Despite the speedy development of effective vaccines, therapeutic antibodies, and antivirals, the emergence of viral variants continues to prolong the COVID-19 pandemic.

A new study featured in the journal Biochemistry shows that the convergent evolution of three different SARS-CoV-2 mutations balances positive and negative selection pressures to increase the likelihood of the selection of all three mutations together. The researchers show the co-evolution of three mutations improves viral fitness, although the individual mutations when present on their own exert deleterious effects.

Krishna Mallela, PhD, a professor in the department of pharmaceutical sciences at the University of Colorado Skaggs School of Pharmacy and Pharmaceutical Sciences at the University of Colorado Anschutz Medical Campus, and who is the senior author of the paper, said, “Earlier studies, including ours, have focused on explaining the effect of single mutations and not the mechanism underlying the co-evolution of mutations. This study helps explain the concept of convergent evolution by balancing positive and negative selection pressures.”

The findings in the article titled, “Convergent Evolution of Multiple Mutations Improves the Viral Fitness of SARS-CoV‑2 Variants by Balancing Positive and Negative Selection,” provide new insights on current and emerging SARS-CoV-2 variants. They explain why approved antibodies are not effectively neutralizing recent variants of concern (VOCs) such as Omicron and its subvariants and may inform the development of new vaccines and therapeutics to counter such VOCs.

(Left to right) This paper is co-authored by Krishna Mallela, PhD, and his students Vaibhav Upadhyay, Casey Patrick, and Alexandra Lucas.

Mallela said, “Understanding the mechanisms underlying the antibody escape and the location of mutations in the spike protein will help in developing new antibody therapeutics that will work against new variants by targeting epitopes with minimal mutations or developing broad neutralizing antibodies that target multiple epitopes.”

In this study, Mallela’s team investigated the convergent evolution of three residues in the receptor-binding domain (RBD) of the virus’ spike protein: lysine-417 to threonine (K417T), glutamic acid-484 to lysine (E484K), and asparagine-501 to tyrosine (N501Y). Co-evolution of these mutations occurs in Beta, Gamma, and Omicron VOCs and nearly 2.15 million SARS-CoV-2 variants in the GISAID database (as of April 2022).

Through cloning and expression of the individual and combined mutants, and wildtype RBD, protein purification, fluorescence and circular dichroism spectroscopy, and isothermal titration calorimetry, the scientists found that although the individual mutations render deleterious effects that induce negative selection of the variants, when present together they make distinct contributions to improve viral fitness and promote positive selection. The researchers examined the individual and collective effects of the mutations on binding to angiotensin converting enzyme 2 receptor (ACE2), immune escape from neutralizing antibodies, RBD protein stability, and expression.

They found that the mutation K417T helps the virus evade class 1 antibodies and increases stability and RBD protein expression but it reduces binding of RBD to ACE2. The E484K mutation helps the virus escape class 2 antibodies but decreases receptor binding, stability, and expression. In addition, the N501Y mutation increases receptor binding but decreases stability and expression.

The triple mutant (K417T/E484K/N501Y) embodies compensatory effects whereby it shows an overall increase in receptor binding, evasion of both class 1 and class 2 antibodies, and stability and expression that are comparable to the wildtype RBD.

“These results show that the convergent evolution of multiple mutations enhances viral fitness on different fronts by balancing both positive and negative selection and improves the chances of selection of mutations together,” the authors concluded.

In future studies, Mallela and his team intend to continue to unravel the molecular mechanisms of SARS-CoV-2 evolution with the goal of facilitating the design and development of robust, new therapeutics that emerging variants cannot escape.

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