseudovirus infection of both Vero-E6 and 293T-ACE2 cells ( Figure 5B , 5D, and S4). The B.1.617.2 pseudovirses were neutralized less effectively than the D614G pseudoviruses by the convalescent patient sera and the vaccinee sera. Neutralization of the B.1.1.529 pseudoviruses by all samples decreased dramatically, with only two samples from Moderna vaccinees having a detectable titer in the Vero-E6 cells. These results indicate that the B.1.1.529 S glycoprotein is significantly less sensitive to neutralization by antibodies elicited to earlier SARS-CoV-2 variant S glycoproteins, either by vaccination or following natural infection. A comparison of the binding of the convalescent patient and vaccinee sera to the S2P and RBD proteins with the neutralizing activity of these sera suggests differences in the targeted S glycoprotein epitopes. The vaccinees elicit higher titers of antibodies against the SARS-CoV-2 spike and against the RBD region; although this binding J o u r n a l P r e -p r o o f correlates better with virus-neutralizing ability, the targeted sites are less conserved between the D614G and B.1.1.529 S glycoproteins. By contrast, many of the S2P epitopes recognized by the convalescent patient sera, although conserved between the D614G and B.1.1.529 S glycoproteins, are not available as neutralization targets on the functional virus spike glycoprotein (Brewer et al., 2022) . Figure 1A) . Although SARS-CoV-2 components other than S can potentially influence transmission, pathogenesis and immune evasion, the concentration of variation in the B.1.1.529 spike suggests its importance. Indeed, S changes render the virus less sensitive to some therapeutic antibodies and antibodies elicited by current vaccines (Grabowski et al., 2022; Pulliam et al., 2022; Scott et al., 2021) . This has raised concerns about the potentially reduced efficacy of vaccines against B.1.1.529 as well as the increased risk for reinfection with this variant (Garcia-Beltran et al., 2022; Gruell et al., 2022; Hoffmann et al., 2022; Liu et al., 2022; Pajon et al., 2022; Planas et al., 2022) . for the original SARS-CoV-2 outbreak, exhibited more efficient S glycoprotein cleavage compared with that of its D614 ancestor (Nguyen et al., 2020; Wang et al., 2021c) . The S glycoprotein cleavage of most of the SARS-CoV-2 variants of concern is not significantly different from that of the D614 variant (Wang et al., 2021c) . Therefore, (e.g., matrix metalloproteases) other than TMPRSS2 to activate cell-cell fusion (Nguyen et al., 2020) . Alternatively, utilization of TMPRSS2 by the B.1.1.529 S glycoprotein may be affected by the different contexts in which virus entry and cell-cell fusion occur. The exposure of native SARS-CoV-2 spike glycoproteins to 0˚C can reveal strain-dependent differences in the stability and functional integrity of the S glycoprotein trimer. The infectivity of B.1.1.529 pseudoviruses decayed similarly to those of D614G and B.1.617.2 pseudoviruses at room temperature and 37˚C, but exhibited a relatively faster decay at 0˚C ( Figure 4A ). This corresponded to an increase in the spontaneous shedding of the S1 glycoprotein from the B.1.1.529 spike trimer during prolonged incubation at 0˚C, compared with the D614G and B.1.617.2 spikes ( Figure 4B and 4C). The cold resistance of enveloped viruses is related to the ability of their oligomeric envelope glycoproteins to resist the destabilizing effects of ice crystal formation (Privalov, 1990) . The sensitivity of human immunodeficiency viruses (HIV-1) to inactivation at 0˚C is related to the propensity of the viral envelope glycoprotein trimer to undergo conformational transitions from the pretriggered conformation to more "open" receptor-bound conformations (Kassa et al., 2009 ). Likewise, we found a correlation between cold sensitivity of SARS-CoV-2 variants and the occupancy of the S glycoprotein trimers in a conformation in which one RBD is in the "up" position and the other two RBDs are in the "down" position ( Figure 4D and 4E). Thus, the cold sensitivity of the B.1.1.529 and P.1 variants may reflect