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How SARS-CoV-2 first adapted in humans

Viruses need input proteins to enter the cells where they will replicate. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) version is called spike or S protein. The S protein, also the target of current vaccines, is rapidly adapting to its new human hosts. It took its first major step in this direction in early 2020, when its amino acid 614 (from 1297) switched from an aspartic acid (D) to a glycine (G). Viruses carrying this D614G mutation are more rapidly transmitted in humans and now make up the majority in circulation. On page 525 of this issue, Zhang et al. (1) use careful structural analysis to reveal how D614G altered the S protein to accelerate the pandemic.

Early in the pandemic, in the quarrel to create tools to study SARS-CoV-2, investigators developed pseudovirus systems to measure infection in a safe, easily quantifiable way. These systems express a viral entry protein on the surface of a reporter virus used to monitor cell entry and have been used for years to examine many such proteins, including the S-protein of “classical”

; SARS-CoV-1. Frustratingly, pseudoviruses built from the SARS-CoV-2 S protein produced much lower signals than those based on the very similar SARS-CoV-1 S protein. This was confusing because biochemical studies of SARS-CoV-1 and SARS-CoV-2 S protein receptor binding domains (RBDs) made it clear that SARS-CoV-2 RBD bound their common receptor, angiotensin-converting enzyme 2 ( ACE2), with higher affinity than SARS-CoV-1 (2, 3). Faced with ineffective assays, many virologists landed on the same solution as their structural biology counterparts: Mutating the S-protein site, which is cleaved by furin-like proteases in virus-producing cells (2). This change kept the S protein S1 domain, which contains RBD and binds ACE2, covalently bound to its S2 domain, which anchors the S protein to the virion. In particular, some – but not all – of these furin-site mutations significantly improved cell pseudovirus infection (4).

This solution solved a technical problem, but it deepened a mystery. Although a number of distantly related coronaviruses carry furin cleavage sites at their S1-S2 boundaries, the SARS-CoV-1 S protein and the all known bat-derived viruses from the same Sarbecovirus genus lack this site. Instead of being cleaved into virus-producing cells, their S-proteins are cleaved by various proteases, while the virus engages ACE2 in the next but not yet infected cell (5). As it happened, mutations at the furin site that improved the SARS-CoV-2 S protein function in pseudovirus allowed the modified S protein to work with these later-stage enzymes, as did the SARS-CoV-1 version. Why did the SARS-CoV-2 furrow site continue, even though it made cell culture infection less effective? In fact, viruses that were passed on in culture regularly lost this place. Does it somehow improve viral transmission? Would it eventually disappear during the pandemic?

In the summer of 2020 Korber et al. sounded the alarm about a “concern mutation”, namely D614G (6). In the laboratory, this change avoided the need to eliminate the S-protein furin site, apparently correcting a design error associated with this unusual cleavage site (4, 6). Animal studies with otherwise identical viruses showed significantly greater replication of the D614G variant in the upper respiratory tract, a site important for transmission (7, 8). In contrast, no significant differences were seen between the two lower respiratory tract viruses, a site responsible for more serious illness (7). These observations are consistent with the current consensus that D614G, now present in most circulating viruses, enhances viral transmission, but in contrast to recently acquired mutations in S1[egAsn[egAsn[fxAsn[egAsn501→ Taurus (N501Y)]it does not change the number of admissions.

The underlying mechanism of this fitness benefit remained a controversial point. Here, another unusual property of the S protein, in this case shared with SARS-CoV-1, became relevant. The SARS-CoV-2 S protein, like most entry-level proteins from viruses with a lipid membrane, accumulates in trimmers. Typically, during the virion collection process, viral entry proteins subtly change their conformations, but it is unusual for these proteins to break their triple symmetry before binding their receptor. However, the mature SARS-CoV-2 S protein often assumes an asymmetric arrangement, with one of its three RBDs assuming an open or “up” conformation (1, 9). Only RBDs in this upconformation can bind ACE2. Once done, the S1 domains differ from S2, and S2 undergoes a pronounced rearrangement to a “postfusion” state. The energy released by this rearrangement drives viral and cell membranes to fuse and gives the virus access to the cell device.

