key: cord-0311441-mil4hx08
authors: Yang, Ziwei; Han, Yang; Ding, Shilei; Finzi, Andrés; Mothes, Walther; Lu, Maolin
title: SARS-CoV-2 variants exhibit increased kinetic stability of open spike conformations as an evolutionary strategy
date: 2021-10-12
journal: bioRxiv
DOI: 10.1101/2021.10.11.463956
sha: 2797200479381e9291fd879d016ed23b48455ba6
doc_id: 311441
cord_uid: mil4hx08

SARS-CoV-2 variants of concern harbor mutations in the Spike (S) glycoprotein that confer more efficient transmission and dampen the efficacy of COVID-19 vaccines and antibody therapies. S mediates virus entry and is the primary target for antibody responses. Structural studies of soluble S variants have revealed an increased propensity towards conformations accessible to receptor human Angiotensin-Converting Enzyme 2 (hACE2). However, real-time observations of conformational dynamics that govern the structural equilibriums of the S variants have been lacking. Here, we report single-molecule Förster Resonance Energy Transfer (smFRET) studies of S variants containing critical mutations, including D614G and E484K, in the context of virus particles. Investigated variants predominantly occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited decelerated transitions in hACE2-accessible/bound states. Our finding of increased S kinetic stability in the open conformation provides a new perspective on SARS-CoV-2 adaptation to the human population.

Since the beginning of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic, variants of concern (VOCs) that are more transmissible and possibly more pathogenic have emerged and caused devastating consequences worldwide. The currently administrated interventions, vaccines and antibody therapy are directed against the virus surface spike (S) protein as the primary target of antibody response 1-6 . [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] . While current vaccines remain efficient against severe disease induced by these VOCs 17, 18 , additional mutation in S could compromise this protection. Similarly, some VOCs are now resistant to first generation antibody therapies and impacted several molecular tests 11, 12, [19] [20] [21] . For instance, D614G is a spike variant that first emerged in January 2020 and became a dominant form as the pandemic spread, reaching >74% global prevalence by June 2020. D614G has been associated with increased infectivity and transmissibility of SARS-CoV-2 22, 23 . E484K has been identified as an escape mutation that facilitates virus immune evasion 24 . E484K was first identified in South Africa (B.1.351) and Brazil (P.1), but it was also gradually adopted by the U.K. (B.1.1.7) strain since, indicating an associated selective advantage 23 . Other mutations on S variants such as L452R, N501Y, and P681R also attracted research attention due to enhanced ACE2 interaction and partial escape from vaccine-elicited antibodies 25 . Therefore, it is of great importance that we understand how these mutations affect the conformational landscape of S, and how this connects to the enhanced immune evasion, infectivity, and transmissibility of SARS-CoV-2 variants.

The S protein is a trimer of S1/S2 heterodimers 26, 27 . The surface-exposing S1 contains the receptor-binding domain (RBD) that engages with cellular receptor human angiotensin-converting enzyme 2 (hACE2) [28] [29] [30] [31] , an event which, upon occurrence, triggers the transmembrane S2 to mediate the virus-cell membrane fusion process that enables virus entry [30] [31] [32] [33] [34] . The S glycoprotein is structurally flexible and conformationally dynamic to facilitate both virus entry and antibody evasion. Each spike protomer samples two well-characterized RBD-orientation-based conformations, the hACE2accessible "RBD-up" and the hACE2-inaccessible "RBD-down" conformations ( Figure   1 ). Consequently, the S trimer can exist in an equilibrium of four possible combinations of the protomers -four conformational states of S trimer: "one-RBD-up," "two-RBD-up," "all-RBD-up," and "all-RBD-down." Extensive structural studies of truncated soluble and virus-associated parental/original strain SD614 have confirmed the existence of the above-mentioned major S trimer configurations [29] [30] [31] [35] [36] [37] [38] [39] [40] [41] [42] [43] . As S continuously evolves, accelerated efforts have been implemented to characterize emerging S variants genetically, virologically, and structurally 25, [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] . However, real-time monitoring of structural/conformational dynamics of S variants that connect structures has been lacking. The mechanism by which spike proteins adapt their conformations and dynamics during virus evolution could facilitate the understanding of pathogenesis, antibody neutralization, and vaccine efficacy of these S variants.