the propensity of the "closed" spike trimer to sample spontaneously the next more "open" state, a conformation with one RBD "up". This propensity to undergo conformational changes could assist the triggering of conformational transitions in the B.1.1.529 spike and help compensate for a lower spike density on the virion. For the P.1 SARS-CoV-2 variant, which has a more typical spike density, increased triggerability could account for the efficiency with which target cells with low levels of ACE2 can be infected (Wang et al., 2021c ) . Relative to the D614G and B.1.617.2 spikes on pseudoviruses, the B.1.1.529 spike demonstrated marked resistance to S1 shedding after exposure to soluble ACE2 at 0˚C (Figure 3D and 3E) . The B.1.1.529 spike shares this property with the P.1 spike but not with the S glycoproteins of other SARS-CoV-2 variants studied (Cerutti et al., 2022; Wang et al., 2021a; Wang et al., 2021c) . Figure 5B, 5D and S4) . These results indicate that most of the neutralizing antibodies generated to previous SARS-CoV-2 variants will be less effective against B.1.1.529. Resistance to neutralizing antibodies is clearly a major factor driving the evolution of SARS-CoV-2 variants (Choi et al., 2020) . This conclusion is consistent with the increased number of vaccine breakthrough infections involving B.1.1.529 compared with earlier SARS-CoV-2 variants (Andrews et al., 2022; Kuhlmann et al., 2022; Pulliam et al., 2022) . The B.1.1.529 spike is apparently well adapted so that any negative consequences of an increased propensity to sample the one-RBD-up conformation, which potentially could result in greater exposure to neutralizing antibodies, are compensated during naturally encountered circumstances. Although both convalescent patient sera and vaccinee sera failed to neutralize B.1.1.529 pseudoviruses, the binding of the convalescent patient sera to recombinant spike trimer (S2P) of B.1.1.529 was only modestly decreased compared with the binding to D614G S2P. By contrast, the difference in binding of the vaccinee sera to these two recombinant spike trimer was much greater. The magnitude of the decrease in binding to D614G and B.1.1.529 RBDs between the convalescent patient sera and vaccinee sera was not as significant as that seen for binding to the S2P glycoproteins ( Figure 5A and 5C) . The vaccinee sera apparently recognize S2P and RBD epitopes that differ between D614G and B.1.1.529, but which represent targets for neutralization on the functional virus spike. A significant fraction of antibodies in the sera of convalescent patients recognize spike epitopes that are conserved between the D614G and B.1.1.529 S2P glycoproteins but are not available as neutralization targets on the native virus spike. For example, spikes that shed S1 during natural infection could present S2 trimers to the host immune system; many of the antibodies directed against these S2 trimers would fail to bind intact S trimer (Brewer et al., 2022) . By contrast, the S2P trimers used for vaccination are uncleaved and stabilized in a pre-fusion conformation, properties that may decrease trimer opening or exposure of epitopes for non-neutralizing antibodies. In conclusion, we demonstrate that the B.1.1.529 spike glycoprotein has a combination of properties differentiating it from previous SARS-CoV-2 variants. In addition to the resistance of the B.1.1.529 spike to antibodies reactive with other SARS-CoV-2 spike glycoproteins, other notable features include the relatively low level of B.1.1.529 S processing and virus spike density, decreased syncytium-forming ability, decreased ability to utilize TMPRSS2 to support virus entry, and the spontaneous and ACE2-regulated stability of the spike trimer, which is temperaturedependent and relates to spike conformational states. A better understanding of the spike-determined biological characteristics of this newly emerged SARS-CoV-2 variant should assist the design of vaccines and other interventions. The assays conducted in our study used viruses pseudotyped by the SARS-CoV-2 S glycoproteins rather than authentic