key: cord-0723486-cz8nf0sp
authors: Lu, Maolin; Uchil, Pradeep D.; Li, Wenwei; Zheng, Desheng; Terry, Daniel S.; Gorman, Jason; Shi, Wei; Zhang, Baoshan; Zhou, Tongqing; Ding, Shilei; Gasser, Romain; Prévost, Jérémie; Beaudoin-Bussières, Guillaume; Anand, Sai Priya; Laumaea, Annemarie; Grover, Jonathan R.; Liu, Lihong; Ho, David D.; Mascola, John R.; Finzi, Andrés; Kwong, Peter D.; Blanchard, Scott C.; Mothes, Walther
title: Real-time Conformational Dynamics of SARS-CoV-2 Spikes on Virus Particles
date: 2020-11-13
journal: Cell Host Microbe
DOI: 10.1016/j.chom.2020.11.001
sha: 9fef46ca5afecc5f5c34dc2a4a208613897ba32b
doc_id: 723486
cord_uid: cz8nf0sp

SARS-CoV-2 spike (S) mediates viral entry into cells and is critical for vaccine development against COVID-19. Structural studies have revealed distinct conformations of S, but real-time information that connects these structures, is lacking. Here we apply single-molecule Fluorescence (Förster) Resonance Energy Transfer (smFRET) imaging to observe conformational dynamics of S on virus particles. Virus-associated S dynamically samples at least four distinct conformational states. In response to human receptor Angiotensin-Converting Enzyme 2 (hACE2), S opens sequentially into the hACE2-bound S conformation through at least one on-path intermediate. Conformational preferences observed upon expsoure to convalescent plasma or antibodies suggest mechanisms of neutralization involving either competition with hACE2 for binding to the receptor-binding domain (RBD) or allosteric interference with conformational changes required for entry. Our findings inform on mechanisms of S recognition and conformations for immunogen design.

SARS-CoV-2 spike (S) mediates viral entry into cells and is a main target for antibody responses against the virus (Gao et al., 2020; Jackson et al., 2020; Mercado et al., 2020; Yu et al., 2020) . S is synthesized as a precursor, processed into S1 and S2 by furin proteases, and activated for fusion when human Angiotensin-Converting Enzyme 2 (hACE2) engages the receptor-binding domain (RBD) and when the N-terminus of S2 is proteolytically processed (Hoffmann et al., 2020b; Walls et al., 2020; Wrobel et al., 2020) . Structures of soluble ectodomains and native virus particles have revealed distinct conformations of S, including a closed trimer with all RBD oriented downward, trimers with one or two RBDs up, and hACE2-stabilized conformations with up to three RBD oriented up Cai et al., 2020; Henderson et al., 2020; Hsieh et al., 2020; Ke et al., 2020; Lan et al., 2020; McCallum et al., 2020; Shang et al., 2020; Walls et al., 2020; Wang et al., 2020; Wrapp et al., 2020b; Wrobel et al., 2020; Yao et al., 2020; Zhou et al., 2020b) . Real-time information that connects these structures, however, has been lacking. Single-molecule Fluorescence (Förster) Resonance Energy J o u r n a l P r e -p r o o f Transfer (smFRET) is well suited to inform on conformational dynamics of proteins reporting domain movements in the millisecond to second range, and has previously been applied to study HIV-1, influenza A, and Ebola spike glycoproteins, via measurements of the distance-dependent energy transfer from an excited donor to a nearby acceptor fluorophores in real-time (Das et al., 2020; Das et al., 2018; Lu et al., 2019a; Ma et al., 2018; Munro et al., 2014) .

To probe dynamics of SARS-CoV-2 spikes, we used available high-resolution structures of the SARS-CoV-2 S trimer to identify sites of fluorophore pair labeling that have the potential to inform on distance changes expected to accompany conformational changes between the RBD-down and receptor hACE2-induced RBD-up trimer structures (Lan et al., 2020; Wrapp et al., 2020b) (Figure 1A and 1B). Accordingly, we engineered A4 and Q3 labeling peptides before and after the receptor-binding motif (RBM) to allow site-specific introduction of donor and acceptor fluorophores at these positions ( Figures 1B and 1C ). We optimized retroviral and lentiviral pseudoviral particles carrying the SARS-CoV-2 S protein ( Figure S1A ) to test the impact of these peptides on infectivity, and found that they were well tolerated, both individually and in combination (Figures S1B and S1C). To measure conformational dynamics of the SARS-CoV-2 S trimer on the surface of virus particles, we applied the S protein carrying the Q3 and A4 labeling peptides at positions 427 and 556, respectively ( Figure 1C ). We established two types of particles, lentiviral particles carrying S, as well as coronaviruslike particles generated by expression of S, membrane (M), envelope (E) and nucleocapsid (N) protein (S-MEN) (Gordon et al., 2020; Siu et al., 2008) (Figures 1A) .

S-MEN particles co-express coronavirus surface proteins M and E. Particle quality and the presence of the corona-like S proteins on both particle surfaces were confirmed by cryo-electron microscopy ( Figures 1C and 1D ).

For smFRET, lentivirus particles and S-MEN particles were generated (see STAR METHODS) by transfecting HEK293T cells with an excess of plasmid-encoding wildtype, doped with trace amounts of plasmid expressing labeling peptide-carrying S (427-Q3/556-A4) to ensure the production of virus particles that contain, on average, only a single engineered S protein. As for analogous investigations of HIV-1 Envelope protein (Lu et al., 2019a; Munro et al., 2014) , donor (Cy3B(3S)) and acceptor fluorophore (LD650) were enzymatically conjugated to the engineered S proteins presented on the virus particle surface in situ (see STAR METHODS). A biotinylated lipid was then incorporated into the virus particle membrane to allow their immobilization within passivated microfluidic devices coated with streptavidin to enable imaging by total internal reflection microscopy ( Figure 1A ). Donor fluorophores on single, immobilized virus particles were excited by a single-frequency 532 nm laser and fluorescence emission from both donor and acceptor fluorophores were recorded at 25 Hz ( Figure   2A ). From the recorded movies, we computationally extracted hundreds of smFRET traces exhibiting anti-correlated donor and acceptor fluorescence intensities, the telltale signature of conformational changes within the S protein on individual virus particles.