Using single-molecule Förster Resonance Energy Transfer (smFRET), we have experimentally revealed that virus-associated SD614 dynamically samples at least four distinct conformational states, transitioning in time from milliseconds to seconds 55 . On the faster dynamic scale, a remarkably long ~ 130 µs sampling simulation of the SD614 opening process suggested the existence of five conformations between the transition from "RBD-down" to "RBD-up," 56 despite the temporal limitation that existed in simulation. Nevertheless, real-time experimental observations of conformational landscapes and the dynamics that govern the conformational equilibriums of recently emerged S variants on the viruses have not been performed. It is unclear whether arising S variants differ from the original SD614 on viruses regarding conformational dynamics/kinetics. Here, we further deployed our smFRET approach to reveal the dynamic details and the mechanistic of the conformational shifts in different S variants at the surface of lentivirus particles. Spike variants that contain our mutations of interest, D614G or/and E484K, as well as their parental strains were analyzed and compared side-by-side in this study. Using smFRET, we investigated how these mutations affect the conformational landscape and dynamics of the S glycoprotein by characterizing the overall movements within S in the millisecond to the second range. We discuss how such influences contribute to the selective advantage that variants harboring these mutations confer, and provide insights into the mechanisms by which mutations in S could increase the severity of SARS-CoV-2 variants.

To perform smFRET investigations of spike variants, lentivirus particles that carry only a single FRET-paired dyes-labeled SARS-CoV-2 Spike protomer among unlabeled wildtype Spikes were imaged on a prism-based total internal reflection fluorescence TIRF microscope (Figure 1 ). Lentivirus particles contain the HIV-1 core and the SARS-CoV-2 S glycoproteins on the virus surface. As previously described 55 , Q3 and A4 peptide tags were inserted before and after the receptor-binding motif of tested S variants at positions 427 and 556 (427-Q3/556-A4, Figure 1 ) to allow the site-specific introduction of donor and acceptor dyes/fluorophores onto S1 (see METHODS). We previously validated that Q3 and A4 introduced at the specified locations didn't affect viral infectivity 55 . Lentivirus particles used for smFRET imaging were prepared by transfecting 293T cells with a 20-fold excess of plasmid encoding SD614, SG614, SAlpha, or SAlpha+E484K over their corresponding 427-Q3/556-A4 plasmid. Statistically, this strategy similarly used for the analogous investigations of HIV-1 envelope 57,58 ensures the production of virus particles that contain, on average, only a single Q3/A4-tagged FRET-engineered S protomer within a hybrid trimer (tagged -wildtype -wildtype) among elsewhere wildtype S trimers on the virus. Enzymes 59,60 that site-specifically recognize amino acids in respective Q3 and A4 tags transfer customized dyeconjugated substrates to these tags 55 , so that the donor and acceptor dyes (LD555, green; LD655, red) were introduced into the S1 of S variants ( Figure 1A ). Fluorescently labeled lentivirus particles were then immobilized on a quartz slide and imaged on a prism-based TIRF microscope. Donor fluorophores were directly excited by a singlefrequency (532 nm) continuous-wave laser, and fluorescence emissions from both donor and acceptor were separately recorded at 25 Hz ( Figures 2B, 3C ,E, and S1A-B).

Conformational motions in the Spike will lead to changes in the inter-dye distance exampled by structural switching from "RBD-down" to "RBD-up" (Figure 1B) , causing the donor-to-acceptor energy transfer efficiency (FRET efficiency) changes. FRET values can be derived by fluorescence signals of FRET-paired dyes in real-time. In smFRET experiments. We extracted fluorescence traces from the recorded movies that demonstrate anti-correlated levels of donor and acceptor fluorescence and derived FRET traces ( Figures 2B, 3C , E, and S1A-B) to reflect conformational changes in the Spike structures.

We first tested S-dependent infectivity of lentivirus particles on hACE2 expressing 293T cells (293T-ACE2). Virus particles bearing SG614, SG614+N501Y, SG614+E484K, SG614+E484K+N501Y, SAlpha, or E484K carrying SAlpha (SAlpha+E484K) variants have a discernible increase in infectivity compared to viruses bearing the parental SD614 ( Figure   2A ) in agreement with previous results 49, 50, 53, 61, 62 .

To know whether D614G substitution on S would influence the conformational profiles of Spikes, we next performed smFRET analyses of SG614 on lentivirus particles conformation from newly-generated conformational population-indicated FRET histogram ( Figure 2C ), compiled of hundreds of FRET traces. In contrast, the FRET histogram of unbound SG614 on lentiviral particles demonstrate that SG614 inherits all four conformations from the SD614 with a shift in the conformational landscape ( Figure 2D ). SG614 exhibits a propensity to occupy the 0.3-FRET indicated "one/two-RBD-up" partially open states, in which either one or two RBD is exposed for hACE2-binding ( Figure 2D ). can be directly observed from the individual FRET traces ( Figure S1 ).