SARS-CoV-2 viruses. We used cell lines to study infection by the pseudoviruses and did not study natural target cells for SARS-CoV-2 infection. Although these experimental systems allow more precise control of variables, there may be differences from natural SARS-CoV-2 infection. In this study, we did not define the S glycoprotein determinants of the distinct B.1.1.529 S phenotypes. analyzed the data and wrote the manuscript. All authors reviewed and edited the manuscript. The authors declare no competing interests. For statistical analysis of the data in A, B and F, a Student's unpaired t test was used to compare the values to those obtained for the wild-type D614G S glycoprotein (*p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001; ns -not significant). by Western blotting with antibodies against S1, S2, huACE2-Fc, and VSV NP. The intensities of the S1 and S2 glycoprotein bands in (D) were measured and the S1/S2 ratios for each concentration of sACE2 are shown. (B) VSV particles pseudotyped with the variant SARS-CoV-2 S glycoproteins were incubated at 0˚C, at RT, and at 37˚C for various times. Pelleted VSV particles were analyzed by Western blotting with antibodies against S1, S2, and VSV NP. The D614G, B.1.617.2, and B.1.1.529 S1 bands were exposed for 10s, 10s, and 30s, respectively; the D614G, B.1.617.2, and B.1.1.529 S2 bands were exposed for 5s, 2.5s, and 15s, respectively. The exposure time of the NP bands was 45s. The results shown are representative of those obtained in two independent experiments. The intensities of the S1 and S2 glycoprotein bands in (B) were measured. The S1/S2 ratios are shown for each variant relative to the ratio for pseudoviruses at time 0. (D) Relative populations of SARS-CoV-2 variant spikes with one RBD in the "up" position (Cerutti et al., 2022) and S1/S2 ratios after a 48-hour incubation at 0˚C, RT and 37˚C ((Wang et al., 2021c) and C above). (E) Correlation between the percentage of spike populations with one RBD in the "up" position and the S1/S2 ratio after a 48-hour incubation at 0˚C, 4°C, RT and 37˚C for spike glycoproteins from different SARS-CoV-2 variants. The r and p values for each curve were obtained by fitting the data with simple linear regression. A significant correlation between the percentage of spikes with one RBD up and the S1/S2 ratio was observed at the 0°C temperature. Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Joseph G. Sodroski (joseph_sodroski@dfci.harvard.edu). All requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr. Joseph G. Sodroski (joseph_sodroski@dfci.harvard.edu). This includes selective cell lines, plasmids, antibodies, viruses, serum and proteins. All reagents will be made available on request after completion of a Material Transfer Agreement. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Data reported in this paper will be shared by the lead contact upon request. This paper does not report original codes. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. The codon-optimized SARS-CoV-2 spike (S) gene (Sino Biological, Wayne, PA) encoding the S glycoprotein lacking 18 amino acids at the carboxyl terminus was cloned into the pCMV3 vector. The gene encoding the B.1.617.2 SARS-CoV-2 spike variant was made by introducing additional mutations into the wild-type (D614) S gene using a site-directed mutagenesis kit (Agilent, Santa Clara, CA). The B.1.1.529 S gene was generated by a high-throughput template-guided gene synthesis approach . Plasmids expressing the SARS-CoV-2 M (Cat# 141386) and E (Cat# 141385) proteins with 2XStrep tags at their C termini were from Addgene. The SARS-CoV-2 N gene cloned in pCAGFLAG was kindly provided by Dr. Pei-Hui Wang . The studies conducted in this manuscript used the BA.1 subvariant of B.1.1.529. The genes encoding the SARS-CoV-2 D614G and B.1.1.529 S trimer ectodomains with 2P and furin cleavage-site changes, and containing a C-terminal 8x His tag (Wrapp et al., 2020) , were synthesized and then cloned into the paH vector. The soluble SARS-CoV-2 S2P spike trimer proteins of the D614G and B.1.1.529 