Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity

Abstract

SARS-CoV-2 from alpha to epsilon As battles to contain the COVID-19 pandemic continue, attention is focused on emerging variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus that have been deemed variants of concern because they are resistant to antibodies elicited by infection or vaccination or they increase transmissibility or disease severity. Three papers used functional and structural studies to explore how mutations in the viral spike protein affect its ability to infect host cells and to evade host immunity. Gobeil et al. looked at a variant spike protein involved in transmission between minks and humans, as well as the B1.1.7 (alpha), B.1.351 (beta), and P1 (gamma) spike variants; Cai et al. focused on the alpha and beta variants; and McCallum et al. discuss the properties of the spike protein from the B1.1.427/B.1.429 (epsilon) variant. Together, these papers show a balance among mutations that enhance stability, those that increase binding to the human receptor ACE2, and those that confer resistance to neutralizing antibodies. Science, abi6226, abi9745, abi7994, this issue p. eabi6226 , p. 642, p. 648 How SARS-CoV-2 variants gain enhanced infectivity and evade host immune responses is elucidated. INTRODUCTION Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been circulating worldwide since the beginning of the pandemic. Some are termed Variants of Concern (VOC) because they show evidence for increased transmissibility, higher disease severity, resistance to neutralizing antibodies elicited by current vaccines or from previous infection, reduced efficacy of treatments, or failure of diagnostic detection methods. VOCs accumulate mutations in the spike (S) glycoprotein. Some VOCs that arose independently in different geographical locations show identical changes, implying convergent evolution and selective advantages of the acquired variations. A set of three amino acid substitutions in the receptor-binding domain (RBD)—Lys417 → Asn (K417N), Glu484 → Lys (E484K), and Asn501 → Tyr (N501Y)—occurred in the B.1.1.28 and B.1.351 lineages that originated in Brazil and South Africa, respectively. The P.1 lineage that branched off B.1.1.28 harbored a Lys417 → Thr (K417T) substitution while retaining the E484K and N501Y changes. The E484K substitution has attracted attention as a result of its location within the epitope of many potent neutralizing antibodies. The N501Y substitution also occurred in the B.1.1.7 variant that originated in the UK and was implicated in increased receptor binding and higher transmissibility of the variant. The B.1.1.7 variant, in turn, shares the His69/Val70 spike deletion mutation with spike from a variant that was implicated in transmission between humans and minks (ΔFVI). RATIONALE Global sequencing initiatives and in vitro neutralization and antibody binding assays have rapidly provided critical and timely information on the VOCs. Here, by combining cryo–electron microscopy (cryo-EM) structural determination with binding assays and computational analyses on the variant spikes, we sought to visualize the impact of the amino acid substitutions on spike conformation to understand how these changes affect their biological function. RESULTS We measured angiotensin-converting enzyme 2 (ACE2) receptor and antibody binding for 19 SARS-CoV-2 S ectodomain constructs harboring amino acid changes found in circulating variants. These included a variant involved in interspecies SARS-CoV-2 transmission between humans and minks, as well as several VOCs including the B.1.1.7, B.1.1.28/P.1, and B.1.351 variants. Consistent with published neutralization data, B.1.1.7 showed decreased binding to N-terminal domain (NTD)–directed antibodies, whereas P.1 and B.1.351 showed reduced binding to both NTD- and RBD-directed antibodies. All variants showed increased binding to ACE2, which was mediated by higher propensity for RBD-up states, and affinity-enhancing mutations in the RBD. We observed spike instability in the mink-associated variant, highlighted by the presence of a population in the cryo-EM dataset with missing density for the S1 subunit of one protomer. Modulation of contacts between the SD1 and HR1 regions led to increased RBD-up states of the B.1.1.7 spike, with the protein stability maintained by a balance of stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 and B.1.351 variants to some potent RBD-directed neutralizing antibodies. CONCLUSION Our study revealed details of how amino acid substitutions affect spike conformation in circulating SARS-CoV-2 VOCs. We define communication networks that modulate spike allostery and show that the S protein uses different mechanisms to converge upon similar solutions for altering the RBD up/down positioning. Cryo-EM structures of SARS-CoV-2 spike ectodomains. Naturally occurring amino acid variations are represented by colored spheres. Spike mutations from a mink-associated (ΔFV) (top left), B.1.1.7 (top right), B.1.351 (bottom right), and a spike with three RBD mutations (bottom left) are shown. Relative proportions of the RBD down and up populations are indicated for each. The three amino acid substitutions in the RBD—K417N/T, E484K, and N501Y—were found in the B.1.1.28 variant and are shared with the P.1 and B.1.351 lineages. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo–electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)–up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.