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Structural basis for enhanced infectivity and immune evasion of SARS-CoV-2 variants

Three papers use functional & structural studies to explore how mutations in the #SARSCoV2 #coronavirus spike protein affect its ability to infect host cells & to evade host immunity

#Variant #variants #DeltaVariant


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.


Abstract

Several fast-spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have become the dominant circulating strains in the COVID-19 pandemic. We report here cryo–electron microscopy structures of the full-length spike (S) trimers of the B.1.1.7 and B.1.351 variants, as well as their biochemical and antigenic properties. Amino acid substitutions in the B.1.1.7 protein increase both the accessibility of its receptor binding domain and the binding affinity for receptor angiotensin-converting enzyme 2 (ACE2). The enhanced receptor engagement may account for the increased transmissibility. The B.1.351 variant has evolved to reshape antigenic surfaces of the major neutralizing sites on the S protein, making it resistant to some potent neutralizing antibodies. These findings provide structural details on how SARS-CoV-2 has evolved to enhance viral fitness and immune evasion.


The COVID-19 pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (1), has led to millions of lives lost and devastating socioeconomic disruptions worldwide. Although the mutation rate of the coronavirus is relatively low because of the proofreading activity of its replication machinery (2), several variants of concern have emerged—including the B.1.1.7 lineage first identified in the United Kingdom, the B.1.351 lineage in South Africa, and the B.1.1.28 lineage in Brazil—within a period of several months (35). These variants not only appear to spread more efficiently than the virus from the initial outbreak [i.e., the strain Wuhan-Hu-1; (1)] but also may be more resistant to immunity elicited by the Wuhan-Hu-1 strain after either natural infection or vaccination (68). The B.1.1.7 variant is of particular concern because it has been reported to be more deadly (9, 10). Thus, understanding the underlying mechanisms of the increased transmissibility, risk of mortality, and immune resistance of new variants may facilitate development of intervention strategies to control the crisis.

SARS-CoV-2 is an enveloped positive-stranded RNA virus that depends on fusion of viral and target cell membranes to enter a host cell. This first key step of infection is catalyzed by the virus-encoded trimeric spike (S) protein, which is also a major surface antigen and thus an important target for development of diagnostics, vaccines, and therapeutics. The S protein is synthesized as a single-chain precursor and is subsequently cleaved by a furin-like protease into the receptor-binding fragment S1 and the fusion fragment S2 [fig. S1 and (11)]. Binding of the viral receptor angiotensin-converting enzyme 2 (ACE2) on the host cell surface to the receptor-binding domain (RBD) of S1, together with a second proteolytic cleavage by another cellular protease in S2 [S2′ site; fig. S1 and (12)], induces dissociation of S1 and irreversible refolding of S2 into a postfusion structure, ultimately leading to membrane fusion (13, 14). In the prefusion conformation, S1 folds into four domains—NTD (N-terminal domain), RBD, and two CTDs (C-terminal domains)—that wrap around the prefusion S2 structure. The RBD can adopt two distinct conformations: “up” for a receptor-accessible state and “down” for a receptor-inaccessible state (15). Rapid progress in structural biology of the S protein has advanced our knowledge of the SARS-CoV-2 entry process (1528). We have previously identified two structural elements, the FPPR (fusion peptide proximal region) and the 630 loop, which appear to modulate the S protein stability as well as the RBD conformation and thus the receptor accessibility (22, 28).

The S protein is the basis of almost all the first-generation COVID-19 vaccines, which were developed using the Wuhan-Hu-1 sequence (29, 30). Several have received emergency use authorization by various regulatory agencies throughout the world because of their impressive protective efficacy and minimal side effects (31, 32). These vaccines appear to have somewhat lower efficacy against the B.1.351 variant than against its parental strain (68, 33), and this variant became completely resistant to many convalescent serum samples in vitro (8). How to address genetic diversity has therefore become a high priority for developing next-generation vaccines. In this study, we have characterized the full-length S proteins from the B.1.1.7 and B.1.351 variants and determined their structures by cryo–electron microscopy (cryo-EM), providing a structural basis for understanding the molecular mechanisms of the enhanced infectivity of B.1.1.7 and the immune evasion of B.1.351.

