As a major surface glycoprotein of enveloped viruses, the virus spike protein is a primary target for vaccines and anti-viral treatments. Current vaccines aiming at controlling the COVID-19 pandemic ...are mostly directed against the SARS-CoV-2 spike protein. To promote virus entry and facilitate immune evasion, spikes must be dynamic. Interactions with host receptors and coreceptors trigger a cascade of conformational changes/structural rearrangements in spikes, which bring virus and host membranes in proximity for membrane fusion required for virus entry. Spike-mediated viral membrane fusion is a dynamic, multi-step process, and understanding the structure-function-dynamics paradigm of virus spikes is essential to elucidate viral membrane fusion, with the ultimate goal of interventions. However, our understanding of this process primarily relies on individual structural snapshots of endpoints. How these endpoints are connected in a time-resolved manner, and the order and frequency of conformational events underlying virus entry, remain largely elusive. Single-molecule Förster resonance energy transfer (smFRET) has provided a powerful platform to connect structure-function in motion, revealing dynamic aspects of spikes for several viruses: SARS-CoV-2, HIV-1, influenza, and Ebola. This review focuses on how smFRET imaging has advanced our understanding of virus spikes' dynamic nature, receptor-binding events, and mechanism of antibody neutralization, thereby informing therapeutic interventions.
The global pandemic of COVID-19 caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has significantly affected every human life and overloaded the health care system worldwide. ...Limited therapeutic options combined with the consecutive waves of the infection and emergence of novel SARS-CoV-2 variants, especially variants of concern (VOCs), have prolonged the COVID-19 pandemic and challenged its control. The Spike (S) protein on the surface of SARS-CoV-2 is the primary target exposed to the host and essential for virus entry into cells. The parental (Wuhan-Hu-1 or USA/WA1 strain) S protein is the virus-specific component of currently implemented vaccines. However, S is most prone to mutations, potentially shifting the dynamics of virus-host interactions by affecting S conformational/structural profiles. Scientists have rapidly resolved atomic structures of S VOCs and elucidated molecular details of these mutations, which can inform the design of S-directed novel therapeutics and broadly protective vaccines. Here, we discuss recent findings on S-associated virus transmissibility and immune evasion of SARS-CoV-2 VOCs and experimental approaches used to profile these properties. We summarize the structural studies that document the structural flexibility/plasticity of S VOCs and the potential roles of accumulated mutations on S structures and functions. We focus on the molecular interpretation of structures of the S variants and its insights into the molecular mechanism underlying antibody evasion and host cell-receptor binding.
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) mediates viral entry into cells and is critical for vaccine development against coronavirus disease 2019 (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 exposure 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.
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•SARS-CoV-2 S protein dynamically samples at least 4 distinct conformational states•hACE2 activates S from the ground state to the activated state via an intermediate•Proteolytic processing of S accelerates hACE2-dependent activation•Antibodies can antagonize S by two different mechanisms of neutralization
The SARS-CoV-2 spike protein has been observed to adopt different structural states. Lu et al. directly visualize the conformational dynamics of spike protein on the surface of virus particles and describe how the conformational landscape changes upon activation by the host receptor or antagonism by antibodies.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) 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, with structural studies of soluble S variants revealing an increased propensity toward conformations accessible to the human angiotensin-converting enzyme 2 (hACE2) receptor. 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 critical mutations observed in VOCs, including D614G and E484K, in the context of virus particles. Investigated variants predominately occupied more open hACE2-accessible conformations, agreeing with previous structures of soluble trimers. Additionally, these S variants exhibited slower 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.
SARS-CoV-2 surface S glycoprotein-the target of antibodies and vaccines-is responsible for binding to the cellular receptor hACE2. The interactions between S and hACE2 trigger structural rearrangements of S from closed to open conformations prerequisite for virus entry. Under the selection pressure imposed by adaptation to the human host and increasing vaccinations and convalescent patients, SARS-CoV-2 is evolving and has adopted numerous mutations on S variants. These promote virus spreading and immune evasion, partially by increasing the propensity of S to adopt receptor-binding competent open conformations. Here, we determined a time dimension, using smFRET to delineate the temporal prevalence of distinct structures of S in the context of virus particles. We present the first experimental evidence of decelerated transition dynamics from the open state, revealing increased stability of S open conformations to be part of the SARS-CoV-2 adaption strategies.
Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively ...pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3-Cy5-labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling.
Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating ...continued refinement of antibody therapies and immunogen design. Here, we elucidate the structural basis and mode of action for two potent SARS-CoV-2 spike (S)-neutralizing monoclonal antibodies, CV3-1 and CV3-25, which remain effective against emerging variants of concern in vitro and in vivo. CV3-1 binds to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the “RBD-up” position and triggers potent shedding of the S1 subunit. In contrast, CV3-25 inhibits membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among β-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in the RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.