Analyses of smFRET data from ligand-free S protein on lentiviral particles revealed that the SARS-CoV-2 S protein is dynamic, sampling at least four distinct conformational states characterized by low-(~0.1), intermediate-(~0.3 and ~0.5), and high-(~0.8) FRET efficiency (FRET). Population FRET histograms, comprised of hundreds of smFRET traces, revealed that the conformation exhibiting intermediate FRET (~0.5) to be the most abundantly occupied ( Figure 2B and Table S1 ). Comparable findings were obtained for S protein incorporated into S-MEN coronavirus-like particles ( Figure 2C) .

Notably, the intermediate-FRET (~0.5) conformation of the S protein was stabilized by a disulfide bridge between amino acids 383 and 985 . Because electron microscopy (EM) methods have identified the disulfide-bridge stabilized state as the three-RBD-down conformation (Henderson et al., 2020; McCallum et al., 2020) , these experiments suggest that the intermediate-(~0.5) FRET S protein conformation J o u r n a l P r e -p r o o f represents the closed trimer with all three RBD oriented downward (RBD-down conformation).

activates S from the ground state to the receptor-bound state through one necessary intermediate

To identify the receptor-bound conformation of the SARS-CoV-2 S protein by smFRET, we measured the conformational consequences of soluble, monomeric hACE2 binding.

Addition of the monomeric hACE2 receptor to surface-immobilized virus particles lead to increased occupancy of the low-(~0.1) FRET S protein conformation from ~22% to 48% (Figure 2E and Table S1 ) , which was observed at the single-molecule level ( Figure 2F ). Similar hACE2 receptor impacts on the SARS-CoV-2 S protein were observed in both lentiviral particle and S-MEN coronavirus-like particle contexts (Figures 2E to 2G) .

Dimeric hACE2, a more potent ligand ( Figure S2D ) (Lui et al., 2020) A unique strength of single-molecule imaging is its capacity to directly reveal both the structural and kinetic features that define biological function (Lu et al., 2019b; Roy et al., 2008) . To extract such information for the SARS-CoV-2 S protein, we employed Hidden Markov Modeling (HMM) (McKinney et al., 2006) to idealize individual smFRET traces.

These data allowed quantitative analyses of the thousands of discrete FRET transitions observed for the S protein on the surface of lentiviral and S-MEN particles in the absence and presence of ligands to gain insights into the order and timing of conformational changes across conditions ( Figures 2B, 2C , 2F, 2G, and S3). Transition density plots (TDPs), which conveniently display the nature, order and frequency of the transitions observed in the ensemble of molecules examined (Roy et al., 2008) , immediately revealed two salient features that informed on the nature of S protein dynamics. First, the symmetry of the evidenced transitions in each TDP with respect to the diagonal axis indicated that the unliganded S protein is in equilibrium exchange conformation, which was also modestly stabilized. The energy barriers for reverse transitions towards the RBD-down conformation (~0.5 FRET) were also elevated, explaining receptor-bound conformation accumulation over time ( Figure S3 ). These analyses lead to a qualitative model for hACE2 activation of the SARS-CoV-2 S protein from the ground state to the receptor-bound state through at least one intermediate conformation ( Figure 2H ). The summary of relative state occupancies, transition rates among conformations and errors are listed in Tables S1 and S2, respectively.

In most cell types, the serine protease TMPRSS2 is required for pH-independent SARS-CoV-2 entry (Bestle et al., 2020; Hoffmann et al., 2020a; Hoffmann et al., 2020b) . In J o u r n a l P r e -p r o o f vitro, the actions of TMPRSS2 are mimicked by the serine protease trypsin, which has similar cleavage specificity (Bestle et al., 2020; Hoffmann et al., 2020b) . smFRET analysis of trypsin-treated S protein on lentiviral particles in the absence of receptor revealed a clear shift towards activation ( Figures 3A and 3B ). After trypsin treatment, the addition of hACE2 receptor was more effective at stabilizing the S protein in the RBD-up (~0.1 FRET) conformation ( Figures 3C, 3D , and S4A).

To further validate this finding, we measured the effects of trypsin pre-treatment in viruscell and cell-cell fusion assays using split NanoLuc system consisting of LgBiT and HiBiT ( Figures 3E, 3F , S4B, and S4C). Here, membrane fusion restored luciferase activity between lentiviral particles carrying the S protein as well as a Vpr-HiBiT fusion protein with cells expressing the LgBiT counterpart fused to a PH domain. This assay revealed fusion to be strictly dependent on the hACE2 receptor and to be stimulated by trypsin treatment (Figures 3E and 3F ). Nearly identical results were observed for cellcell fusion between donor cells expressing S and target cells expressing hACE2 ( Figures S4B and S4C ), confirming the activating role of trypsin treatment.

We next explored the suitability of the smFRET assay to characterize the conformational consequences of antibody binding to the SARS-CoV-2 S protein.

Multiple studies on antibodies generated from COVID-19 patients have shown that one type of antibody often dominates immune responses Brouwer et al., 2020; Ju et al., 2020; Liu et al., 2020; Robbiani et al., 2020; Wu et al., 2020a) . This prompted us to screen plasma from convalescent patients with neutralizing activity that can bind to the S protein on lentiviral particles (Beaudoin-Bussieres et al., 2020) using a modified virus-capture assay (VCA) . Cross-reactive CR3022 (Yuan et al., 2020) , one of the very first reported antibodies from SARS-CoV-1 that also binds to SARS-CoV-2 spike RBD domain, served as a good indicator of RBD binding ( Figure   4A ). We identified two plasma samples (S002 and S006) able to specifically bind the RBD, recognize S expressed at the cell surface and to neutralize viral particles (Figures J o u r n a l P r e -p r o o f 4A-C, and S5). smFRET analysis of antibody-bound S revealed that both CR3022 and plasma from patient S006, stabilized S in the RBD-up (~0.1 FRET) conformation, as analogous to receptor hACE2 ( Figures 4D and 4E ). These data point to the presence of RBD-directed antibodies in patient S006. By contrast, smFRET indicated that plasma from patient S002 contained an activity that stabilized the RBD-down (~0.5 FRET) conformation ( Figure 4F ). Plasma S002 antagonized hACE2 binding, but RBD competition did not affect its recognition of S, suggesting that its neutralization activity does not solely rely on blocking the receptor interface. We then assessed the conformational preference of four RBD-directed antibodies: the neutralization nanobody VHH72, and the potently neutralizing antibodies H4, 2-43 and 2-4, each of which binds RBD in a different way Wrapp et al., 2020a; Wu et al., 2020b) . Figure 4K ). The absence or presence of hACE2 did not appear to affect the RBD-up stabilization evidenced for antibodies CR3022, S006, VHH72, or H4 ( Figure   5 ). However, plasma S002, and to a lesser extent antibody 2-4, reduced the hACE2dependent stabilization of the RBD-up (~0.1 FRET) conformation ( Figure 5 ), suggesting that they may interfere with hACE2 receptor binding via an allosteric mechanism. These findings indicate that SARS-CoV-2 neutralization can be achieved in two ways: 1) antibodies that conformationally mimic hACE2 and directly compete with hACE2 receptor binding, or 2) by allosterically stabilizing the S protein in its RBD-down conformation.