Next, we wanted to gain insights into the sequence and the frequency of conformational dynamics of SD614G. We applied the Hidden Markov Modeling (HMM) 63 Table S1 ). This finding partially contradicts the cryoEM structural results 52, 53 , which indicate soluble forms of SAlpha being more "open" compared to the same engineered soluble forms of SD614 and SG614. This insight has resulted from a lack of consideration of the activated state -a fully open "three-RBD-up" conformation. To further assess the conformational effects of E484K in the context of SBeta, SGamma and SAlpha, which later adopted the alternation, we next performed smFRET studies using stabilizes the S variants towards the "one/two-RBD-up" conformation by reducing the "all-RBD-down" conformation. The overall conformational landscape of ligand-free SAlpha+E484K ( Figure 3D ) is similar to that of SG614 ( Figure 2D ), with slightly elevated occupancy in the "one/two-RBD-Up" conformations. The observation that E484K renders S variants adopting more "one/two-RBD-up" conformations agrees with all existing high-resolution cryoEM structural results of E484K carrying SBeta and SGamma 52, 53 . The similarity between overall conformational distributions (FRET histograms) of SAlpha+E484K and SG614 is also in line with the structural and biochemical observations that highlighted the consistency between the structural and biochemical profiles of the E484K-containing SBeta and SG614 variants 53 . Interestingly, structural characterization of hACE2-bound S variants has been rarely reported. In our study, smFRET investigations of ligand-free and hACE2-bound S variants were done in parallel in a lentivirus context.

demonstrates that the parental Spike and variants are unambiguously predominated by the "all-RBD-up" (0.1-FRET) activated state upon binding to hACE2 receptors ( Figure   3G ). The quantifications of relative conformation-occupancy for all inspected S variants are summarized in Figure 3G , and the determining parameters are listed in Table S1 . Our results of transition rates among four different conformations imply that the spike protomer is prone to be more conformationally stable in the "RBD-up" conformation than in the "RBD-down." Thus, it is not surprising to observe that hACE2-bound spikes ("all-RBD-up") show least propensity to shuttle back to the upstream conformations on the fusion path ( Figure 4B) . Similarly, the "all-RBD-up", hACE2-bound conformation shows the lowest relative free energy in the derived quantitative model for hACE2 activation 

SARS-CoV-2 variants of concern -VOCs ( Figure 1C ) pose significant challenge to public health worldwide. All four variants feature an initially world-wide adopted D614G mutation compared to the original Wuhan strain, while three of them contain an E484K mutation (E484K was also later adopted by the Alpha variant according to the World Health Organization, https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/).

As these variants have out-competed the original Wuhan strain and became the globally dominant strains, it is of great importance that we understand how these mutations within the spike influence the behavior and functionality of the spike. In this study, we have uncovered various aspects of such influence, which furthered our understanding of SARS-CoV-2's survival strategy during the pandemic that started in 2019.

The controversy of D614G on spike conformations and receptor-binding: enhancing conformational flexibility or stability? Lower or higher binding affinity to hACE2?

D614G mutation in the S protein is associated with enhanced transmissibility of SARS-Cov-2 viruses 66, 67 . We showed that lentiviruses that contain D614G spike exhibit significant increase in infectivity compare to the original Wuhan strain. High-resolution cryoEM structures of truncated soluble SG614 suggest that the naturally occurring SG614 is more conformationally flexible and favors the "open" conformations that are more accessible to hACE2, which likely accounts for enhanced virus transmissibility [49] [50] [51] 61 . A most recent cryoEM work shows that full-length SG614 exhibits higher S trimer stability and lower binding affinity to hACE2 than the parental D614 48 , which contradicts several studies 49, 50, 67 . The coexistence of enhanced transmissibility and reduced hACE2 binding was rationalized by attributing the transmissibility primarily to the effect of D614G substitution on preventing S1 from shedding off on the virion, which consequently increases the number of functional spikes on the viral surface 48 . This explanation was supported by observing a disordered-to-ordered transition of a "630 loop" caused by the D614G substitution, which seemingly stabilizes S trimer in the "one-RBD-up conformation", preventing premature S1 shedding 48 .

Using smFRET, we have determined that the SG614 variant exhibits a higher number of The original S strain adopted a predominately "all-RBD-down" closed conformation in order to conceal the RBD from host humoral responses 29, 31, 42, 55 , thus allowing the SARS-CoV-2 to better survive within the host. While S can only bind to hACE2 when the RBD is exposed, the concealment of RBD can be an important immune evasion strategy for the virus, as RBD is a primary target of neutralizing antibodies. As a cost of this protective trait, the receptor-binding ability of S has also suffered reduction due to the concealment of RBD. However, the subsequent variants carrying the D614G mutation within S have adopted another strategy to compromise this. D614G reduces S1 shedding and exhibits more functional spikes on the surface predominantly in hACE2-accessible conformations, which significantly increases the hACE2-binding competence, as discussed above. While this may contribute to an enhanced transmission rate, the increased number of surface S combined with their RBD-exposed conformation could render the viruses markedly more susceptible to host antibody attacks. It indicates that higher receptor-binding competence and faster transmission rate confer more fitness advantage for the spread of SARS-CoV-2 than a higher degree of protection against host immunity. Therefore, early SARS-CoV-2 evolution to the human host from late 2019 to early 2020 likely prioritizes receptor-binding competence to enhance transmissibility instead of the defense against host immunity.

