Those antibodies include CD4 binding site-directing bnAbs (VRC01 and 3BNC117), V1V2 glycan site-targeting bnAbs (PG9, PG16, PGT145), and V3-glycan patch bnAbs (10C1074, PGT128, PGT122) [22,25,101]

Those antibodies include CD4 binding site-directing bnAbs (VRC01 and 3BNC117), V1V2 glycan site-targeting bnAbs (PG9, PG16, PGT145), and V3-glycan patch bnAbs (10C1074, PGT128, PGT122) [22,25,101]. 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 F?rster resonance energy transfer (smFRET) has provided a Aconine powerful platform to connect structureCfunction 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. Keywords: single-molecule imaging, F?rster resonance energy transfer (FRET), virusChost interactions, spike proteins, virus entry, viral membrane fusion, conformational dynamics, SARS-CoV-2, HIV-1, influenza and Ebola 1. Introduction Virus spikes on the surface of enveloped viruses are often also viral fusion proteins that mediate the fusion between viral membranes and cellular membranes (Figure 1) essential for virus entry [1,2,3]. The merging of virus and lipid bilayers progresses through a hemifusion intermediate, followed by a fusion pore widening, content mixing, and the delivery of virus capsids into the CAGH1A host cytoplasm [4]. Viral fusion proteins respond to the binding of cellular receptors or acidic pH to undergo conformational rearrangements, which eventually promote membrane fusion. Viral fusion proteins have been categorized into three classes [1,2], of which Class I viral fusion proteins include the medically important SARS-CoV-2 spike (S) protein, the HIV-1 envelope (Env) protein, influenza hemagglutinin (HA), and Ebola glycoprotein (GP). These virus spikes are first synthesized as trimers of a single-chain polypeptidean immature precursor, then go through proteolytical processing by host proteases to form mature spikestrimers of heterodimers (Figure 1A). Mature spikes are highly metastable on the virus surface. Upon interacting with hosts, mature spikes undergo conformational changes from pre-fusion conformations to the lowest-energy post-fusion conformation (a common hairpin-like or the analogous coiled-coil conformation) through hypothetical intermediates in which the fusion peptide extends and inserts into the host target membrane (Figure 1B). Numerous pre-fusion and post-fusion structures of virus spikes have provided unprecedented details of conformations at individual steps during the viral membrane fusion [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21]. The recent dynamic studies on virus spikes established platforms to connect these structural snapshots in real time, revealed the order and the kinetics of transitional events, and guided developing interventions aiming to arrest or block viral membrane fusion, thus stopping viral infection [22,23,24,25,26,27,28,29,30,31]. Open in a separate window Figure 1 Class I viral fusion proteins and proposed model of viral membrane fusion. (A) Schematic drawing of spike precursor and cleaved spike. The spike protein is initially synthesized as a single-chain polypeptide (spike precursor) and later cleaved into a trimer of covalently or non-covalently Aconine linked heterodimer. The heterodimer consists of the surface receptor-binding subunit (gray) and the fusion subunit (fusion peptide or fusion loop (FP/FL), dark yellow; N-terminal domain, cyan; C-terminal domain, dark blue). (B) Proposed conformational events of virus spikes during viral membrane fusion. These events are as follows, involving conformational changes in the surface subunit (top row) and changes in the fusion subunit (low row, simplified by only showing the fusion subunit [1]). (1) Prefusionconformations of the spike in closed and open forms. Spike activation proceeds through an opening of the trimer, usually in response to binding to receptor or due to a cellular cue such as low pH. For non-covalently linked spikes, dissociating/decoupling between the surface/exterior subunit with the fusion subunit has been observed/suggested after the spike opens, such as HIV-1 and SARS-CoV-2 spikes. FP/FL remains sequestered in this process. (2) Exposing, extending, and inserting the FP/FL into the cellular membrane leads to the formation of an extended prehairpin intermediate. (3) Folding back the C-terminal segment of the fusion subunit back on the N-terminal segment core brings viral and cellular membranes into proximity. (4) Further folding and dragging two membranes into contact promotes two membranes merging to form a hemifusion stalk. (5) The Aconine fusion subunit folds into a stable post-fusion conformation, allowing a fusion pore to form. The intermediate steps from (2) to (4) remain elusive. This proposed model does not specify or speculate the number of spikes required for fusion pore formation. As spikes are highly exposed to our immune system, they are main targets of neutralizing antibodies and thus are critical for developing vaccines Aconine and anti-spike therapeutics. Most vaccines or vaccine candidates for HIV-1/AIDS and SARS-CoV-2/COVID-19 are based on their spike proteins to trigger the immune system to produce neutralizing antibodies. Interestingly, in the face of immune pressure, many viral spike proteins use conformational masking of vulnerable antibody-targeted epitopes. In addition to glycan shields and hypermutations, this strategy of.