The strength of the presented smFRET approach is revealed by the capacity to examine the dynamic properties of the S protein in real time, including: 1) the distinct conformational states that it spontaneously transits under physiological conditions; 2) J o u r n a l P r e -p r o o f the impact of sequence alterations on S protein dynamics; and 3) the responses of the S protein to cognate hACE2 receptor and antibody recognition.

The present analyses of dynamic S protein molecules provides three lines of evidence that indicate that the intermediate-(~0.5) FRET state observed represents the RBDdown, ground state conformation of the S protein, in which all three RBD domains are oriented towards the viral particle membrane ( Figure 6 ). First, in line with previous electron microscopy (EM) investigations (Cai et al., 2020; Henderson et al., 2020; Hsieh et al., 2020; Ke et al., 2020; Lan et al., 2020; McCallum et al., 2020; Shang et al., 2020; Walls et al., 2020; Wang et al., 2020; Wrobel et al., 2020; Yao et al., 2020) , the RBDdown state is the most populated. In further agreement with recent EM studies, both a disulfide bridge (S383C, D985C) (Henderson et al., 2020; McCallum et al., 2020) and antibody 2-4 stabilized the S protein in a conformation with all three RBD oriented down . While our smFRET observations highlight considerable conformational flexibility in these contexts compared to EM of soluble trimers, these distinctions may be attributed to a tendency of our analysis approach to over-emphasize dynamic features, while EM may over-emphasize static conformations rigidified by cryogenic temperatures that may be more readily identified and classified . Multiple lines of evidence also facilitated assignment of the RBD-up (~0.1 FRET) conformation of the S protein with all three RBD domains oriented away from the virus particle membrane ( Figure 6 ). For instance, this conformation was stabilized by soluble monomeric hACE2 receptor, and even further stabilized in the presence of soluble, dimeric hACE2 receptor as well as RBD-targeting antibodies, such as CR3022, that are known to access their epitopes when the S protein is in an activated, RBD-up conformation (Wrapp et al., 2020a; Wu et al., 2020b; Yuan et al., 2020) . The structure of the on-path (~0.3 FRET) intermediate observed during S opening may represent an initial receptor binding conformation that is globally similar in its overall architecture to the all-RBD down ground state. Cryo-EM structures of soluble SARS-CoV-2 S trimers (Wrapp et al., 2020b) , which are asymmetrically engaged with only one or two hACE2 molecules receptors -rather than one for each S protomer (Zhou et al., 2020b) , reveal that the distance between the two labeling sites increases in the ligand-free protomers adjacent J o u r n a l P r e -p r o o f to a protomer engaged to hACE2 ( Figure 6 ). The additional, highly compacted S conformation (~0.8 FRET) evidenced, which is also depopulated by activating ligands, remains unknown.

These smFRET analyses are thus in global agreement with the conformational states observed at the single particle EM and cryoET level (Cai et al., 2020; Henderson et al., 2020; Ke et al., 2020; Klein et al., 2020; Lan et al., 2020; McCallum et al., 2020; Shang et al., 2020; Song et al., 2018; Walls et al., 2020; Wang et al., 2020; Wrapp et al., 2020a; Wrapp et al., 2020b; Wu et al., 2020b; Yao et al., 2020; Yuan et al., 2020) , while also revealing dynamic information relevant to S protein activation and ligand recognition. The observed FRET states, interpreted within the framework provided by (A) Virus particles carrying a two-dye labeled SARS-CoV-2 spike protein protomer among wildtype spikes were immobilized on a quartz slide and imaged on a customized prism-based Total internal reflection fluorescence (TIRF) microscope. The quartz slide was passivated with PEG/PEG-biotin to allow coating with streptavidin, and subsequent immobilization of viruses carrying a biotin-lipid (DSPE-PEG-biotin). Two virus particle systems were developed to incorporate SARS-CoV-2 spike proteins on their surfaces. HIV-1 lentivirus particles comprise HIV-1 cores and SARS-CoV-2 spike proteins on the surface. S-MEN virus-like particles consist of four structural proteins, in which S represents spike, M the membrane protein, E the envelope protein, and N the nucleocapsid protein of SARS-CoV-2. (B) Placement of labeling tags for the site-directed introduction of fluorophores (Cy3B, green; LD650, red) was guided by conformational changes in S1 induced by binding of the cellular receptor human angiotensin-converting enzyme 2 (hACE2) from the "RBD-down" to the "RBDup" conformation ( Figure S1 ). RBD, receptor-binding domain; NTD, N-terminal domain. Structures were adapted from RCSB Protein Data Bank accessories 6VSB ('Down' S1/S2 protomer: S1, light cyan; S2, dark blue) and 6VYB/6M0J ('Up' protomer S1/S2 engaged with hACE2: hACE2, magenta). (C) Domain organization of full-length (1273 amino acids) wildtype SARS-CoV-2 spike protein (S1+S2) colored by domain and sequence for the region carrying the peptide-labeling tags. The insertion sites of labeling tags Q3 and A4 for the donor and acceptor fluorophores are indicated. S1/S2, S2', protease cleavage sites; NTD, N-terminal domain; RBD, receptor-binding domain; RBM, receptor binding motif; SD1, subunit domain 1; SD2, subunit domain 2; FP, fusion peptide; HR1/HR2, heptad repeat 1/heptad repeat 2; TM, transmembrane domain; CT, cytoplasmic tail. Peptide insertion sites into RBD and SD1 before and after RBM for the dually tagged S protein applied in these studies. Table S1 . (H) Relative free energy model of conformational landscapes of SARS-CoV-2 spikes in response to the hACE2 binding. The differences in free energies between states were roughly scaled based upon relative state occupancies of each state. Table S1 . (E, F) Trypsin enhances SARS-CoV-2 spike-mediated hACE2-dependent virus-cell fusion. (E) Assay design to monitor virus-cell fusion using the HiBit and LgBiT split NanoLuc system (Yamamoto et al., 2019) . Vpr-HiBit was packaged into lentiviral particles carrying SARS-CoV-2 spike (LV_Spike). HEK293 target cells transiently expressing LgBiT tagged to PH domain of human phospholipase Cδ at the N-terminus alone or together with hACE2. hACE2-dependent virus-cell fusion was determined by monitoring reconstituted NanoLuc activity in target cells 24 h after infection. (F) Normalized relative luciferase units (RLU; mean ± s.d., two replicates with quadruplicates) measured 24 h post infection to quantify virus-cell fusion in stated target cells after treating viruses with or with indicated amounts of trypsin for 15-20 min at 37 o C. NanoLuc activities were normalized to luciferase activity detected in uninfected target cells. p values derived from unpaired t-test; **** corresponds to p < 0.0001. Figure 2B ). FRET histograms represent mean ± s.e.m., determined from three randomly assigned populations of FRET traces. For state occupancies see Table S1 . 