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•Antibodies CV3-1 and CV3-25 neutralize emerging SARS-CoV-2 variants•CV3-1 binds to 485-GFN-487 loop of RBD on S and triggers S1 shedding•CV3-25 binds the stem helix of S2 and inhibits membrane fusion•Conserved epitopes are candidates for pan-coronavirus vaccines
Li et al. elucidate the structural basis and mode of action for two potent anti-S neutralizing monoclonal antibodies that remain effective against SARS-CoV-2 emerging variants of concern. Vaccine immunogen designs based on both conserved epitopes are candidates to elicit pan-coronavirus protective immune responses
HIV-1 entry into cells requires binding of the viral envelope glycoprotein (Env) to receptor CD4 and coreceptor. Imaging of individual Env molecules on native virions shows Env trimers to be dynamic, ...spontaneously transitioning between three distinct well-populated conformational states: a pre-triggered Env (State 1), a default intermediate (State 2) and a three-CD4-bound conformation (State 3), which can be stabilized by binding of CD4 and coreceptor-surrogate antibody 17b. Here, using single-molecule Fluorescence Resonance Energy Transfer (smFRET), we show the default intermediate configuration to be asymmetric, with individual protomers adopting distinct conformations. During entry, this asymmetric intermediate forms when a single CD4 molecule engages the trimer. The trimer can then transition to State 3 by binding additional CD4 molecules and coreceptor.
Latic acid bacteria (LAB) are exploited for development of gene expression system owing to its health promoting properties and a high degree of safety status. Most of the expression systems were ...constructed in Lactobacillus lactis with inducible promoters. It is necessary to exploit novel promoters to develop LAB host platforms which are indispensable in dairy and health application to satisfy the production demand of increased number of target-genes. Previously, promoter P.sub.srfA had been displayed broad host range and used to construct auto-inducible expression system in B. subtilis and E. coli. In this work, the feasibility of P.sub.srfA in LAB was estimated. Plasmid with the green fluorescent protein (GFP) inserting downstream of P.sub.srfA was transformed into L. casei 5257, L. plantarum 97, L. fermentum 087 and Weissella confusa 10, respectively. The recombinant strains grew well and displayed different fluorescence which could be detected by spectrophotometer and laser scanning confocal microscope. Moreover, the promoter activity was strain- specifically influenced by particular carbon and nitrogen sources. Heterologous laccase CotA could be expressed by P.sub.srfA in L. casei 5257-05 and L. plantarum 97-06. By adjusting the pH value from 4.5 to 6.5 during incubation, the CotA activity detected from L. plantarum 97-05 and L. casei 5257-05 was increased by 137.7% and 61.5%, respectively. Finally, the fermentation pH was variably up-regulated along with the production of NADH oxidase which was controlled by the P.sub.srfA and its derivative mutated with core regions. These data suggested that P.sub.srfA was valid for gene expression in different species of LAB. Moreover, P.sub.srfA could be used as an attractive candidate for fine-tuning gene expression in a broad range of prokaryotic expression plants.
The trimeric HIV-1 Envelope protein (Env) mediates viral-host cell fusion via a network of conformational transitions, with allosteric elements in each protomer orchestrating host receptor-induced ...exposure of the co-receptor binding site and fusion elements. To understand the molecular details of this allostery, here, we introduce Env mutations aimed to prevent CD4-induced rearrangements in the HIV-1 BG505 Env trimer. Binding analysis and single-molecule Förster Resonance Energy Transfer confirm that these mutations prevent CD4-induced transitions of the HIV-1 Env. Structural analysis by single-particle cryo-electron microscopy performed on the BG505 SOSIP mutant Env proteins shows rearrangements in the gp120 topological layer contacts with gp41. Displacement of a conserved tryptophan (W571) from its typical pocket in these Env mutants renders the Env insensitive to CD4 binding. These results reveal the critical function of W571 as a conformational switch in Env allostery and receptor-mediated viral entry and provide insights on Env conformation that are relevant for vaccine design.
The host proteins SERINC3 and SERINC5 are HIV-1 restriction factors that reduce infectivity when incorporated into the viral envelope. The HIV-1 accessory protein Nef abrogates incorporation of ...SERINCs via binding to intracellular loop 4 (ICL4). Here, we determine cryoEM maps of full-length human SERINC3 and an ICL4 deletion construct, which reveal that hSERINC3 is comprised of two α-helical bundles connected by a ~ 40-residue, highly tilted, "crossmember" helix. The design resembles non-ATP-dependent lipid transporters. Consistently, purified hSERINCs reconstituted into proteoliposomes induce flipping of phosphatidylserine (PS), phosphatidylethanolamine and phosphatidylcholine. Furthermore, SERINC3, SERINC5 and the scramblase TMEM16F expose PS on the surface of HIV-1 and reduce infectivity, with similar results in MLV. SERINC effects in HIV-1 and MLV are counteracted by Nef and GlycoGag, respectively. Our results demonstrate that SERINCs are membrane transporters that flip lipids, resulting in a loss of membrane asymmetry that is strongly correlated with changes in Env conformation and loss of infectivity.
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