variants were generated by transfecting Expi293 cells with the trimer proteinexpressing constructs using FectPRO DNA transfection reagent (Polyplus, New York, NY). The soluble S2P spike trimers were purified from cell supernatants 5 days later using Ni-NTA resin (Invitrogen), according to the manufacturer's protocol . Two µg of each S2P trimer and RBD protein was analyzed on a NuPAGE Bis-Tris protein gel (Invitrogen, Waltham, MA) run at 200 V using MES buffer, after which the gel was stained with Coomassie Blue dye. To generate VSV-based vectors pseudotyped with SARS-CoV-2 S glycoproteins, 6 X 10 6 HEK293T cells were plated in a 10-cm dish one day before transfection. Fifteen µg of the SARS-CoV-2 S glycoprotein plasmid was transfected into the HEK293T cells using Polyethylenimine (Polysciences, Warrington, PA). Twenty-four hours later, the cells were infected at a multiplicity of infection of 3 to 5 with rVSV-∆G pseudovirus bearing a luciferase gene (Kerafast, Boston, MA) for 2 h at 37˚C and then washed three times with PBS. Cell supernatants containing pseudoviruses were harvested after another 24 h, clarified by low-speed centrifugation (2000 rpm for 10 min) and filtered through a 0.45-µM filter. Viruses were then aliquoted and stored at -80˚C until use Wang et al., 2021a; Wang et al., 2021b) . To generate HIV-based vectors pseudotyped with SARS-CoV-2 S glycoproteins, 6 X 10 6 HEK293T cells were plated in a 10-cm dish one day before transfection. Then, 7.5 µg of SARS-CoV-2 S glycoprotein expressor plasmid and 7.5 µg pHIV-1NL4-3∆Env-NanoLuc reporter construct were cotransfected into the HEK293T cells using S, S1, and S2 band intensities from unsaturated Western blots were calculated using ImageJ Software. S1/S2 ratios represent the ratios of the intensities of the S1 and S2 glycoprotein bands in the Western blots. The values for the processing index of the B.1.617.2 and B.1.1.529 variant S glycoproteins were calculated as follows: Processing index = (S1/S × S2/S)variant ÷ (S1/S × S2/S)D614G For production of SARS-CoV-2 virus-like particles (VLPs), a plasmid expressing the variant SARS-CoV-2 S glycoprotein was transfected into 293T cells in J o u r n a l P r e -p r o o f 6-well plates in combination with equal amounts of plasmids expressing the SARS-CoV-2 M, E and N proteins. Two days after transfection, cell lysates and VLP pellets were prepared as described above. Samples were analyzed by Western blotting with antibodies against S1 and S2, as described above. Samples were also analyzed by Western blotting with rabbit anti-SARS-CoV-2 N antibody (GeneTex, Cat# GTX135357) and then developed with HRP-conjugated anti-rabbit antibody (Cytiva, Marlborough, MA (NA934-1ML)). Strep-tagged SARS-CoV-2 M and E proteins were analyzed by Western blotting with HRP-conjugated Strep tag antibody (Sigma-Aldrich, Cat# 71591). SARS-CoV-2 S glycoproteins in cell lysates or on pseudovirus particles were prepared as described above. Protein samples were boiled in 1 X denaturing buffer and incubated with PNGase F or Endo Hf (New England Biolabs, Ipswich, MA) for 1 h at 37˚C according to the manufacturer's protocol. The samples were then analyzed by SDS-PAGE and Western blotting as described above. The pseudoviruses were freshly prepared as described above, without freezing and thawing. The pseudovirus preparations were incubated with target cells seeded in 96well plates at a density of 3 X 10 4 cells/ well. For VSV-based pseudoviruses, target cells were cultured for 16-24 hours after infection and then harvested to measure the luciferase activity (Promega, Madison, WI (Cat# E4550)). For HIV-1-based pseudoviruses, target cells were cultured for 2-3 days after infection and then cells were harvested to measure the NanoLuc luciferase activity (Promega, Cat# N1120). To measure viral stability at different temperatures, pseudoviruses were incubated in an ice water bath (0˚C), at 4˚C, at room temperature and at 37˚C for different lengths of time prior to measuring their infectivity in Vero-E6 cells. To measure the infectivity of pseudovirus variants on target cells