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Walther Mothes (walther.mothes@yale.edu).

All other unique reagents generated in this study are available from the Lead Contact with a completed Materials Transfer Agreement.

The data that support the findings of this study are available from the corresponding authors upon reasonable request. The full source code of SPARTAN, which was used for analysis of smFRET data, is publicly available (http://www.scottcblanchardlab.com/software). Custom MATLAB code are available upon request from the Lead Contact.

Cell Lines 293 (or HEK293) cells, 293T (or HEK293T) cells, 293T-ACE2, Expi293F, and Huh7.5 cells were cultured in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin/ streptomycin, 2 mM L-glutamine, and in the presence of 5% CO 2 . Cell culture media was exchanged before transfection. 293 cells are human embryonic kidney cells obtained from ATCC (cat # CRL-1573™). Expi293F cells are derived from the 293 cell line and were from ThermoFisher Scientific Inc (cat # A14528; RRID: CVCL_D615). 293T cells were derived from 293 cells, into which the simian virus 40 T-antigen was inserted. Huh7.5 is an immortal cell line of epithelial-like adult hepatocellular carcinoma cells expressing hACE2 on cell surfaces. 293T-ACE2 cells stably expressing human ACE2 is derived from 293T cells .

All SARS-CoV-2-postive patient serum work was conducted in accordance with the Declaration of Helsinki in terms of informed consent and approval by an appropriate institutional board (CHUM, 19.381). The donors met all donor eligibility criteria: previous confirmed COVID-19 infection by PCR and complete resolution of symptoms for at least 14 days.

Donor S002: Male, 65 years old, sample recovered 25 days after symptoms onset. Donor S006: Male, 30 years old, sample recovered 41 days after symptoms onset.

A full-length wild-type pCMV3-SARS-CoV-2 Spike (S1+S2)-long (termed as pCMV-S, codon-optimized, Sino Biological, cat # VG40589-UT) plasmid was used as a template to generate tagged pCMV-S. The translated amino acid sequence of pCMV-S is identical to QHD43416.1 (GenBank). Labeling tags Q3 (GQQQLG) and A4 (DSLDMLEM) (Lin and Ting, 2006; Yin et al., 2006) were placed before and after the receptor-binding motif (RBM) where the cellular receptor ACE binds ( Figure S1 ). The short peptide labeling tag Q3 was inserted at either of two positions in the receptorbinding domain (RBD) to generate the pCMV-S Q3-1 and Q3-2 constructs. The labeling tag A4 was inserted at either of two sites in subunit domain 1 (SD1) to generate the constructs pCMV-S A4-1 and A4-2, respectively. Insertion of single tags into S proteins did not affect viral infectivity. Four different combinations of double-tagged S (designated as pCMV-S Q3-1 A4-1, Q3-1 A4-2, Q3-2 A4-1, Q3-2 A4-2) were constructed to permit smFRET imaging. Each pair of inserted tags did not compromise S-dependent lentivirus infectivity.

The infectivity of lentivirus particles carrying SARS-CoV-2 spike proteins was determined using a vector containing an HIV-1 or MLV long terminal repeat (LTR) that expresses a Gaussia luciferase reporter (Gluc) (Jin et al., 2009; Zhong et al., 2013) 293T cells were cultured in DMEM media, supplemented with 10% FBS, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, and in the presence of 5% CO 2 . Cell culture media was exchanged before transfection. Cells were transfected at 60-80 % confluency with the plasmid encoding respective spike protein, 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). Variants of plasmids encoding spike included the wild-type pCMV-S, single or dually tagged pCMV-S, pCMV-S ∆19 (S lacking the last 19 amino acids of the cytoplasmic tail), and an additional S ∆19 expressing plasmid (a gift from Marc Johnson, University of Missouri). To generate retroviral particles with an MLV core carrying full-length S, an MLV GagPol construct, and an intron-regulated Gaussia luciferase reporter construct (MLV-inGluc) were used (Jin et al., 2009 ). An HIV-1 GagPol-InGluc construct (Johnson et al., 2020 ) (a gift from Marc Johnson, University of Missouri) in which HIV-1 GagPol and HIV-1-inGluc genes are expressed from a single plasmid were also tested. Viruses were harvested 40 hours post-transfection, filtered, and concentrated 7-fold by ultracentrifugation at 25,000 rpm in an SW28 rotor for 2 hours at 4 °C. Pelleted viruses were resuspended in above DMEM media and titered on Huh7.5 cells that endogenously express hACE2. Gluc activity was measured in the cell supernatant at 48 hours using a Pierce TM Gaussia luciferase flash assay kits (ThermoFisher Scientific, # 16158).

Antibody CR3022, VHH74, H4, 2-43, 2-4, and human ACE2 expression constructs were synthesized (Gene Universal Inc, Newark DE) and subcloned into corresponding pVRC8400 vectors. To express antibodies (Kong et al., 2019) , 0.15 ml of Turbo293 transfection reagent (Speed BioSystems) were added to 2.5 ml Opti-MEM medium (Life Technology) and incubated for 5 min at room temperature (RT). Meanwhile, 25 µg of heavy chain and 25 µg of light chain plasmid DNA were added to 2.5 ml of Opti-MEM medium in another tube. The Opti-MEM medium containing Turbo293 were then mixed with the medium containing plasmid DNAs, incubated for 15 min at RT, and added to 40 ml of Expi293 cells (Life Technology) at 2.5 million cells/ml. The transfected cells were cultured in shaker incubator at 120 rpm, 37 °C, 9% CO 2 for 5 days. Culture supernatants were harvested and purified over 0.5 ml Protein A (GE Healthcare) resin in columns. Each antibody was eluted with IgG elution buffer (Pierce), immediately neutralized with one tenth volume of 1M Tris-HCL pH 8.0. The antibodies were then buffer exchanged in PBS by dialysis. Monomeric (residues 1-615) and dimeric (residues 1-732) human ACE2 proteins were produced as described previously (Zhou et al., 2020a) . In brief, DNA sequence encoding monomeric or dimeric ACE2 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 transient transfection of 293F cells and 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 with a Superdex 200 16/60 column in 5 mM HEPES, pH7.5 and 150 mM NaCl.

