Biophysical evolution of the receptor binding domains of SARS-CoVs (preprint)

With hundreds of coronaviruses (CoVs) identified in bats that are capable of infecting humans, it is important to understand how CoVs that affected the human population have evolved. Seven known coronaviruses have infected humans, of which three CoVs caused severe disease with high mortality rates: SARS-CoV emerged in 2002, MERS-CoV in 2012, and SARS-CoV-2 in 2019. Both SARS-CoV and SARS-CoV-2 belong to the same family, follow the same receptor pathway, and use their receptor binding domain (RBD) of spike protein to bind to the ACE2 receptor on the human epithelial cell surface. The sequence of the two RBDs is divergent, especially in the receptor binding motif (RBM) that directly interacts with ACE2. We probed the biophysical differences between the two RBDs in terms of their structure, stability, aggregation, and function. Since RBD is being explored as an antigen in protein subunit vaccines against CoVs, determining these biophysical properties will also aid in developing stable protein subunit vaccines. Our results show that despite RBDs having a similar three-dimensional structure, they differ in their thermodynamic stability. RBD of SARS-CoV-2 is significantly less stable than that of SARS-CoV. Correspondingly, SARS-CoV-2 RBD shows a higher aggregation propensity. Regarding binding to ACE2, less stable SARS-CoV-2 RBD binds with a higher affinity than more stable SARS-CoV RBD. In addition, SARS-CoV-2 RBD is more homogenous in terms of its binding stoichiometry towards ACE2, compared to SARS-CoV RBD. These results indicate that SARS-CoV-2 RBD differs from SARS-CoV RBD in terms of its stability, aggregation, and function, possibly originating from the diverse RBMs. Higher aggregation propensity and decreased stability of SARS-CoV-2 RBD warrants further optimization of protein subunit vaccines that use RBD as an antigen either by inserting stabilizing mutations or formulation screening.

Mild SARS-CoV-2 infection results in long-lasting microbiota instability (preprint)

Viruses targeting mammalian cells can indirectly alter the gut microbiota, potentially compounding their phenotypic effects. Multiple studies have observed a disrupted gut microbiota in severe cases of SARS-CoV-2 infection that require hospitalization. Yet, despite demographic shifts in disease severity resulting in a large and continuing burden of non-hospitalized infections, we still know very little about the impact of mild SARS-CoV-2 infection on the gut microbiota in the outpatient setting. To address this knowledge gap, we longitudinally sampled 14 SARS-CoV-2 positive subjects who remained outpatient and 4 household controls. SARS-CoV-2 cases exhibited a significantly less stable gut microbiota relative to controls, as long as 154 days after their positive test. These results were confirmed and extended in the K18-hACE2 mouse model, which is susceptible to SARS-CoV-2 infection. All of the tested SARS-CoV-2 variants significantly disrupted the mouse gut microbiota, including USA-WA1/2020 (the original variant detected in the United States), Delta, and Omicron. Surprisingly, despite the fact that the Omicron variant caused the least severe symptoms in mice, it destabilized the gut microbiota and led to a significant depletion in Akkermansia muciniphila. Furthermore, exposure of wild-type C57BL/6J mice to SARS-CoV-2 disrupted the gut microbiota in the absence of severe lung pathology.

Biophysical fitness landscape of the SARS-CoV-2 Delta variant receptor binding domain (preprint)

