Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is initiated by virus binding to the ACE2 cell-surface receptors
, followed by fusion of the virus and cell membranes to ...release the virus genome into the cell. Both receptor binding and membrane fusion activities are mediated by the virus spike glycoprotein
. As with other class-I membrane-fusion proteins, the spike protein is post-translationally cleaved, in this case by furin, into the S1 and S2 components that remain associated after cleavage
. Fusion activation after receptor binding is proposed to involve the exposure of a second proteolytic site (S2'), cleavage of which is required for the release of the fusion peptide
. Here we analyse the binding of ACE2 to the furin-cleaved form of the SARS-CoV-2 spike protein using cryo-electron microscopy. We classify ten different molecular species, including the unbound, closed spike trimer, the fully open ACE2-bound trimer and dissociated monomeric S1 bound to ACE2. The ten structures describe ACE2-binding events that destabilize the spike trimer, progressively opening up, and out, the individual S1 components. The opening process reduces S1 contacts and unshields the trimeric S2 core, priming the protein for fusion activation and dissociation of ACE2-bound S1 monomers. The structures also reveal refolding of an S1 subdomain after ACE2 binding that disrupts interactions with S2, which involves Asp614
and leads to the destabilization of the structure of S2 proximal to the secondary (S2') cleavage site.
SARS-CoV-2 is thought to have emerged from bats, possibly via a secondary host. Here, we investigate the relationship of spike (S) glycoprotein from SARS-CoV-2 with the S protein of a closely related ...bat virus, RaTG13. We determined cryo-EM structures for RaTG13 S and for both furin-cleaved and uncleaved SARS-CoV-2 S; we compared these with recently reported structures for uncleaved SARS-CoV-2 S. We also biochemically characterized their relative stabilities and affinities for the SARS-CoV-2 receptor ACE2. Although the overall structures of human and bat virus S proteins are similar, there are key differences in their properties, including a more stable precleavage form of human S and about 1,000-fold tighter binding of SARS-CoV-2 to human receptor. These observations suggest that cleavage at the furin-cleavage site decreases the overall stability of SARS-CoV-2 S and facilitates the adoption of the open conformation that is required for S to bind to the ACE2 receptor.
Influenza virus remains a threat because of its ability to evade vaccine-induced immune responses due to antigenic drift. Here, we describe the isolation, evolution, and structure of a broad-spectrum ...human monoclonal antibody (mAb), MEDI8852, effectively reacting with all influenza A hemagglutinin (HA) subtypes. MEDI8852 uses the heavy-chain VH6-1 gene and has higher potency and breadth when compared to other anti-stem antibodies. MEDI8852 is effective in mice and ferrets with a therapeutic window superior to that of oseltamivir. Crystallographic analysis of Fab alone or in complex with H5 or H7 HA proteins reveals that MEDI8852 binds through a coordinated movement of CDRs to a highly conserved epitope encompassing a hydrophobic groove in the fusion domain and a large portion of the fusion peptide, distinguishing it from other structurally characterized cross-reactive antibodies. The unprecedented breadth and potency of neutralization by MEDI8852 support its development as immunotherapy for influenza virus-infected humans.
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•Binding to all influenza A subtypes neutralizing seasonal and pandemic strains•Utilizes a rare VH (VH6-1) and carries a low level of somatic mutations•Highly conserved epitope encompassing fusion peptide and hydrophobic groove•Superior therapeutic window compared to oseltamivir in animals
Identification of a human monoclonal antibody that reacts effectively with all influenza A hemagglutinin subtypes paves the way for developing immunotherapy for people infected with the flu virus.
The majority of currently circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses have mutant spike glycoproteins that contain the D614G substitution. Several studies have ...suggested that spikes with this substitution are associated with higher virus infectivity. We use cryo-electron microscopy to compare G614 and D614 spikes and show that the G614 mutant spike adopts a range of more open conformations that may facilitate binding to the SARS-CoV-2 receptor, ACE2, and the subsequent structural rearrangements required for viral membrane fusion.
Considerable progress has been made toward understanding the structural basis of the interaction of the two major surface glycoproteins of influenza A virus with their common ligand/substrate: ...carbohydrate chains terminating in sialic acid. The specificity of virus attachment to target cells is mediated by hemagglutinin, which acquires characteristic changes in its receptor-binding site to switch its host from avian species to humans. Anti-influenza drugs mimic the natural sialic acid substrate of the virus neuraminidase enzyme but utilize the much tighter binding of the drugs for efficacy. Resistance to one of the two main antiviral drugs is differentially acquired by the two distinct subsets of neuraminidase as a consequence of structural differences in the enzyme active site between the two phylogenetic groups.