NanoLuc-expressing SARS-CoV-2 decorated lentiviruses were first generated by transfecting a plasmid mixture containing 5 µg of pCMV-S, 5 µg of pCMV delta R8.2 (expresses HIV-1 GagPol), 2 µg of pLL3.7 NanoLuc (packaging reporter to express NanoLuc) into 60-70 % HEK293 cells using FuGENE 6 (3ul of FuGENE 6 for every µg of DNA) in a 10 cm TC dish. The culture supernatants were harvested at 48 h after transfection, clarified using 0.45 µm filter, and viruses partially purified by sedimentation through a layer of 15 % sucrose solution made in 1X PBS. The virus pellet was resuspended in OptiMEM to achieve a 20-fold concentration over original culture volume. Infectivity inhibition assays were performed by incubating NanoLuc expressing lentiviruses either alone or with soluble hACE2 (monomeric and dimeric) at indicated concentrations in triplicates for 90 mins at room temperature prior to infection of the 293T cells stably expressing hACE2 seeded in a tissue-culture treated 96-well solid white assay plate (Costar Inc, catalog #3917). The culture supernatants were completely removed 24 h p.i. and cells lysed using 50 µl NanoLuc assay buffer. Luciferase activity was then measured using Tristar multiwell luminometer (Berthold Technology, Bad Wildbad, Germany) for 2.5 seconds by adding 20 µl of Nano-Glo® substrate (Promega Inc, WI, USA; diluted 1:40 in NanoLuc assay buffer). HEK293T cells (not expressing hACE2) infected similarly served as controls to determine basal luciferase activity for obtaining normalized relative light units in infected samples. Infectivity obtained in mock-treated samples were set to 100 %. The data were processed and plotted using GraphPad Prism 8 v8.4.3.

In the case of VSV-G, SARS-CoV-1, and SARS-CoV-2 parallel neutralization assay ( Figure S5B ), experiments were performed as follows. Target cells were infected with single-round luciferase-expressing lentiviral particles. Briefly, 293T cells were transfected by the calcium phosphate method with the pNL4.3 R-E-Luc plasmid (NIH AIDS Reagent Program) and a plasmid encoding for SARS-CoV-2 Spike, SARS-CoV-1 Spike or VSV-G at a ratio of 5:4. Two days post-transfection, cell supernatants were harvested and stored at -80 °C until use. 293T-ACE2 target cells were seeded at a density of 1×10 4 cells/well in 96-well luminometer-compatible tissue culture plates (Perkin Elmer) 24 hours before infection. Recombinant viruses in a final volume of 100 µl were incubated with the indicated plasma dilutions (1/50; 1/250; 1/1250; 1/6250; 1/31250) for 1 hour at 37°C and were then added to the target cells followed by incubation for 48 hours at 37°C; cells were lysed by the addition of 30 µl of passive lysis buffer (Promega) followed by one freeze-thaw cycle. An LB941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 µl of luciferin buffer (15 mM MgSO 4 , 15 mM KH 2 PO 4 [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 µl of 1 mM d-luciferin potassium salt (Prolume). The neutralization half-maximal inhibitory dilution (ID 50 ) represents the plasma dilution to inhibit 50 % of the infection of 293T-ACE2 cells by recombinant viruses bearing the indicated surface glycoproteins. The plasmids expressing the human coronavirus Spikes of SARS-CoV-2 and SARS-CoV-1 were previously described (Hoffmann et al., 2020b; Hoffmann et al., 2013; Hofmann et al., 2005; Prevost et al., 2020) . The pNL4.3 R-E-Luc was obtained from NIH AIDS Reagent Program. The vesicular stomatitis virus G (VSV-G)-encoding plasmid (pSVCMV-IN-VSV-G) was previously described (Lodge et al., 1997) .

We used the split nanoluc assay to monitor virus-cell fusion (Yamamoto et al., 2019) . For the preparation of Vpr-HiBiT-containing SARS CoV-2 decorated lentiviruses, a plasmid mixture containing 4 µg of pCMV-S, 4 µg of psPAX2 (Gag-pol, Rev, and Tat expression vector; does not express Vpr), 2 µg of pLL3.7 (packaging reporter to express GFP), and 2 µg of pCMV-Vpr-HiBiT (Vpr-HiBiT expression vector)) was transfected into 60-70 % HEK293 cells using FuGENE 6 (3ul of FuGENE 6 for 1µg of DNA). The culture supernatants were harvested at 48 hours after transfection, clarified using 0.45 µm filter, and viruses partially purified by sedimentation through a layer of 15 % sucrose solution made in 1X PBS. The virus pellet was resuspended in Opti-MEM to achieve a 20-fold concentration over original culture volume. Virus preparations were mock-treated or treated with trypsin (50 and 150 µg/ml) at 37 °C for 15 min. Neat serum at a final concentration of 10 % was added to terminate both trypsin and mock-treated samples. 2.5 × 10 5 HEK293 cells in 24-wells were transfected with 400 ng of hACE2-expressing plasmid or pcDNA3.1 vector along with 100 ng PH-LgBiT (LgBiT-tagged to pleckstrin homology domain of human phospholipase Cδ at the N-terminus) expressing plasmid using FuGENE 6 for use as target cells. 24 hours post-transfection, 2 x 10 5 cells were seeded in tissue-culture treated 96-well solid white assay plate (Costar Inc, # 3917) in 100 µl RPMI complete medium. They were infected with 25 µl of prepared Vpr-HiBiTcontaining viruses in quadruplicates for 24 hours at 37 °C. Light signals derived from reconstituted HiBiT/LgBiT association were measured using Tristar multiwell luminometer (Berthold Technology, Bad Wildbad, Germany) for 2.5 seconds by adding 20 µl of Nano-Glo® substrate (Promega Inc, WI, USA; diluted 1:40 in PBS). Mockinfected cells served as controls to determine basal luciferase activity for obtaining normalized relative light units in infected samples. The data were processed and plotted using GraphPad Prism 8 v8.4.3.

