2023-06-28 15:06:49
The conquest of the world by the variant of SARS-CoV-2, called omicron, began with the spread of the BA.1 viral line. Several mutations occurred on the branch of the evolutionary tree leading to this lineage, allowing the virus to hide from neutralizing antibodies. In a recent study, it was shown that most of these mutations individually impair the binding of the virus to the cellular ACE2 receptor, but together they provided the BA.1 lineage with an evolutionary advantage over other lines: BA.1 viruses attach to the cell more efficiently than the reference Wuhan variant, similar to Delta lines. Compensatory mutations and epistasis are involved in this – the influence of mutations on the effects of each other.
Binding to receptors on the surface of cells is one of the most important properties for the fitness of the virus, which is carefully monitored by natural selection. Virus SARS-CoV-2 binds to the receptor ACE2 due to the receptor-binding domain (RBD) of the S-protein. Some time ago, in the journal Nature, an interesting articlethe authors of which experimentally looked at how 15 mutations that distinguish the RBD of the BA.1 line (from which the variant micron) from the Wuhan reference variant, and all possible combinations of these mutations affect ACE2 binding (i.e., RBD affinity for ACE2). All possible 215=32,768 sets of these mutations were analyzed, and scientists also traced all possible evolutionary paths of RBD evolution from the Wuhan strain to BA.1 and established the presence of compensatory interactions between mutations.
The binding of different RBD variants to the ACE2 receptor was tested using yeast display technology (K. V. Teymennet-Ramírez et al., 2022. Yeast Surface Display System: Strategies for Improvement and Biotechnological Applications). The genetic sequence encoding the RBD domain was mutated, each mutant variant was linked to the gene for a special “anchor” protein and introduced into yeast cells so that one cell received only one mutant variant. The foreign gene was involved in the circulation of cellular life, and the resulting protein was sent to the surface of the cell. The design was designed so that the RBD domain was exposed to the extracellular environment and was available for binding to the ACE2 receptor, which was added to the solution.
Several variants of the experiment were carried out, differing in the amount of the added receptor. This eventually allowed the mutant variants to be separated along the binding gradient: the weaker the RBD variant binds the receptor, the greater the concentration of the latter is required for binding to occur. The receptor was bound to a fluorescent label, which made it possible to separate the cells that successfully bound it using flow cytometry and cell sorting. This neat technology is briefly described as follows: a liquid with cells in a thin stream is passed through a laser, which provokes the glow of fluorescent molecules bound to the cells, and a detector, which captures the fluorescence. After detection, each cell is separated from the flow by vibration of the device into a separate droplet, which receives an electrical charge depending on its fluorescence. At the end of the path, cells with different luminescence characteristics are sent to different test tubes in accordance with the charge of their drop.
After sorting the cells according to the degree of binding to the receptor, the mutant RBDs from different tubes were sequenced (read the sequences of their genes), which made it possible to determine for each variant how successfully it performs its function necessary for the virus to infect the cell.
The spread in receptor binding strength for the 32,768 tested RBD variants was three orders of magnitude. The combination of all fifteen mutations observed in the BA.1 lineage resulted in a slight increase in binding compared to the “Wuhan variant” hCoV-19/Wuhan/WIV04/2019 (WIV04), the sequence of which is considered to be ancestral to the entire SARS-CoV-2.
Interestingly, many of the mutations individually impair RBD binding to ACE2 compared to WIV4. And, it seems, the logical consequence of this fact is this: in total, all mutations should have made the affinity of RBD lower than that of the Wuhan virus. What is the reason that individually negative mutations in the aggregate had a positive (for the virus) effect? This happened thanks to epistasis (T. J. VanderWeele, 2010. Epistatic Interactions). In general, epistasis can be defined as a deviation of the real consequences of a combination of mutations from what we expect, knowing the effects of individual mutations. The epistatic interaction of a pair of mutations is called second-order epistasis, three mutations is called third-order epistasis, and so on. The importance of the phenomenon of epistasis in molecular evolution has been shown in many works, however, the relative prevalence of epistatic interactions of different orders is still unknown (J. Zhou et al., 2022. Higher-order epistasis and phenotypic prediction).
The authors of the discussed work evaluated the contribution of epistatic interactions between mutations using a mathematical model. Not only paired epistasis was found, but also interactions of higher orders – the third and fourth. It turned out that most often epistasis occurs between mutations in close positions of the protein. This is quite an expected result, since adjacent RBD positions are likely to bind to the same site of the cellular receptor. The greatest contribution to the elimination of the negative effect of other mutations on receptor binding was made by two mutations: N501Y (aspargine to tyrosine at position 501) and Q498R (glutamine to arginine at position 498). Five BA.1 mutations previously shown to be involved in protecting the virus from antibodies (K417N, G446S, E484A, Q493R, and G496S) impair RBD binding to ACE2, especially in combination with each other. However, the Q498R and N501Y mutations neutralize this effect.
An analysis of the evolutionary tree of the virus confirmed the findings. Mutations that disrupt the binding of RBD to the receptor in the experiment rarely occur on the evolutionary tree. They are likely to be swept away by natural selection, since maintaining a high affinity of the S protein for ACE2 is important for the virus. It turned out that mutations, whose negative effect on binding is compensated in the experiment by the N501Y mutation, occur more often in evolutionary lines containing it than in those where it is absent.
The authors of the work believe that the combination of mutations inherent in the BA.1 line, which provides avoidance of immunity while maintaining high affinity for ACE2, might not be formed in a series of acute infections, when there are few mutations between transmissions, and selection once morest mutations that reduce the efficiency of binding to the cellular receptor, too strong. A more likely origin of the line is a chronic infection. It is also likely to form in animals, where mutations might have had a different effect, causing selection to look at them differently.
Источник: Alief Moulana, Thomas Dupic, Angela M. Phillips, Jeffrey Chang, Serafina Nieves, Anne A. Roffler, Allison J. Greaney, Tyler N. Starr, Jesse D. Bloom & Michael M. Desai. Compensatory epistasis maintains ACE2 affinity in SARS-CoV-2 Omicron BA.1 // Nature. 2022. DOI: 10.1038/s41467-022-34506-z.
Galina Klink
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