Mechanistic insights of host cell fusion of SARS-CoV-1 and SARS-CoV-2 from atomic resolution structure and membrane dynamics.
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ABSTRACT:
SUBMITTER: Chakraborty H
PROVIDER: S-EPMC7375304 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cell entry by SARS-CoV-2 is accomplished by the S2 subunit of the spike S protein on the virion surface by capture of the host cell membrane and fusion with the viral envelope. Capture and fusion require the prefusion S2 to transit to its potent fusogenic form, the fusion intermediate (FI). However, the FI structure is unknown, detailed computational models of the FI are unavailable, and the mechanisms and timing of membrane capture and fusion are not established. Here, we constructed a full-length model of the SARS-CoV-2 FI by extrapolating from known SARS-CoV-2 pre- and postfusion structures. In atomistic and coarse-grained molecular dynamics simulations the FI was remarkably flexible and executed giant bending and extensional fluctuations due to three hinges in the C-terminal base. The simulated configurations and their giant fluctuations are quantitatively consistent with SARS-CoV-2 FI configurations measured recently using cryo-electron tomography. Simulations suggested a host cell membrane capture time of ∼2 ms. Isolated fusion peptide simulations identified an N-terminal helix that directed and maintained binding to the membrane but grossly underestimated the binding time, showing that the fusion peptide environment is radically altered when attached to its host fusion protein. The large configurational fluctuations of the FI generated a substantial exploration volume that aided capture of the target membrane, and may set the waiting time for fluctuation-triggered refolding of the FI that draws the viral envelope and host cell membrane together for fusion. These results describe the FI as machinery that uses massive configurational fluctuations for efficient membrane capture and suggest novel potential drug targets.
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Project description:During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. The viral spike (S) protein mediates both the initial contact with the host cell and the subsequent membrane fusion. Proteolytic cleavage of S at the S2' site exposes its fusion peptide (FP) as the new N-terminus. By binding to the host membrane, the FP anchors the virus to the host cell. The reorganization of S2 between virus and host then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 μs long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP, we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than 10-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.
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Project description:During viral entry, enveloped viruses require the fusion of their lipid envelope with host cell membranes. For coronaviruses, this critical step is governed by the virally-encoded spike (S) protein, a class I viral fusion protein that has several unique features. Coronavirus entry is unusual in that it is often biphasic in nature, and can occur at or near the cell surface or in late endosomes. Recent advances in structural, biochemical and molecular biology of the coronavirus S protein has shed light on the intricacies of coronavirus entry, in particular the molecular triggers of coronavirus S-mediated membrane fusion. Furthermore, characterization of the coronavirus fusion peptide (FP), the segment of the fusion protein that inserts to a target lipid bilayer during membrane fusion, has revealed its particular attributes which imparts some of the unusual properties of the S protein, such as Ca2+-dependency. These unusual characteristics can explain at least in part the biphasic nature of coronavirus entry. In this review, using severe acute respiratory syndrome coronavirus (SARS-CoV) as model virus, we give an overview of advances in research on the coronavirus fusion peptide with an emphasis on its role and properties within the biological context of host cell entry.
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Project description:The membrane fusion mechanism of SARS-CoV-2 offers an attractive target for the development of small molecule antiviral inhibitors. Fusion involves an initial binding of the crown-like trimeric spike glycoproteins of SARS-CoV-2 to the receptor angiotensin II-converting enzyme 2 (ACE2) on the permissive host cellular membrane and a prefusion to post-fusion conversion of the spike trimer. During this conversion, the fusion peptides of the spike trimer are inserted into the host membrane to bring together the host and viral membranes for membrane fusion in highly choreographic events. However, geometric constraints due to interactions with the membranes remain poorly understood. In this study, we build structural models of super-complexes of spike trimer/ACE2 dimers based on the molecular structures of the ACE2/neutral amino acid transporter B(0)AT heterodimer. We determine the conformational constraints due to the membrane geometry on the enzymatic activity of ACE2 and on the viral fusion process. Furthermore, we find that binding three ACE2 dimers per spike trimer is essential to open the central pore as necessary for triggering productive membrane fusion through an elongation of the central stalk. The reported findings thus provide valuable insights for targeting the membrane fusion mechanism for drug design at the molecular level.
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Project description::There are seven known coronaviruses that infect humans: four mild coronaviruses, including HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1, only cause mild respiratory diseases, and three severe coronaviruses, including SARS-CoV, MERS-CoV and SARS-CoV-2, can cause severe respiratory diseases even death of infected patients. Both infection and death caused by SARS-CoV-2 are still rapidly increasing worldwide. In this study, we demonstrate that viral coding proteins of SARS-CoV-2 have distinct features and are most, medium and least conserved with SARS-CoV, MERS-CoV, and the rest four mild coronaviruses (HCoV-229E, HCoV-OC43, HCoV-NL63, and HCoV-HKU1), respectively. Moreover, expression of host responsive genes (HRG), HRG-enriched biological processes, and HRG-enriched KEGG pathways upon infection of SARS-CoV-2 show slightly overlapping with SARS-CoV and MERS-CoV but distinctive to the four mild coronaviruses. Interestingly, enrichment of overactivation of neutrophil by HRGs is only and commonly found in infections of severe SARS-CoV-2, SARS-CoV, and MERS-CoV but not in the other four mild coronaviruses, and the related gene networks show different patterns. Clinical data supports that overactivation of neutrophil for severe patients can be one major factor for the similar clinical symptoms observed in SARS-CoV-2 infection compared to infections of the other two severe coronavirus (SARS-CoV, and MERS-CoV). Taken together, our study provides a mechanistic insight into SARS-CoV-2 epidemic via revealing the conserved and distinct features of SARS-CoV-2, raising the critical role of dysregulation of neutrophil for SARS-CoV-2 infection. SUPPLEMENTARY INFORMATION:Supplementary data are available at Bioinformatics online.
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Project description:Entry of SARS-CoV-2 into a host cell is mediated by spike, a class I viral fusion protein responsible for merging the viral and host cell membranes. Recent studies have revealed atomic-resolution models for both the postfusion 6-helix bundle (6HB) and the prefusion state of spike. However, a mechanistic understanding of the molecular basis for the intervening structural transition, important for the design of fusion inhibitors, has remained elusive. Using nuclear magnetic resonance spectroscopy and other biophysical methods, we demonstrate the presence of α-helical, membrane-bound, intermediate states of spike’s heptad repeat (HR1 and HR2) domains that are embedded at the lipid-water interface while in a slow dynamic equilibrium with the postfusion 6HB state. These results support a model where the HR domains lower the large energy barrier associated with membrane fusion by destabilizing the host and viral membranes, while 6HB formation actively drives their fusion by forcing physical proximity.
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