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Halo scientists reveal Achilles’ heel of deadly coronaviruses from SARS-CoV to Omicron


As coronavirus disease 2019 (COVID-19) persists, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor binding domain (RBD) comprises a free fatty acid (FFA)–binding pocket. FFA binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic β-coronaviruses (β-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2, and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold–causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation, while the open, infectious conformation is devoid of LA. Electron tomography of SARS-CoV-2–infected cells reveals that LA treatment inhibits viral replication, resulting in fewer deformed virions. Our results establish FFA binding as a hallmark of pathogenic β-CoV infection and replication, setting the stage for FFA-based antiviral strategies to overcome COVID-19.


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19) pandemic with millions of lives lost, damaging communities and economies. Human coronaviruses were previously only known to cause mild diseases of the upper respiratory tract until the emergence of the pathogenic coronaviruses SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV) in 2002 and 2012, respectively. Both cause severe pneumonias with a high incidence of mortality. Pathogenic SARS-CoV-2, SARS-CoV, MERS-CoV, and the endemic common cold–causing HCoV-OC43 and HCoV-HKU1 viruses all belong to the β-coronavirus (β-CoV) genus of the family Coronaviridae. During the present pandemic, numerous variants of concern (VOCs) have emerged, exhibiting increased transmissibility, increased risk of reinfection, and reduced vaccine efficiency (1), highlighting the urgent need for effective antiviral treatment strategies. These VOCs include the SARS-CoV-2 lineages B.1.1.7 (Alpha), B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), and, most recently, B.1.1.529 (Omicron) (2).
The trimeric spike (S) glycoprotein decorates the surface of coronaviruses and mediates entry into host cells. S is the major antigen recognized by neutralizing antibodies and the main target for vaccine development (3). SARS-CoV S and SARS-CoV-2 S both bind to human angiotensin-converting enzyme 2 (ACE2) receptor on the host cell surface (46), MERS-CoV S binds to dipeptidyl-peptidase-4 (DPP4) (57), while the HCoV-HKU1 and HCoV-OC43 S proteins bind to the N-acetyl-9-O-acetylneuraminic acid receptor (8). SARS-CoV-2 S is cleaved by host cell proteases into the receptor binding fragment S1 and the partially buried fusion fragment S2 (4). S1 is composed of the N-terminal domain (NTD), the receptor binding domain (RBD) with a receptor binding motif (RBM), and two C-terminal domains (CTDs). S2 mediates fusion of the viral envelope with host cell membranes and is composed of the fusion peptide, heptad repeats, transmembrane domain, and cytoplasmic C terminus (9). In the prefusion conformation, the RBDs in the S trimer can alternate between closed (“down”) and open (“up”) conformations. SARS-CoV and SARS-CoV-2 S require RBD up conformations for interaction with ACE2 (6910) for cell entry.
In our previous SARS-CoV-2 S structure, we discovered a free fatty acid (FFA) bound to a hydrophobic pocket in the RBD (11). Mass spectroscopy identified this ligand as linoleic acid (LA), an essential omega-6 polyunsaturated fatty acid (PUFA) that the human body cannot synthesize (1112). LA binding stabilizes S in a compact, locked conformation that is incompatible with ACE2 receptor binding (11). In immunofluorescence assays, synthetic mini-virus particles decorated with LA-bound S showed reduced docking to ACE2-expressing host cells as compared to mini-virus with LA-free S (13), corroborating that LA interferes with receptor binding and subsequent host cell entry mediated by S. S protein sequence alignments suggest conservation of the hydrophobic pocket in the RBDs of SARS-CoV, SARS-CoV-2, MERS-CoV, and the corresponding B domains in hCoV-OC43 and hCoV-HKU1 (11), indicating that the pocket may be a hallmark shared by all human β-CoVs. Intriguingly, all SARS-CoV-2 VOCs stringently maintain this pocket, notably including Omicron, which accumulated a wide range of mutations in S elsewhere, suggesting that the pocket provides a selective advantage for the virus.
Here, we sought to unveil whether LA binding and the functional consequences of LA binding are conserved in S glycoproteins of pathogenic β-CoVs SARS-CoV, MERS-CoV, and SARS-CoV-2 VOCs (Alpha, Beta, Gamma, Delta, and Omicron), as compared to HCoV-HKU1, a β-CoV causing only mild disease (common cold). We demonstrate that all comprise a hydrophobic pocket capable of binding LA, except common cold–causing HCoV-HKU1 S that cannot bind LA. At the same time, we demonstrate that a single–amino acid substitution of a residue lining the entrance of the hydrophobic pocket in HCoV-HKU1 S is sufficient to restore LA binding. We analyze SARS-CoV S by cryogenic electron microscopy (cryo-EM) showing that LA-bound SARS-CoV S adopts a hitherto elusive locked structure sharing characteristics with LA-bound locked SARS-CoV-2 S (11), incompatible with ACE2 receptor binding. In contrast, in the open conformation of SARS-CoV S, the pocket in the RBDs is devoid of LA. Molecular dynamics (MD) simulations corroborate spontaneous LA binding in the respective hydrophobic pockets in the RBDs of SARS-CoV, MERS-CoV, and SARS-CoV-2 VOCs, while no LA binding to HCoV-HKU1 S is observed. Using correlative light EM (CLEM) followed by electron tomography of SARS-CoV-2–infected cells, we provide evidence that LA, beyond counteracting infection at the S protein level (1113), also interferes with viral replication inside infected cells. This likely occurs through inhibition of cytoplasmic phospholipase A2 (cPLA2), a key enzyme implicated in viral replication via formation of intracellular replication compartments (14) and in the cytokine storm causing systemic inflammation in COVID-19 (1517).