Cell-cell fusion assays 2.5 × 10 5 HEK293 cells seeded per well of 24-well plate were transfected with 400 ng of hACE2 and 100 ng PH-LgBiT expressing plasmid (Yamamoto et al., 2019) using FuGENE 6 (3 ul of FuGENE 6 for 1 µg of DNA). The second set of HEK293 cells for coculture were transfected with 400 ng of SARS CoV-2 spike-expressing plasmid or pcDNA 3.1 vector along with 100 ng of Vpr-HiBiT (HiBiT tagged to HIV-1 Vpr at the Nterminus) expressing plasmid. 24 hours later, HEK293 cells expressing Vpr-HiBiT alone or co-expressing spike and Vpr-HiBiT were either untreated or treated with 50 µg/ml trypsin solution for 10 min at 37 °C. Trypsin was neutralized by adding serum-containing medium and cells washed once before resuspending in complete RPMI medium. 1 x 10 5 cells from various Vpr-HiBiT and PH-LgBiT expressing populations were mixed (1:1 ratio, 50 µl each population) in tissue culture treated 96-well solid white assay plate (Costar Inc, catalog #3917) in quadruplicates. Membrane fusion was initiated by spinning down the mixed cells in a 96-well plate (1600 rpm for 5 min, swing-out buckets, Heraeus, Sorvall centrifuge). The cells were then incubated at 37 °C in tissue culture incubator for 4 hours or 24 hours before measuring reconstituted HiBiT/LgBiT activity using a Tristar multiwell Luminometer (Berthold Technology, Bad Wildbad, Germany) for 2.5 seconds by adding 20 µl of Nano-Glo® substrate (Promega Inc, WI, USA; diluted 1:40 in PBS). Individual unmixed populations of cells were treated identically and served as controls to determine basal luciferase activity to obtain normalized relative light units. The data were processed and plotted using GraphPad Prism 8 v8.4.3.

Lentiviruses carrying SARS-CoV-2 spikes were prepared similarly as previously described for HIV-1 (Alsahafi et al., 2019; Lu et al., 2019a; Ma et al., 2018; Munro et al., 2014) . Two short peptides labeling tags (Q3: GQQQLG; A4: DSLDMLEM), either single or in pair-wise combinations were introduced into indicated positions in the S1 subunit on the plasmid pCMV-S (see Figure S1 ). Viruses carrying 100 % single-tagged or double-tagged pCMV-S showed no noticeable defect in virus infectivity. The spike carrying one pair-wise combination of peptide tags (designated as pCMV-S Q3-1 A4-1) was used to make lentiviruses or S-MEN coronavirus like particles with SARS-CoV-2 spikes incorporated in the virus surface.

Lentivirus particles carrying SARS-CoV-2 spikes were produced by pseudotying an HIV-1 core with full-length spike proteins. To generate lentivirus particles that carry only one double tagged S on average among all the S trimers on the virus surface, plasmids encoding wildtype pCMV-S, pCMV-S Q3-1 A4-1, and pCMV delta R8.2 were transfected at a ratio of 20:1:21, respectively. Under these conditions, the vast majority of virus particles carry wildtype spikes. 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 deployed in our previous studies (Alsahafi et al., 2019; Lu et al., 2019a; Ma et al., 2018; Munro et al., 2014) . 293T cells were transfected using FuGENE 6 with above indicated plasmids to express spikes on the HIV-1. When virus particles were generated that contained the disulfide bridge between S383C and D985C S, both plasmids, the plasmid encoding the spike protein, as well as plasmids encoding the dually tagged spike protein contained the S383C and D985C modification. To generate S-MEN particles, plasmids encoding wildtype pCMV-S, dual-tagged pCMV-S Q3-1 A4-1, pLVX-M (SARS-CoV-2 membrane protein expressing plasmid), an pLVX-E (SARS-CoV-2 envelope expressing plasmid, and pLVX-N (SARS-CoV-2 nucleocapsid expressing plasmid) were transfected at a ratio of 20:1:21:21:21. pLVX plasmids are gifts from Nevan Krogan, UCSF.

Lentivirus or S-MEN particles carrying dually tagged S proteins were harvested 40 hour post-transfection, filtered through 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 50 mM pH 7.5 HEPES buffer supplied with 10 mM MgCl 2 and 10 mM CaCl 2 .

Cryo-electron tomography 6 nm gold tracer was added to the concentrated S-decorated HIV-1 lentivirus and S-MEN particles viruses at 1:3 ratio, and 5 µl of the mixture was placed onto freshly glow discharged holey carbon grids for 1 min. Grids were blotted with filter paper, and rapidly frozen in liquid ethane using a homemade gravity-driven plunger apparatus. Cryo-grids were imaged on a cryo-transmission electron microscope (Titan Krios, Thermo Fisher Scientific) that was operated at 300 kV, using a Gatan K3 direct electron detector in counting mode with a 20 eV energy slit. Tomographic tilt series between −51° and +51° were collected by using SerialEM (Mastronarde, 2005) in a dose-symmetric scheme (Hagen et al., 2017) with increments of 3°. The nominal magnification was 64,000 X, giving a pixel size of 1.346 Å on the specimen. The raw images were collected from single-axis tilt series with accumulative dose of ~50 e− per Å 2 . The defocus was -3 µm and 8 frames were saved for each tilt angle. Frames were motion-corrected using Motioncorr2 (Zheng et al., 2017) to generate drift-corrected stack files, which were aligned using gold fiducial makers by IMOD/etomo (Mastronarde and Held, 2017) . Tomograms were reconstructed by weighted back projection and tomographic slices were visualized with IMOD.