expressing different levels of human ACE2, serial dilutions (from 2 µg to 24.7 ng) of the human ACE2 expressor plasmid (Addgene, Watertown, MA (Cat# 1786)) were transfected into 293T cells in 12-well plates using 1 mg/ml PEI. Two days after transfection, cells were trypsinized and used as target cells for measuring the infectivity of VSV pseudotypes (Wang et al., 2021c) . For the alpha-complementation assay measuring cell-cell fusion, COS-1 effector cells were plated in black-and-white 96-well plates and then cotransfected with a plasmid expressing ⍺-gal and either pCMV3 or the SARS-CoV-1 S glycoprotein variant at a 1:1 ratio, using lipofectamine 3000 (Thermo Fisher) according to the manufacturer's protocol. At the same time, 293T target cells in 6-well plates were cotransfected with plasmids expressing ω-gal and human ACE2 at a 1:1 ratio, using lipofectamine 3000. Forty-eight hours after transfection, target cells were scraped and resuspended in medium. Medium was removed from the effector cells, and target cells were then added to effector cells (one target-cell well provides sufficient cells for 50 effector-cell wells). Plates were spun at 500 x g for 3 min and then incubated at 37˚C in 5% CO2 for 4 h. Medium was aspirated and cells were lysed in Tropix lysis buffer (Thermo Fisher Scientific). The -galactosidase activity in the cell lysates was measured using the Galacto-Star Reaction Buffer Diluent with Galacto-Star Substrate (Thermo Fisher Scientific), following the manufacturer's protocol. To evaluate S glycoprotein expression in COS-1 cells, cells in 6-well plates were cotransfected with a plasmid expressing ⍺-gal and either pCMV3 or a plasmid expressing the SARS-CoV-2 S glycoprotein variant at a 1:1 ratio, using lipofectamine 3000 (Thermo Fisher) according to the manufacturer's protocol. To evaluate the S glycoprotein in cell lysates, cells were lysed with 1.5% Cymal-5 containing a protease inhibitor two days after transfection. To evaluate the S glycoprotein on the cell surface, cells were incubated with EZ-link Sulfo-NHS-SS-Biotin. The biotinylated cell surface proteins were then precipitated by NeutrAvidin Agarose using a Pierce™ Cell Surface Protein Isolation Kit (Thermo Scientific, Cat# 89881). The expression of the S glycoproteins in cell lysates and on the cell surface was analyzed by Western blotting as described above. SPR assays for human ACE2-Fc (SinoBiological, Cat# 10108-H02H) binding to SARS-CoV-2 D614G or B.1.1.529 spike trimers were performed using a Biacore T200 biosensor, equipped with a Series S CM5 chip (Cytiva, Cat# BR100530), at 25°C, in a running buffer of 10 mM HEPES pH 7.4, 150 mM NaCl, 0.2 mg/mL BSA and 0.01% (v/v) Tween-20 at 25°C. Each spike was captured through its C-terminal His6 tag on an anti-His6 antibody surface, generated using the His-capture kit (Cytiva, Cat# 28995056) according to the instructions of the manufacturer. During a binding cycle, each spike was captured over individual flow cells at approximately 500-700 RU and an anti-His6 antibody surface was used as a reference flow cell. Human ACE2-Fc was prepared at six concentrations using a three-fold dilution series in running buffer, ranging from 1.1 to 90 nM. Samples were tested in order of increasing protein concentration. Blank buffer cycles were performed by J o u r n a l P r e -p r o o f injecting running buffer instead of the analyte, after two ACE2 injections. The association and dissociation rates were each monitored for 90s and 300s respectively, at 50 μL/min. Bound spike/ACE2 complexes were removed using a 10s pulse of 15 mM H3PO4 at 100 μL/min, thus regenerating the anti-His6 surface for a new cycle of recapturing of each spike, followed by a 60s buffer wash at 100 μL/min. The data were processed and fit to a 1:1 interaction model using the Scrubber 2.0 (BioLogic Software). VSV particles pseudotyped with S glycoprotein variants were prepared as described above. To evaluate spontaneous S1 shedding at different temperatures, the cell supernatants containing pseudoviruses were incubated in an ice water bath (0˚C), at 4˚C, room