Among the five known SARS-CoV-2 variants of concern, Delta is the most virulent leading to severe symptoms and increased number of deaths. Our study seeks to examine how the biophysical parameters of the Delta variant correlate to the clinical observations. Receptor binding domain (RBD) is the first point of contact with the human host cells and is the immunodominant form of the spike protein. Delta variant RBD contains two novel mutations L452R and T478K. We examined the effect of single mutations as well as the double mutation on RBD expression in human Expi293 cells, RBD stability using urea and thermal denaturation, and RBD binding to angiotensin converting enzyme 2 (ACE2) receptor and to neutralizing antibodies using isothermal titration calorimetry. Delta variant RBD showed significantly higher expression compared to the wild-type RBD, and the increased expression is due to L452R mutation. Despite their non-conservative nature, none of the mutations significantly affected RBD structure and stability. All mutants showed similar binding affinity to ACE2 and to Class 1 antibodies (CC12.1 and LY-CoV016) as that of the wild-type. Delta double mutant L452R/T478K showed no binding to Class 2 antibodies (P2B-2F6 and LY-CoV555) and a hundred-fold weaker binding to a Class 3 antibody (REGN10987), and the decreased antibody binding is determined by the L452R mutation. These results indicate that the immune escape from neutralizing antibodies, rather than receptor binding, is the main biophysical parameter determining the fitness landscape of the Delta variant RBD and is determined by the L452R mutation.

Convergent evolution of multiple mutations improves the viral fitness of SARS-CoV-2 variants by balancing positive and negative selection (preprint)

ABSTRACT Multiple mutations have been seen to undergo convergent evolution in SARS-CoV-2 variants of concern. One such evolution occurs in Beta, Gamma, and Omicron variants at three amino acid positions K417, E484, and N501 in the receptor binding domain of the spike protein. We examined the physical mechanisms underlying the convergent evolution of three mutations K417T/E484K/N501Y by delineating the individual and collective effects of mutations on binding to angiotensin converting enzyme 2 receptor, immune escape from neutralizing antibodies, protein stability and expression. Our results show that each mutation serves a distinct function that improves virus fitness supporting its positive selection, even though individual mutations have deleterious effects that make them prone to negative selection. Compared to the wild-type, K417T escapes Class 1 antibodies, has increased stability and expression; however, it has decreased receptor binding. E484K escapes Class 2 antibodies; however, it has decreased receptor binding, stability and expression. N501Y increases receptor binding; however, has decreased stability and expression. When these mutations come together, the deleterious effects are mitigated due to the presence of compensatory effects. Triple mutant K417T/E484K/N501Y has increased receptor binding, escapes both Class 1 and Class 2 antibodies, and has similar stability and expression as that of the wild-type. These results show the implications of presence of multiple mutations on virus evolution that enhance viral fitness on different fronts by balancing both positive and negative selection and improves the chances of selection of mutations together.

Interplay between protein stability, binding to ACE2 and escape from neutralizing antibodies determines the natural selection of SARS-CoV-2 receptor binding domain variants (preprint)

Emergence of new SARS-CoV-2 variants has raised concerns at the effectiveness of vaccines and antibody therapeutics developed against the unmutated wild-type virus. It is thus important to understand the emergence of mutants from an evolutionary viewpoint to be able to devise effective countermeasures against new variants. We examined the effect of 12 most commonly occurring mutations in the receptor binding domain (RBD) on its expression, stability, activity, and antibody escape potential- some of the factors that may influence the natural selection of mutants. Recombinant proteins were expressed in human cells. Stability was measured using thermal denaturation melts. Activity and antibody escape potential were measured using isothermal titration calorimetry in terms of binding of RBD variants to ACE2 and to a neutralizing human antibody CC12.1, respectively. Our results show that variants differ in their expression levels, suggesting that mutations can impact the availability of proteins for virus assembly. All variants have similar or higher stability than the wild-type, implying that increased RBD stability might be another important factor in virus evolution. In terms of ACE2 binding, when compared to the wild-type, only 3 out of 7 expressed variants show stronger affinity, 2 have similar affinity, whereas the other 2 have weaker affinity, indicating that increased affinity towards ACE2 is an important but not the sole factor in the natural selection of variants. In terms of CC12.1 binding, when compared to the wild-type, 4 out of 7 variants have weaker affinity, 2 have a similar affinity, and 1 variant has a stronger affinity. Taken together, these results indicate that multiple factors contribute towards the natural selection of variants, and all of these factors have to be considered in order to understand the natural selection of SARS-CoV-2 variants. In addition, since not all variants can escape a given neutralizing antibody, antibodies to treat new variants can be chosen based on the specific mutations in that particular variant.