Biochemical and antigenic properties of the intact S proteins from the new variants To characterize the full-length S proteins with the sequences derived from natural isolates of the B.1.1.7 (hCoV-19/England/MILK-C504CD/2020) and B.1.351 (hCoV-19/South Africa/KRISP-EC-MDSH925100/2020) variants (fig. S1), we first transfected human embryonic kidney (HEK) 293 cells with the respective expression constructs and compared their membrane fusion activities with those of the full-length S constructs of their parental strains [Wuhan-Hu-1: D614 (Asp at position 614) and its early D614G variant: G614 (Asp-to-Gly mutation at position 614) (34)]. All S proteins expressed at comparable levels (fig. S2A), and the cells producing these S proteins fused efficiently with ACE2-expressing cells (fig. S2B). Consistent with our previous findings (22, 28), the G614 and B.1.351 variant S constructs showed slightly higher fusion activity than the D614 and B.1.1.7 variants, but the differences diminished when the transfection level increased. To produce the full-length S proteins, we added a C-terminal strep-tag to the B.1.1.7 and B.1.351 S (fig. S3A) and expressed and purified these proteins under the conditions established for producing the D614 and G614 S trimers (22, 28). The B.1.1.7 protein eluted in three distinct peaks, representing the prefusion S trimer, postfusion S2 trimer, and dissociated S1 monomer, respectively (22), consistent with Coomassie-stained SDS–polyacrylamide gel electrophoresis (SDS-PAGE) analysis (fig. S3B). Nonetheless, the prefusion trimer was the predominant form, accounting for >70% of the total protein, indicating that this trimer is more stable than D614, where the prefusion trimer was only <25%. Like the G614 trimer (28), B.1.351 protein eluted in a single major peak, corresponding to the prefusion S trimer (fig. S3B), with no obvious peaks for dissociated S1 and S2. SDS-PAGE analysis showed that the prefusion trimer peaks contained primarily the cleaved S1/S2 complex for both the proteins, with the cleavage level moderately higher for B.1.351 than for B.1.1.7. These results indicate that the B.1.351 and G614 S proteins have almost identical biochemical properties, whereas the B.1.1.7 trimer is slightly less stable. To assess antigenic properties of the prefusion variant S trimers, we measured their binding to soluble ACE2 and S-directed monoclonal antibodies isolated from COVID-19 convalescent individuals by bio-layer interferometry (BLI). These antibodies target various epitopic regions on the S trimer, as defined by clusters of competing antibodies and designated RBD-1, RBD-2, RBD-3, NTD-1, NTD-2, and S2 [fig. S4A; (35)]. All but the last two clusters contain neutralizing antibodies. The B.1.1.7 variant bound stronger to the receptor than did its G614 parent, regardless of the ACE2 oligomeric state (Fig. 1, fig. S4B, and table S1). The B.1.351 trimer had higher affinity for monomeric ACE2, but slightly lower affinity for dimeric ACE2, than the G614 trimer. In both cases, affinity for ACE2 of the B.1.351 protein was lower than that of the B.1.1.7 variant. These results suggest that the mutation in the RBD of the B.1.1.7 variant [N501Y (Asn501→Tyr)] enhances receptor recognition, whereas the additional mutations in the B.1.351 variant [K417N (Lys417→Asn) and E484K (Glu484→Lys)] reduce ACE2 affinity to a level close to that of the G614 protein, consistent with the previous data (36, 37). All selected monoclonal antibodies bound G614 S with reasonable affinities, and the B.1.1.7 variant showed a similar pattern but with substantially stronger binding to almost all the antibodies (Fig. 1, fig. S4B, and table S1). By contrast, the B.1.351 variant completely lost binding to the two RBD-2 antibodies, G32B6 and C12A2, as well as to the two NTD-1 antibodies, C12C9 and C83B6, whereas the affinities for the rest of the antibodies were the same as those of the G614 trimer. The BLI data were also consistent with the binding results with the membrane-bound S trimers measured by flow cytometry (fig. S5).

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https://science.sciencemag.org/content/373/6555/642




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