Virus particles were labeled through site-specifically enzymatic labeling, as previously described (Lu et al., 2019a; Munro et al., 2014) . Transglutaminase transferred Cy3B(3S) from the cadaverine conjugate to the central glutamine residue of the Q3 (GQQQLG) tag in S1. The AcpS enzyme mediated the addition of the Cy5 derivative (LD650-CoA) to the serine residue of the A4 tag (DSLDMLEM). For these reactions to occur, Cy3B(3S)-cadaverine (0.5 µM, Lumidyne Technologies), LD650-CoA (0.5 µM, Lumidyne Technologies), transglutaminase (0.65 µM, Sigma Aldrich), and AcpS (5 µM, home-made) were added to the above suspensions of viruses. The labeling reaction mix was incubated at room temperature overnight. PEG2000-biotin (0.02 mg/ml, Avanti Polar Lipids) was added to the reaction mix and incubated for 30 min at room temperature with rotation. Free dyes and lipids were then purified away from labeled virus particles by ultracentrifugation for one hour at 4 °C at 40,000 rpm over a 6%-18% Optiprep (Sigma Aldrich) gradient. Purified virus particles were stored at -80 °C for future use in smFRET imaging.

smFRET imaging data acquisition All smFRET imaging data acquisition was performed on a home-built prism-based total internal reflection fluorescence (TIRF) microscope. Lentiviruses or S-MEN coronaviruslike particles carrying fluorescently tagged spike protein, as well as lipid-biotin in the viral membranes, were immobilized on polyethylene glycol (PEG)-passivated, streptavidin-coated quartz slides for imaging. The evanescent field was generated at the interface between the quartz slide and the virus sample solution by prism-based total internal reflection, with laser excitation from a single-frequency 532-nm laser. Donor fluorophores labeled on viruses were directly excited by the generated evanescent field. Fluorescence signals from both donor and acceptor fluorophores were collected through a 1.27-NA 60 x water-immersion objective (Nikon). Collected signals were optically separated by passing through a 650 DCXR dichroic filter (Chroma) mounted on MultiCam LS image splitter (Cairn Research). Separated donor (ET590/50, Chroma) and acceptor fluorescent (ET690/50, Chroma) signals were simultaneously recorded on two synchronized ORCA-Flash4.0 V3 sCMOS cameras (Hamamatsu) at 25 frames per second for 80 seconds. Unless otherwise noted, all virus samples were imaged in pH 7.4, 50 mM Tris buffer with 50 mM NaCl, a cocktail of triplet-state quenchers, and an oxygen-scavenger system (Aitken et al., 2008) . Where indicated, the conformational effects of different ligands/antibodies/plasma on SARS-CoV-2 spike were tested by preincubating fluorescently labeled viruses for 90 mins at room temperature before imaging in the continued presence of the ligands. In experiments with two different ligands presented, the second ligand (hACE2) was added to first-ligand-bound viruses and incubated similarly. The specific concentration of ligands incubated with viruses in smFRET imaging are as follows: 200 µg/ml hACE2 (human hACE2); 200 µg/ml dimeric hACE2, 50 µg/ml trypsin; 200 µg/ml CR3022; 200 µg/ml VHH72; 200 µg/ml H4; 200 µg/ml 2-4; 200 µg/ml 2-43; 100 fold dilution of a SARS-CoV-2 positive patient plasma.

The assay was modified from a previous published method and recently described in detail elsewhere . Briefly, pseudoviral particles were produced by transfecting 2×10 6 HEK293T cells with pNL4.3 Luc R-E-(3.5 µg), plasmids Prevost et al., 2020) encoding for SARS-CoV-2 Spike or SARS-CoV-1 Spike (3.5 µg) protein and VSV-G (pSVCMV-IN-VSV-G, 1 µg) using the standard calcium phosphate protocol. Forty-eight hours later, supernatantcontaining virion was collected and cell debris was removed through centrifugation (1,500 rpm for 10 min). To immobilize antibodies on ELISA plates, white MaxiSorp ELISA plates (Thermo Fisher Scientific) were incubated with 5 µg/ml of antibodies or 1:500 diluted plasma in 100 µl phosphate-buffered saline (PBS) overnight at 4 o C. Unbound antibodies or plasma were removed by washing the plates twice with PBS. Plates were subsequently blocked with 3% bovine serum albumin (BSA) in PBS for 1 hour at room temperature. After two washes with PBS, 200 µl of virus-containing supernatant was added to the wells. For sACE2 competition, 10 µM sACE2 was incubated with the supernatant at 37 o C for 1 hour before adding to the coated wells.

After 4 to 6 hours incubation, supernatant was removed and the wells were washed with PBS 3 times. Viral capture by any given antibody was visualized by adding 10×10 4 SARS-CoV-2-resistant Cf2Th cells (not shown) in full DMEM medium per well. Fortyeight hours post-infection, cells were lysed by the addition of 30 µl of passive lysis buffer (Promega) and three freeze-thaw cycles. An LB941 TriStar luminometer (Berthold Technologies) was used to measure the luciferase activity of each well after the addition of 100 µl of luciferin buffer (15 mM MgSO 4 , 15 mM KH 2 PO 4 [pH 7.8], 1 mM ATP, and 1 mM dithiothreitol) and 50 µl of 1 mM D-luciferin potassium salt (Prolume).

Using the standard calcium phosphate method, 10 µg of Spike expressor (SARS-CoV-1, SARS-CoV-2, NL63, 229E, OC43) and 2 µg of a green fluorescent protein (GFP) expressor (pIRES-GFP) was transfected into 2 × 10 6 293T cells. At 48 hours post transfection, 293T cells were stained with plasma from SARS-CoV-2-infected or uninfected individuals (1:250 dilution). The percentage of transfected cells (GFP+ cells) was determined by gating the living cell population based on the basis of viability dye staining (Aqua Vivid, Invitrogen). Samples were acquired on a LSRII cytometer (BD Biosciences, Mississauga, ON, Canada) and data analysis was performed using FlowJo vX.0.7 (Tree Star, Ashland, OR, USA). Alternatively, convalescent plasma was incubated with 20 µg/mL of SARS-CoV-2 RBD prior cellsurface staining in order to compete for RBD-specific antibodies.

The SARS-CoV-2 RBD ELISA assay used was recently described Prevost et al., 2020) . Briefly, recombinant SARS-CoV-2 S RBD proteins (2.5 µg/ml), or bovine serum albumin (BSA) (2.5 µg/ml) as a negative control, were prepared in PBS and were adsorbed to plates (MaxiSorp; Nunc) overnight at 4 °C. Coated wells were subsequently blocked with blocking buffer (Tris-buffered saline [TBS] containing 0.1% Tween20 and 2% BSA) for 1 hour at room temperature. Wells were then washed four times with washing buffer (Tris-buffered saline [TBS] containing 0.1% Tween20). CR3022 mAb (50 ng/ml) or serial dilutions of plasma from SARS-CoV-2infected or uninfected donors (1/100; 1/250; 1/500; 1/1000; 1/2000; 1/4000) were prepared in a diluted solution of blocking buffer (0.1 % BSA) and incubated with the RBD-coated wells for 90 minutes at room temperature. Plates were washed four times with washing buffer followed by incubation with anti-IgG secondary Abs (diluted in a diluted solution of blocking buffer [0.4% BSA]) for 1 hour at room temperature, followed by four washes. HRP enzyme activity was determined after the addition of a 1:1 mix of Western Lightning oxidizing and luminol reagents (Perkin Elmer Life Sciences). Light emission was measured with a LB941 TriStar luminometer (Berthold Technologies). Signal obtained with BSA was subtracted for each plasma and was then normalized to the signal obtained with CR3022 mAb present in each plate.