In addition to D614G, E484K is another mutation adopted by most of the dominating strains, appearing in 3 of the 4 VOCs (Alpha, Beta and Gamma), of which Alpha variant later was similarly observed to adopt E484K according to the World Health

Organization. The appearance of both E484K and N501Y mutations appear to increase the hACE2-binding affinity of S 53,62 . This characteristic trait indicates that E484K coexisting with N501Y also serves to increase the transmissibility of SARS-CoV-2. Our results suggest that, like D614G, E484K achieves this by promoting the S conformation to shift from predominantly "all-RBD-down" hACE2-inaccessible closed state to "one/two-RBD-up" hACE2-accessible partially open states, thus exposing the RBD and facilitating receptor binding of S. Similar to that of D614G, emerged E484K carrying S variants likely promote transmissibility by adopting more dynamics-decelerated or implied stability-enhanced fusion-promoting hACE2-accessible conformations, revealed by our smFRET results. Notably, E484K was also identified as an escape mutation, as it enables the virus to escape host antibody recognition 9,24,68-70 . This immune-evasive trait could be caused by the reversal of electric charge at position 484 due to the E-to-K mutation, which creates a disadvantaged electrostatic interaction site that likely weaken the binding efficiency of anti-RBD antibodies. The shift of electric charge could potentially cause local conformational changes in the RBD 16 , which exceeds the capacity of our current smFRET setup. It is possible that this unique gain-of-function mutation was selected to reconcile the cost of exposing the RBD, which is a primary target of highly potent neutralizing antibodies. It could explain why the original Alpha variant, identified in the UK, did not contain the E484K but has since adopted it. By the time of performing this study, SARS-CoV-2 has modified its survival strategy by adopting E484K in addition to D614G to confer higher transmissibility (likely complemented with N501Y) and protect S against antibodies. As the delta variant currently out-compete others as the dominant pandemic form, SARS-CoV-2 could have shifted its survival strategy again, which requires further investigation beyond the scope of this study.

In conclusion, we used smFRET to reveal real-time conformational populations adopted by virus-associated S variants and experimentally identified conformationinterconverting temporal dynamics. We found that S evolves in favor of the hACE2- 

The authors declare no competing interests.

No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment.

293T (or HEK293T) cells, 293T-ACE2, and Expi293F cells were cultured in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, 2 mM Lglutamine, and in the presence of 5% CO2. Cell culture media was exchanged with fresh media before transfection. 293 cells, human embryonic kidney cells, were obtained from ATCC (cat # CRL-1573™). 293T cells were 293 cells derivative with the simian virus 40 T-antigen inserted. 293T-ACE2 cells, derived from 293T, stably express human ACE2 71 . Expi293F cells, derived from the 293 cells, were purchased from ThermoFisher Scientific (cat # A14528; RRID: CVCL_D615).

Our current FRET-engineered system uses enzymatic labeling that introduces peptide tags located sterically further from any of accumulated mutations on S variants. Untagged and double-tagged SARS-CoV-2 spike variants were generated based on a template full-length pCMV3-SARS-CoV-2 Spike (codon-optimized, Sino Biological, cat # VG40589-UT) plasmid that has translated amino acid sequence identical to QHD43416.1 (GenBank). As described previously 55 , peptide-based labeling tags Q3 (GQQQLG) and A4 (DSLDMLEM) 59,60 were engineered into the parental S D614 (SD614, first identified in Wuhan) and tested variants in this study at positions before and after the receptor-binding motif (RBM) to avoid interfering with the binding of cellular receptor human ACE (hACE2) ( Figure 1C ). Insertion of a pair of 427-Q3/556-A4 into S did not compromise S-dependent lentivirus infectivity and this double-tagged FRET-engineered SD614 (designated as SD614 Q3/A4) were constructed for smFRET imaging of SD614 55 . D614G point mutation was introduced into both untagged full-length pCMV3 SD614 and double-tagged SD614 Q3/A4 constructs to generate both untagged and tagged SG614 variants. E484K and N501Y were further introduced into the untagged SG614 individually and together. In the case of S Alpha (pcDNA3.1 SAlpha, codon-optimized) variant, DNA sequences encoding untagged or double-tagged (with tags positioning at the exact positions as in tagged SD614 or SG614) were synthesized by GeneScript and cloned into the pcDNA3.1 vectors. E484K point mutation was introduced into both untagged and tagged pcDNA3.1 SAlpha by site-specific mutagenesis.