To explain D614G suitability, some investigators focused on the effect of D614G on the frequency with which this one-up conformation could be found, suggesting that more efficient involvement of the receptor accounted for the improved transmissibility of viruses carrying this mutation. (10, 11). Others noted that S proteins of D614G-expressing viruses fell apart less frequently, an effect perhaps amplified in the challenging environment of a living organism. They observed that D614G helped the S1 domain to cling to S2, preventing S2 from prematurely and unproductively assuming its conformation after fusion (4, 9, 12). Thus, the virus had more functional S-proteins that could bind and infect the next cell.

Improving viral transmission

The Gly614 (G614) mutation in tip (S) increases the order of the 630 loop compared to wild-type Asp614 (D614). This prevents premature S1 shedding, which is often seen with wild-type S proteins, ensuring that more S protein remains in a fusion-ready “one-up” state with a receptor binding domain (RBD) exposed in the trimer, ready to bind angiotensin -converting enzyme 2 (ACE2) on host cells, which increases infection efficiency.


To cut through this controversy, Zhang et al. solved the structure and provided detailed analyzes of both D614 and G614S proteins in several states. They first noted that, as they and others had previously observed, the loss of D614 in S1 breaks an ionic bond to a proximal lysine, K854, in S2 (9). Loss of this salt bridge is initially contradictory because it would loosen the connection between S1 and S2, although it might facilitate the movement of the RBD to the up configuration. However, structures from Zhang et al. shows that a major difference between S proteins with and without D614G is the visibly greater order of G614 S proteins in a region spanning residues 620 to 640, which the authors call the “630 loop.” This loop is just downstream of the G614. It is therefore possible that either the loss of the D614-K854 salt bridge or the greater flexibility of the backbone provided by a glycine helps the 630 loop to nest closer in a gap formed by two major S-protein domains (the amino-terminal domain and carboxyl -terminal domain 1). Regardless, this loop is found in a more rigid and stable arrangement between these domains when residue 614 is a G than when it is a D.

The key is that both RBD-up conformation and dissociation of S1 from S2 – activated by furin cleavage – require splitting of the 630 loop. Thus, one can more easily access the RBD-up conformation with the original D614 S protein, but once this conformation is achieved, this S protein is more likely to fall apart due to premature shedding of its S1 domain. . Conversely, with G614, more energy is required to achieve an RBD-up state, but dissociation of S1 from S2 also becomes less favorable because the remaining folded 630 loops continue to hold the trimmer together. Thus, the D614G variants have more S proteins in up-orientation because the next, irreversible step toward inactivation is slower. Infection with D614G is more effective because it prevents premature S1 shedding (see figure).

These structural studies have real effects. All current vaccines are based on the original, unstable D614 form of the S protein (13). Fortunately, most vaccine developers, including Moderna and Pfizer-BioNTech, took a lesson from studies of SARS-CoV-1 and Middle Eastern Respiratory Syndrome (MERS) coronavirus to reduce S protein excretion by introducing non-native prolines into S2 (14). Those who developed the Johnson and Johnson and Novavax vaccines had prior to removing the furrow site as well. In contrast, the developers of the University of Oxford chose the AstraZeneca vaccine wild-type S protein (containing D614), as is the case with the inactivated virus vaccine produced by Sinovac. To be clear, other variables, particularly antigen delivery systems, are likely to account for differences in efficacy between these vaccines. However, studies with apples to apples on animals make it clear that both engineered proline and ablination at the furin site contribute to vaccine efficacy (15). It is almost certain that the next round of vaccines that better reflect the S-protein variants currently in circulation will include D614G. Vaccines expressing unmodified S proteins with G614 may enjoy a relatively sharp jump because this change compensates for the lack of engineered stabilizing mutations.

Zhang’s work et al. also reveals more about the natural history of the virus. The remarkable emergence of D614G suggests that the acquisition of a destabilizing furin site was a recent event. The virus can easily lose this place, as it often does in cell culture systems, suggesting that it somehow facilitates human transmission. This is not a conclusion that most students of human coronavirus would have expected, given that SARS-CoV-1, which transmits with reasonable efficiency, lacks this site, while the more distantly related MERS coronavirus carries this site and transmits bad. How the SARS-CoV-2 furin site promotes new infections in humans remains a central open question in the field.

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