J o u r n a l P r e -p r o o f Statistical significance (in Figures 3F, 4B , and S4C) was analyzed using GraphPad Prism version 8.0.2 by applying unpaired Student's t-test to obtain p values. p values lower than 0.05 were considered statistically significant. P values were indicated as *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. smFRET data analysis smFRET data analysis was performed using MATLAB (MathWorks)-based customized SPARTAN software package (Juette et al., 2016) . For each labeled virus recorded over 80 seconds, the background signal at the single-molecule level was first identified based on the fluorophore bleaching point and subtracted. Donor and acceptor fluorescence intensity time-trajectory or traces were then extracted for each recorded virus and corrected for donor to acceptor crosstalk. The energy transfer efficiency (termed as FRET in graphs) from the donor to the neighboring acceptor was evaluated as FRET= I A /(γI D +I A ), where I D and I A are the fluorescence intensities of donor and acceptor, respectively, and γ is the correlation coefficient compensating for the difference in quantum yields and detection efficiencies of donor and acceptor.

FRET efficiencies of each FRET pair report on the relative distance between the donor and the acceptor over time and ultimately reveal the conformational dynamics of host molecules (spikes on the virus in our case) in real-time. Viruses lacking donor or acceptor fluorophores, containing multiple labeled protomers, or containing more than one labeled spike on a single virus were automatically excluded from further analysis. FRET traces (FRET values as a function of real-time) were then manually selected if they displayed sufficient signal-to-noise (S/N) ratio and anti-correlated fluctuations in donor and acceptor fluorescence intensity between clearly defined FRET states, which are indicative of fully active molecules. N (or N m ), number of traces/molecules as indicated in each FRET histogram, was compiled into the associated FRET histogram. Based on visual inspection of traces that revealed direct observations of state-to-state transitions, FRET histograms were fitted into the sum of four Gaussian distributions using the least-squares fitting algorithm in Matlab. FRET histograms represent mean ± s.e.m., determined from three randomly assigned populations of FRET traces, as indicated in corresponding figure legends. Each gaussian represents one FRETindicated conformational state, and the area under each Gaussian curve estimates each state's occupancy. The occupancy of each state was used to evaluate the difference in free energies between states i and j, according to ∆G°i j =-k B Tln(P i /P j ), where P i and P j are the ith and jth state's occupancy, respectively, k B is the Boltzmann constant, and T is the temperature in kelvin. Relative state occupancies are presented as mean ± s.e.m., derived from histograms, and the determining parameters are listed in. Table S1 .

The idealization of each FRET trace using a 4-state Hidden Markov Model was performed in the SPARTAN software package using a segmental K-means algorithm (McKinney et al., 2006) . The state-to-state transition tracing, indicative of the locations and the frequencies of transitions, was displayed in a transition density plot (termed as TDP). Dwell times (the time during which a molecule occupies one specific conformation before transitioning to any other conformations) distributions were compiled into survival probability plots and were fitted to the sum of two exponential distributions (y = A 1 exp -k1t + A 2 exp -k2t ). The transition rates were weighted from averaging the two rate constants by their amplitudes.

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

An asymmetric opening of HIV-1 Env is required for anti-cluster A antibody binding

Nef proteins from HIV-1 elite controllers are inefficient at preventing antibody-dependent cellular cytotoxicity

Structures of human antibodies bound to SARS-CoV-2 spike reveal common epitopes and recurrent features of antibodies

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Potent neutralizing antibodies from COVID-19 patients define multiple targets of vulnerability

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A SARS-CoV-2 protein interaction map reveals targets for drug repurposing

Implementation of a cryo-electron tomography tiltscheme optimized for high resolution subtomogram averaging

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

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

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An mRNA Vaccine against SARS-CoV-2 -Preliminary Report

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

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

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SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography

Antibody lineages with vaccine-induced antigen-binding hotspots develop broad HIV neutralization

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

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

The membrane-proximal intracytoplasmic tyrosine residue of HIV-1 envelope glycoprotein is critical for basolateral targeting of viral budding in MDCK cells

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

Illuminating the virus life cycle with single-molecule FRET imaging

Trimeric SARS-CoV-2 Spike interacts with dimeric ACE2 with limited intra-Spike avidity

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

Automated electron microscope tomography using robust prediction of specimen movements

Automated tilt series alignment and tomographic reconstruction in IMOD

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

Analysis of single-molecule FRET trajectories using hidden Markov modeling

Single-shot Ad26 vaccine protects against SARS-CoV-2 in rhesus macaques

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

Cross-sectional evaluation of humoral responses against SARS-CoV-2 Spike

Convergent antibody responses to SARS-CoV-2 in convalescent individuals

A practical guide to single-molecule FRET

Structural basis of receptor recognition by SARS-CoV-2

The M, E, and N structural proteins of the severe acute respiratory syndrome coronavirus are required for efficient assembly, trafficking, and release of virus-like particles

Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2

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Longitudinal bioluminescent imaging of HIV-1 infection during antiretroviral therapy and treatment interruption in humanized mice

Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein

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SARS-CoV-2 and bat RaTG13 spike glycoprotein structures inform on virus evolution and furin-cleavage effects

Neutralizing antibody responses to SARS-CoV-2 in a COVID-19 recovered patient cohort and their implications

A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2

Cell-cell and virus-cell fusion assay-based analyses of alanine insertion mutants in the distal alpha9 portion of the JRFL gp41 subunit from HIV-1

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Site-specific protein labeling by Sfp phosphopantetheinyl transferase

DNA vaccine protection against SARS-CoV-2 in rhesus macaques

A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV

MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy

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

Cryo-EM structures delineate a pH-dependent switch that mediates endosomal positioning of SARS-CoV-2 spike receptor-binding domains