The infectivity of lentivirus particles carrying S proteins (including variants) on the surface was evaluated using a vector containing an HIV-1 long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (HIV-1-inGluc) 72, 73 . 293T cells were transfected at 60-80 % confluency with the plasmid encoding indicated full-length SARS-CoV-2 S glycoproteins, the plasmid encoding an intron-regulated Gluc (HIV-1-inGluc), and a plasmid pCMV delta R8.2 encoding HIV-1 GagPol (Addgene, plasmid # 12263) using FuGENE 6 (Promega, # E2311). 40 hours post-transfection, lentivirus particles were harvested, filtered through a 0.45 mM filter (Pall Corporation), and titered on target cells 293T-ACE2 that endogenously express hACE2. Lentivirus infectivity was determined 48 hours post-infection by measuring Gaussia luciferase activity in the 293T-ACE2 supernatant using a Pierce TM Gaussia luciferase flash assay kits (ThermoFisher Scientific, cat# 16158).

The human ACE2 (hACE2) expression construct was synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors. Monomeric (residues 1-615) human ACE2 proteins were produced as described previously 74 . Briefly, DNA sequence encoding monomeric hACE2 followed by an HRV3C cleavage site, monomeric Fc tag, and 8xHisTag at the 3'-end were synthesized and cloned into the pVRC8400 vector. The proteins were expressed by transiently transfecting Expi293F cells and then purified by Protein A affinity columns. The Fc and 8xHis tags were removed by overnight HRV3C digestion at 4 °C. hACE2 proteins were further purified over a Superdex 200 16/60 column in the pH7.5 buffer solution containing 5 mM HEPES and 150 mM NaCl.

Lentivirus particles carrying S proteins (the ancestor and variants) on the surface were prepared similarly as previously described for the parental SD614 55 and HIV-1 57, 58, 75 . Briefly, lentivirus particles were produced by pseudo-typing an HIV-1 core with fulllength S proteins. To conduct studies of S variants by smFRET, we introduced the labeling peptide tags Q3 and A4 at the same positions (427 and 556, respectively) in S1 of SG614, SAlpha, E484K carrying SAlpha+E484K variants as in previously reported SD614 55 . Lentivirus particles used for smFRET imaging were made in 293T cells by transfecting a 20-fold excess of plasmid encoding SD614, SG614, SAlpha, or SAlpha+E484K over their corresponding 427-Q3/556-A4 to ensure that, on average, every virus particle carries a single Q3/A4-tagged S protomer. Plasmids encoding indicated untagged wildtype S (or variants), the corresponding double-tagged/FRET-engineered S, and a lentivirus packaging delta R8.2 were used at a ratio of 20:1:21, respectively. 293T cells were transfected using FuGENE 6 with above-indicated plasmids to express spikes on the HIV-1 lentivirus particles. Particles were harvested 40 hours post-transfection, filtered through a syringe filter with 0.45 µm pore size, and sedimented through a 15 % sucrose cushion at 25,000 rpm for 2 hours. The virus pellets were then re-suspended in pH 7.5 50 mM HEPES containing 10 mM MgCl2 and 10 mM CaCl2. Of note, the strategy of using an excess of plasmid-encoding wildtype vs. trace amounts of a plasmid expressing labeling-tag-carrying S ensures the production of virus particles that contain, on average, only a single FRET-engineered S protomer. While most virus particles carry untagged S, among the small portion of virus particles containing tagged S, more than 95 % will carry one dually tagged protomer while the other two protomers remain wildtype. The same strategy has been used in our previous smFRET studies 55, 57, 58, 75 .

Lentivirus particles were fluorescently labeled through site-specific labeling based on enzymatic reactions, as previously described 55, 57, 58, 75 . Briefly, transglutaminase transferred a Cy3B(3S) derivative (LD555-cadaverine) from the cadaverine conjugate to the central glutamine residue of the Q3 (GQQQLG) tag in S1. The AcpS enzyme catalyzed the attachment of the Cy5 derivative (LD655-CoA) to the serine residue of the A4 tag (DSLDMLEM). LD555-cadaverine (0.5 µM, Lumidyne Technologies), LD655-CoA (0.5 µM, Lumidyne Technologies), transglutaminase (0.65 µM, Sigma Aldrich), and AcpS (5 µM, home-made) were added to the above lentivirus particles prepared for smFRET, and incubated at room temperature overnight. LD555-cadaverine and LD655-CoA were synthesized by Scott Blanchard laboratory (Lumidyne Technologies) based on new generation photostable Cy3/Cy5 organic dyes 76 . DSPE-PEG2000-biotin (0.02 mg/ml, Avanti Polar Lipids) was then added to the reaction mix and incubated for 30 min at room temperature with rotation. Labeled lentivirus particles were then purified to eliminate excess free dyes and lipids by ultracentrifugation for one hour at 40,000 rpm over a 6%-18% Optiprep (Sigma Aldrich) gradient. Purified fluorescently-labeled lentivirus particles were stored at -80 °C.