Functional conservation of the S FFA-binding pocket in β-CoVs

LA binding to S can be analyzed by surface plasmon resonance (SPR). We previously determined a binding constant of ~41 nM for LA to SARS-CoV-2 S RBD (11). To corroborate our hypothesis that a functional hydrophobic pocket is evolutionarily conserved, we tested whether other β-CoVs are also capable of LA binding (Fig. 1). On the basis of sequence alignments, the RBDs of SARS-CoV, MERS-CoV, SARS-CoV-2, and VOCs (Alpha, Beta, Gamma, Delta, and Omicron), including current BA.5 and BA.2.75 variants, all maintain the hydrophobic pocket, at least since the emergence of SARS-CoV in 2002 (Fig. 1, A to C). In SPR experiments, LA bound to immobilized SARS-CoV RBD (Fig. 1D). We observed a slow dissociation of LA from the RBD consistent with tight LA binding. LA also bound to immobilized MERS-CoV RBD (Fig. 1E). In contrast, the B domain of HCoV-HKU1 S did not bind LA despite high sequence similarity (Fig. 1, A and F). HCoV-HKU1 S comprises a bulky glutamate E375 located directly in front of the hydrophobic pocket (18), obstructing the pocket entrance (Fig. 1F). We mutated HCoV-HKU1 E375 to alanine and restored LA binding (Fig. 1F). This indicates that the pocket function, while structurally conserved, may have been lost in HCoV-HKU1, a β-CoV that causes mild disease. The RBDs of SARS-CoV-2 VOCs all bound LA, confirming that LA binding is conserved (Fig. 1G) and not affected by the mutations in S that cluster away from the pocket (Fig. 1H). Together, we confirmed full conservation of LA binding in highly pathogenic β-CoV S proteins but not in S of mild disease–causing HCoV-HKU1. HCoV-OC43, which likewise causes common cold, appears to also comprise a hydrophobic pocket (Fig. 1A), as seen in an earlier HCoV-OC43 S cryo-EM structure that displays unassigned density in the B domain (fig. S1) (19). It remains unclear what exactly this unassigned density corresponds to, which appears too small to accommodate the C18 hydrocarbon chain of LA (fig. S1).

Cryo-EM structures of locked and open SARS-CoV S

To elucidate LA binding by SARS-CoV that emerged 2002, we determined the S cryo-EM structure. The S ectodomain was produced as a secreted trimer using MultiBac (20) identically as described for SARS-CoV-2 S (fig. S2) (11). As before, we did not supplement LA during expression or subsequent sample purification and preparation steps. Cryo-EM data collection was performed with purified S protein (fig. S2 and table S1). Three-dimensional (3D) classification and refinement identified one conformation with all three RBDs in the down position and two different open conformations with one or two RBDs in the up position, respectively (fig. S3 and tables S1 and S2). Using 81,242 particles, the “one-RBD up” open conformation reached 3.3-Å resolution and was further analyzed (Fig. 2A and figs. S3 and S4). Analysis of 178,203 particles adopting the three RBD down conformation yielded a 2.48-Å resolution map after applying C3 symmetry (Fig. 2A and figs. S3 and S4). This three-RBD down form of SARS-CoV S exhibits a compact arrangement of the RBDs with fully ordered RBM, similar to our previously identified LA-bound locked S structure of SARS-CoV-2 (11).