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- Cervantes, Marcos, et al.
(författare)
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The ACE2 receptor accelerates but is not biochemically required for SARS-CoV-2 membrane fusion
- 2023
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Ingår i: Chemical Science. - : Royal Society of Chemistry. - 2041-6520 .- 2041-6539. ; 14:25, s. 6997-7004
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Tidskriftsartikel (refereegranskat)abstract
- The SARS-CoV-2 coronavirus infects human cells via the ACE2 receptor. Structural evidence suggests that ACE2 may not just serve as an attachment factor but also conformationally activate the SARS-CoV-2 spike protein for membrane fusion. Here, we test that hypothesis directly, using DNA-lipid tethering as a synthetic attachment factor in place of ACE2. We find that SARS-CoV-2 pseudovirus and virus-like particles are capable of membrane fusion without ACE2 if activated with an appropriate protease. Thus, ACE2 is not biochemically required for SARS-CoV-2 membrane fusion. However, addition of soluble ACE2 speeds up the fusion reaction. On a per-spike level, ACE2 appears to promote activation for fusion and then subsequent inactivation if an appropriate protease is not present. Kinetic analysis suggests at least two rate-limiting steps for SARS-CoV-2 membrane fusion, one of which is ACE2 dependent and one of which is not. Since ACE2 serves as a high-affinity attachment factor on human cells, the possibility to replace it with other factors implies a flatter fitness landscape for host adaptation by SARS-CoV-2 and future related coronaviruses.
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- Sengar, Anjali, et al.
(författare)
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Mechanistic dissection of antibody inhibition of influenza entry yields unexpected heterogeneity
- 2023
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Ingår i: Biophysical Journal. - : Cell Press. - 0006-3495 .- 1542-0086. ; 122:11, s. 1996-2006
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Tidskriftsartikel (refereegranskat)abstract
- Neutralizing antibodies against influenza have generally been classified according to their recognition sites, with antibodies against the head domain of hemagglutinin thought to inhibit attachment and antibodies against the stalk region thought to inhibit fusion. Here, we report the development of a microfluidic assay to measure neutralization of viral entry that can clearly differentiate between effects on attachment and fusion. Testing multiple broadly neutralizing antibodies against the hemagglutinin stalk domain, we obtain a surprising result: some broadly neutralizing antibodies inhibit fusion only, while others inhibit both fusion and viral attachment. Antibodies binding the globular head domain primarily inhibit attachment but can also reduce the fusogenic capability of viral particles that nonetheless bind the receptor. These findings shed light on the unexpectedly heterogeneous mechanisms of antibody neutralization even within similar recognition sites. The assay we have developed also provides a tool to optimize vaccine design by permitting assessment of the elicited antibody response with greater mechanistic resolution.
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| 5. |
- Sengar, Anjali, et al.
(författare)
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Single-Virus Fusion Measurements Reveal Multiple Mechanistically Equivalent Pathways for SARS-CoV-2 Entry
- 2023
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Ingår i: Journal of Virology. - : American Society for Microbiology. - 0022-538X .- 1098-5514. ; 97:5
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Tidskriftsartikel (refereegranskat)abstract
- SARS-CoV-2 can use multiple pathways to infect cells, as demonstrated recently when new viral variants switched dominant infection pathways. Here, we used single-virus fusion experiments together with biochemical reconstitution to show that these multiple pathways coexist simultaneously and specifically that the virus can be activated by different proteases in different cellular compartments with mechanistically identical effects. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) binds to cell surface receptors and is activated for membrane fusion and cell entry via proteolytic cleavage. Phenomenological data have shown that SARS-CoV-2 can be activated for entry at either the cell surface or in endosomes, but the relative roles in different cell types and mechanisms of entry have been debated. Here, we used single-virus fusion experiments and exogenously controlled proteases to probe activation directly. We found that plasma membrane and an appropriate protease are sufficient to support SARS-CoV-2 pseudovirus fusion. Furthermore, fusion kinetics of SARS-CoV-2 pseudoviruses are indistinguishable no matter which of a broad range of proteases is used to activate the virus. This suggests that the fusion mechanism is insensitive to protease identity or even whether activation occurs before or after receptor binding. These data support a model for opportunistic fusion by SARS-CoV-2 in which the subcellular location of entry likely depends on the differential activity of airway, cellsurface, and endosomal proteases, but all support infection. Inhibition of any single host protease may thus reduce infection in some cells but may be less clinically robust.IMPORTANCE SARS-CoV-2 can use multiple pathways to infect cells, as demonstrated recently when new viral variants switched dominant infection pathways. Here, we used single-virus fusion experiments together with biochemical reconstitution to show that these multiple pathways coexist simultaneously and specifically that the virus can be activated by different proteases in different cellular compartments with mechanistically identical effects. The consequences of this are that the virus is evolutionarily plastic and that therapies targeting viral entry should address multiple pathways at once to achieve optimal clinical effects.
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