smFRET imaging data acquisition All smFRET imaging experiments were performed on a home-built prism-based total internal reflection fluorescence (TIRF) microscope. Lentiviruses particles carrying fluorescently labeled spike proteins were immobilized on PEG-passivated quartz slides coated with streptavidin. The evanescent field was generated at the interface between the quartz slide and the virus-containing sample solution by prism-based total internal reflection, with 532-nm CW laser excitation (Laser Quantum). The donor fluorophore (LD555) labeled on the virus is excited by the generated evanescent field and can transfer energy at its excited states to the neighboring acceptor fluorophore (LD655). Fluorescence from both fluorophores was collected through a 1.27-NA 60 x waterimmersion objective (Nikon) and then optically separated by a 650 DCXR dichroic filter (Chroma) mounted on a MultiCam LS image splitter (Cairn Research). Fluorescence of donor (ET590/50, Chroma) and acceptor (ET690/50, Chroma) was separately and simultaneously recorded on two synchronized ORCA-Flash4.0 V3 sCMOS cameras (Hamamatsu) at 25 Hz for 80 seconds. Fluorescently labeled virus samples were imaged in pH 7.4, 50 mM Tris buffer containing 50 mM NaCl, a cocktail of triplet-state quenchers, and an oxygen-scavenger system 77 . Where indicated, the conformational effects of hACE2 on S proteins were conducted by pre-incubating fluorescently labeled viruses with 200 µg/ml hACE2 for 90 mins at room temperature before imaging and smFRET imaging data were taken in the continued presence of 200 µg/ml hACE2.

The analysis of smFRET data was performed using a MATLAB-based customized SPARTAN software package 78 . Both donor and acceptor fluorescence intensity of each labeled virus were recorded over 80 seconds. The background signals at the singlemolecule level were first identified based on the single-step fluorophore bleaching point and subtracted from the recorded signals. For each recorded virus, donor and acceptor fluorescence intensity traces (fluorescence time trajectory) were then extracted and corrected for the donor to acceptor crosstalk. The energy transfer efficiency from the donor to the acceptor (FRET values, simplified as FRET in graphs) was calculated over time according to FRET= IA/(gID+IA), where ID and IA are the fluorescence intensities of donor and acceptor, respectively, and g is the coefficient correcting for the difference in detection efficiencies of donor and acceptor. FRET traces (FRET values as a function of real-time) monitor the relative donor-to-acceptor distance over time, which reflect conformational dynamics of the donor/acceptor-labeled spike on the lentivirus in realtime. Virus particles that fall into any of the following categories were automatically excluded from our data analysis: 1) lacking either donor or acceptor or both fluorophores; 2) containing multiple labeled protomers; 3) containing more than one labeled spike on a single virus. In addition, FRET traces from individual viruses that meet our initial selection criteria and display sufficient signal-to-noise (S/N) ratio and anti-correlated fluctuations in donor and acceptor fluorescence were manually picked for data analysis. The anti-correlated feature directly defines FRET-indicated conformational states, which indicate fully active/dynamic spikes on virus particles. Thus, only one FRET-labeled protomer within one S trimer on one lentivirus particle (1protomer:1-trimer:1-virus particle) showing clear anti-correlated features of donor and acceptor fluorescence and single fluorescence bleaching point passed our filters. FRET traces included in our analysis were then compiled into FRET histograms, which reflect conformational ensembles. Based on visual inspection of fluorescence and FRET traces that revealed direct observations of state-to-state (conformation-to-conformation) transitions, FRET histograms were fitted into the sum of four Gaussian distributions using the least-squares fitting algorithm in MATLAB. Each gaussian represents one FRET-defined conformational state of S proteins, and the area under each Gaussian curve estimates the relative probability of S occupying each conformation. The relative occupancy of each conformational state was used to evaluate the difference in relative free energies between states i and j, according to ΔG°ij=-kBTln(Pi/Pj), where Pi and Pj are the occupancy of ith and jth states, respectively, kB is the Boltzmann constant, and T is the temperature in kelvin. FRET traces compiled in the FRET histogram under indicated experimental conditions were idealized using a segmental Kmeans algorithm 63 with a 4-state Hidden Markov Model -providing the simplest explanation of our data. The frequency and order of idealized state-to-state transitions were displayed in a transition density plot (TDP). Dwell times of S in each states were compiled into survival probability plots which were fitted to the sum of two exponential distributions (y(t) = A1 exp -k1t + A2 exp -k2t ), where y(t) is the probability and t is the dwell time. The transition rates were determined by averaging the two rate constants weighted by their amplitudes. (A) Virus-like particles that carry a single two-dye-labeled SARS-CoV-2 spike protomer among unlabeled wildtype spikes were immobilized on a quartz slide and then imaged on a prismbased total internal reflection fluorescence (TIRF) microscope. The quartz slide was passivated with PEG/PEG-biotin to enable streptavidin coating and the subsequent immobilization of virions that contain a biotin-lipid (DSPE-PEG-biotin). The virus-like particles were composed of HIV-1 cores and SARS-CoV-2 spikes on the virus surface. (B) Positioning of labeling dyes for the site-directed incorporation of fluorophores (LD555cadaverine: a Cy3B derivative, green; LD655-CoA: a Cy5 derivative, red) was elucidated by the S1 conformational change from the ''RBD-down'' to the ''RBD-up'' structures, which are adapted from RCSB PDB: 6ZB5 (''all-RBD-down'') and 7KJ5 (''one-RBD-up''). (C) Domain organization of the parental full-length SARS-CoV-2 spike protein with D614G and E484K mutation. The introduction sites of labeling tags Q3 (green) and A4 (red), where the donor and acceptor fluorophores will be conjugated to respectively, are indicated. SD1, subunit domain 1; SD2, subunit domain 2; S1/S2, S2', protease cleavage sites; FP, fusion peptide; HR1/HR2, heptad repeat 1/heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. Labeling peptides Q3 and A4 are inserted into RBD and SD1 respectively before and after RBM within the S protein in these studies. Emerged variants that are currently classified by CDC as "variants of concern" and their respective key mutations in the spike are specified in the table. Table S1 . Table S1 . 3 -"Up" 3 -"Down" 1 -"Up" 2 -"Up" Unknown 3 -"Up" 3 -"Down" 1 -"Up" 2 -"Up" Figure 5 . Relative free-energy models depict conformational landscapes of different virus-associated spike variants upon activation by the binding of hACE2.