temperature and 37˚C for different times. Virus particles were then pelleted at 18,000 x g for 1 h at 4˚C. The pelleted samples were resuspended in 1 X LDS sample buffer (Invitrogen, Cat# NP0008) and analyzed by Western blotting. To evaluate soluble huACE2-Fc-induced S1 shedding, the cell supernatants containing pseudovirus particles were incubated with soluble huACE2-Fc (Sino Biological Inc (cat# 10108-H02H)) at different concentrations at 0˚C or 37˚C for 1 h. Afterwards, virus particles were pelleted at 18,000 x g for 1 h at 4˚C. The pelleted virus particles were washed once with cold PBS before the samples were resuspended in 1 X LDS sample buffer and analyzed by Western blotting. S1, S2 and NP were detected as described above; huACE2-Fc bound to the pseudovirus particles was detected with Peroxidase AffiniPure goat anti-human IgG (H+L) antibody (Jackson ImmunoResearch). Fifty nanograms of S2P spike trimer or RBD protein (Sino Biological Inc. (Cat: 40592-V08H121 and 40592-V08H)) was coated on each well of an ELISA plate at 4˚C overnight. Then the plates were blocked with 300 mL of blocking buffer (1% BSA and 10% bovine calf serum in PBS) at 37˚C for 2 h. Afterwards, serially diluted convalescent serum or Moderna vaccinee serum was added and incubated at 37˚C for 1 h. Next, 100 µL per well of 10,000-fold diluted Peroxidase AffiniPure goat antihuman IgG (H+L) antibody (Jackson ImmunoResearch) was added and incubated for 1 h at 37˚C. Between each step, the plates were washed with PBST three times. Finally, the TMB substrate (Sigma, St. Louis, MO) was added and incubated for 5 min at room temperature before the reaction was stopped using 1 M sulfuric acid. Absorbance was measured at 450 nm. The serum endpoint dilutions that achieved an OD450 value > 3-fold over background were calculated by fitting the data in five-parameter dose-response curves in GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA). To test the inhibition of virus infection by Camostat and E64d, 293T-ACE2, 293T-ACE2-TMPRSS2, Vero-E6, or Vero-E6-ACE2-TMPRSS2 cells were pre-treated with serial dilutions of Camostat or E64d in triplicate in 96-well plates for 1 h at 37˚C prior to inoculation with VSV pseudotypes. The cultures were maintained for 16-24 h at 37˚C before luciferase activity was measured as described above. Sera were collected from March to June 2020 from New York City patients that recovered from COVID-19; six sera were collected from Moderna vaccinees (Wang et al., 2021b) . Pseudovirus neutralization assays were performed by incubating VSV vectors pseudotyped by S glycoprotein variants with serial dilutions of huACE2-Fc J o u r n a l P r e -p r o o f (Cat# 10108-H02H), sera from convalescent COVID-19 patients or vaccinee sera in triplicate in 96-well plates for 1 h at 37˚C. Approximately 3 x 10 4 target cells (Vero-E6 or 293T-ACE2 cells) per well were then added. The cultures were maintained for an additional 16-24 h at 37˚C before luciferase activity was measured as described above. The % inhibition and neutralization values were calculated from the reduction in luciferase activity compared with mock-treated controls. The concentrations of Camostat, E64d, huACE2-Fc and serum titers that inhibit 50% of infection (the IC50 and ID50 values, respectively) were determined by fitting the data in five-parameter dose-response curves in GraphPad Prism 9 (GraphPad Software Inc., San Diego, CA). Evaluations of statistical significance were performed employing Student's unpaired or paired t test using GraphPad Prism 9 software. Levels of significance are indicated as follows: ns, p>0.05; *p<0.05; **p<0.01; ***p<0.001; and ****p<0.0001. Linear correlation was determined by fitting the data with simple linear regression. EC50, IC50 and ID50 values were determined by fitting the data in five-parameter dose-response curves in GraphPad Prism 9. Western blot data were analyzed by Image Lab and ImageJ software. All data presented is representative or mean data derived from at least two independent experiments. 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