Hemagglutinin (HA) is the receptor-binding and membrane fusion glycoprotein
of influenza virus and the target for infectivity-neutralizing antibodies. The
structures of three conformations of the ...ectodomain of the 1968 Hong Kong
influenza virus HA have been determined by X-ray crystallography: the
single-chain precursor, HA0; the metastable neutral-pH conformation found on
virus, and the fusion pH-induced conformation. These structures provide a
framework for designing and interpreting the results of experiments on the
activity of HA in receptor binding, the generation of emerging and reemerging
epidemics, and membrane fusion during viral entry. Structures of HA in complex
with sialic acid receptor analogs, together with binding experiments, provide
details of these low-affinity interactions in terms of the sialic acid
substituents recognized and the HA residues involved in recognition.
Neutralizing antibody-binding sites surround the receptor-binding pocket on the
membrane-distal surface of HA, and the structures of the complexes between
neutralizing monoclonal Fabs and HA indicate possible neutralization
mechanisms. Cleavage of the biosynthetic precursor HA0 at a prominent loop in
its structure primes HA for subsequent activation of membrane fusion at
endosomal pH (
Figure 1
). Priming involves
insertion of the fusion peptide into a charged pocket in the precursor;
activation requires its extrusion towards the fusion target membrane, as the N
terminus of a newly formed trimeric coiled coil, and repositioning of the
C-terminal membrane anchor near the fusion peptide at the same end of a
rod-shaped molecule. Comparison of this new HA conformation, which has been
formed for membrane fusion, with the structures determined for other virus
fusion glycoproteins suggests that these molecules are all in the
fusion-activated conformation and that the juxtaposition of the membrane anchor
and fusion peptide, a recurring feature, is involved in the fusion mechanism.
Extension of these comparisons to the soluble N-ethyl-maleimide-sensitive
factor attachment protein receptor (SNARE) protein complex of vesicle fusion
allows a similar conclusion.
Infection by enveloped viruses involves fusion of their lipid envelopes with cellular membranes to release the viral genome into cells. For HIV, Ebola, influenza and numerous other viruses, envelope ...glycoproteins bind the infecting virion to cell-surface receptors and mediate membrane fusion. In the case of influenza, the receptor-binding glycoprotein is the haemagglutinin (HA), and following receptor-mediated uptake of the bound virus by endocytosis
, it is the HA that mediates fusion of the virus envelope with the membrane of the endosome
. Each subunit of the trimeric HA consists of two disulfide-linked polypeptides, HA1 and HA2. The larger, virus-membrane-distal, HA1 mediates receptor binding; the smaller, membrane-proximal, HA2 anchors HA in the envelope and contains the fusion peptide, a region that is directly involved in membrane interaction
. The low pH of endosomes activates fusion by facilitating irreversible conformational changes in the glycoprotein. The structures of the initial HA at neutral pH and the final HA at fusion pH have been investigated by electron microscopy
and X-ray crystallography
. Here, to further study the process of fusion, we incubate HA for different times at pH 5.0 and directly image structural changes using single-particle cryo-electron microscopy. We describe three distinct, previously undescribed forms of HA, most notably a 150 Å-long triple-helical coil of HA2, which may bridge between the viral and endosomal membranes. Comparison of these structures reveals concerted conformational rearrangements through which the HA mediates membrane fusion.
Recently emerged variants of SARS-CoV-2 contain in their surface spike glycoproteins multiple substitutions associated with increased transmission and resistance to neutralising antibodies. We have ...examined the structure and receptor binding properties of spike proteins from the B.1.1.7 (Alpha) and B.1.351 (Beta) variants to better understand the evolution of the virus in humans. Spikes of both variants have the same mutation, N501Y, in the receptor-binding domains. This substitution confers tighter ACE2 binding, dependent on the common earlier substitution, D614G. Each variant spike has acquired other key changes in structure that likely impact virus pathogenesis. The spike from the Alpha variant is more stable against disruption upon binding ACE2 receptor than all other spikes studied. This feature is linked to the acquisition of a more basic substitution at the S1-S2 furin site (also observed for the variants of concern Delta, Kappa, and Omicron) which allows for near-complete cleavage. In the Beta variant spike, the presence of a new substitution, K417N (also observed in the Omicron variant), in combination with the D614G, stabilises a more open spike trimer, a conformation required for receptor binding. Our observations suggest ways these viruses have evolved to achieve greater transmissibility in humans.
Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have ...examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.
Coronaviruses of bats and pangolins have been implicated in the origin and evolution of the pandemic SARS-CoV-2. We show that spikes from Guangdong Pangolin-CoVs, closely related to SARS-CoV-2, bind ...strongly to human and pangolin ACE2 receptors. We also report the cryo-EM structure of a Pangolin-CoV spike protein and show it adopts a fully-closed conformation and that, aside from the Receptor-Binding Domain, it resembles the spike of a bat coronavirus RaTG13 more than that of SARS-CoV-2.
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