(A -D) Free-energy models of parental SD614 (A) and variants SG614 (B), SAlpha (C), and SAlpha+E484K (D). The differences in free energies between states were roughly scaled based upon the relative state occupancies of each state. The ligand-free D614G and E484K carrying spikes are dominated by the intermediate-FRET state ("one/two-RBD-up"), which exhibit the lowest relative free energy among all four FRET states. In contrast, all hACE2-bound spikes show the lowest relative free energy at the low-FRET state ("all-RBD-up"). Figure 4B ) were estimated by double exponential-fitting of survival probability plots. Figure S3 . Kinetic analysis of spike variants SAlpha and SAlpha+E484K on lentivirus particles, and modulation by hACE2. (Related to Figure 4 ).

(A, B) Survival probability plots as in Figure S2 for spike variants SAlpha (A) and SAlpha+E484K (B) give rise to the estimation of transition rates among different conformations of spike variants. Transition rates were summarized in Figure 4B . Table S1 . Relative probability and determining parameters of four FRET-defined conformational states occupied by spike variants on lentivirus particles. (Related to Figures 2 -5) .

Conformational distribution-indicated FRET efficiency histograms were fitted into the sum of four distinct Gaussian distributions (μ, mean or expectation; σ, standard deviation) for each conformation. Parameters (μ and σ) were based upon the observation of original FRET efficiency traces and were further determined using hidden Markov modeling. The probability of conformations that spikes occupy was presented as mean ± s.e.m., determined from three independent measurements. R-squared values were evaluated to indicate the goodness of fit. 

Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine

Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine

Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK

Interim results of a phase 1-2a Trial of Ad26.COV2.S Covid-19 vaccine

Monoclonal antibodies for prevention and treatment of COVID-19

Monoclonal antibodies for COVID-19

Resistance of SARS-CoV-2 variants to neutralization by monoclonal and serum-derived polyclonal antibodies

SARS-CoV-2 variants show resistance to neutralization by many monoclonal and serum-derived polyclonal antibodies

Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7

mRNA vaccine-elicited antibodies to SARS-CoV-2 and circulating variants

Neutralizing activity of BNT162b2-elicited serum

Efficacy of the ChAdOx1 nCoV-19 Covid-19 Vaccine against the B.1.351 Variant

SARS-CoV-2 variant B.1.617 is resistant to Bamlanivimab and evades antibodies induced by infection and vaccination

Multiple SARS-CoV-2 variants escape neutralization by vaccineinduced humoral immunity

SARS-CoV-2 variants B.1.351 and P.1 escape from neutralizing antibodies

Increased resistance of SARS-CoV-2 variant P.1 to antibody neutralization

Single-dose mRNA vaccine effectiveness against SARS-CoV-2 in healthcare workers extending 16 weeks post-vaccination: a test-negative design from Quebec

Single-dose mRNA vaccine effectiveness against SARS-CoV-2, including Alpha and Gamma variants: a test-negative design in adults 70 years and older in British Columbia

mRNA-1273 vaccine induces neutralizing antibodies against spike mutants from global SARS-CoV-2 variants. bioRxiv

Impact of Delta on viral burden and vaccine effectiveness against new SARS-CoV-2 infections in the UK. 2021

Tracking Changes in SARS-CoV-2 Spike: Evidence that D614G Increases Infectivity of the COVID-19 Virus

SARS-CoV-2 variants, spike mutations and immune escape

Covid-19: The E484K mutation and the risks it poses

Contribution of single mutations to selected SARS-CoV-2 emerging variants spike antigenicity

SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor

A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells

Structural and functional basis of SARS-CoV-2 entry by using human ACE2

Structure, function, and antigenicity of the SARS-CoV-2 spike glycoprotein

Distinct conformational states of SARS-CoV-2 spike protein

Structures and distributions of SARS-CoV-2 spike proteins on intact virions

Cell entry mechanisms of SARS-CoV-2

Mechanisms of coronavirus cell entry mediated by the viral spike protein

Single-Molecule FRET imaging of virus spike-host interactions

Receptor binding and priming of the spike protein of SARS-CoV-2 for membrane fusion

Controlling the SARS-CoV-2 spike glycoprotein conformation

Structure-based design of prefusion-stabilized SARS-CoV-2 spikes

Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor

Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike

Structure-guided covalent stabilization of coronavirus spike glycoprotein trimers in the closed conformation

Structural basis of receptor recognition by SARS-CoV-2

Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation

Molecular architecture of the SARS-CoV-2 virus

One year of SARS-CoV-2 evolution

An interactive web-based dashboard to track COVID-19 in real time

Establishment and lineage dynamics of the SARS-CoV-2 epidemic in the UK

Network for Genomic Surveillance in South Africa writing, g. A genomics network established to respond rapidly to public health threats in South Africa

Structural impact on SARS-CoV-2 spike protein by D614G substitution

The effect of the D614G substitution on the structure of the spike glycoprotein of SARS-CoV-2

D614G mutation alters SARS-CoV-2 spike conformation and enhances protease cleavage at the S1/S2 junction

Structural and functional analysis of the D614G SARS-CoV-2 spike protein variant

Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity

Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants

Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

Real-Time conformational dynamics of SARS-CoV-2 spikes on virus particles

A glycan gate controls opening of the SARS-CoV-2 spike protein

Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET

Conformational dynamics of single HIV-1 envelope trimers on the surface of native virions

Transglutaminase-catalyzed site-specific conjugation of smallmolecule probes to proteins in vitro and on the surface of living cells

Site-specific protein labeling by Sfp phosphopantetheinyl transferase

SARS-CoV-2 D614G spike mutation increases entry efficiency with enhanced ACE2-binding affinity

Higher infectivity of the SARS-CoV-2 new variants is associated with K417N/T, E484K, and N501Y mutants: An insight from structural data

Analysis of single-molecule FRET trajectories using hidden Markov modeling

The great escape? SARS-CoV-2 variants evading neutralizing responses

Impact of temperature on the affinity of SARS-CoV-2 Spike glycoprotein for host ACE2

Spike mutation D614G alters SARS-CoV-2 fitness

SARS-CoV-2 spike D614G change enhances replication and transmission

SARS-CoV-2 spike E484K mutation reduces antibody neutralisation

Antibody cocktail to SARS-CoV-2 spike protein prevents rapid mutational escape seen with individual antibodies

Escape from neutralizing antibodies by SARS-CoV-2 spike protein variants

Cross-Sectional evaluation of humoral responses against SARS-CoV-2 spike

Assembly of the murine leukemia virus is directed towards sites of cell-cell contact

Cell-to-cell transmission can overcome multiple donor and target cell barriers imposed on cell-free HIV

Structure-Based design with tag-based purification and in-process biotinylation enable streamlined development of SARS-CoV-2 spike molecular probes

HIV-1 Env trimer opens through an asymmetric intermediate in which individual protomers adopt distinct conformations

Tuning the Baird aromatic triplet-state energy of cyclooctatetraene to maximize the self-healing mechanism in organic fluorophores

An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments

Single-Molecule imaging of non-equilibrium molecular ensembles on the millisecond timescale