SARS-CoV-2 has evolved variants with substitutions in the spike receptor-binding domain (RBD) that impact its affinity for ACE2 receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites—a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single amino-acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently N501Y, cause epistatic shifts in the effects of mutations at other sites. These epistatic shifts shape subsequent evolutionary change, for example enabling many of the antibody-escape substitutions in the Omicron RBD. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.
Kinetics studies of dNTP analogues having pyrophosphate-mimicking β,γ-pCXYp leaving groups with variable X and Y substitution reveal striking differences in the chemical transition-state energy for DNA polymerase β that depend on all aspects of base-pairing configurations, including whether the incoming dNTP is a purine or pyrimidine and if base-pairings are right (T•A and G•C) or wrong (T•G and G•T). Brønsted plots of the catalytic rate constant (log(kpol)) versus pKa4 for the leaving group exhibit linear free energy relationships (LFERs) with negative slopes ranging from −0.6 to −2.0, consistent with chemical rate-determining transition-states in which the active-site adjusts to charge-stabilization demand during chemistry depending on base-pair configuration. The Brønsted slopes as well as the intercepts differ dramatically and provide the first direct evidence that dNTP base recognition by the enzyme–primer–template complex triggers a conformational change in the catalytic region of the active-site that significantly modifies the rate-determining chemical step.
INTRODUCTIONSARS-CoV-2 evolution presents an ongoing challenge to public health. Tens of thousands of mutations have arisen in the SARS-CoV-2 genome as the pandemic has progressed. Understanding the relative importance of mutations in viral proteins, particularly those of relevance for antiviral immunity, is key to allocating preparedness efforts. Mutations in the viral Spike protein have received particular attention because Spike is the target of antibody-mediated immunity and is the primary antigen in current vaccines (1). As of December 1st, 2021, there are 10,381 distinct amino acid substitutions, insertions, or deletions in Spike sequences from the GISAID database (2). These mutations occur at all but one position in the protein, in different combinations, creating over 160,000 unique Spike protein sequences. A small subset of these mutations are components of "Variants Being Monitored" (VBMs), "Variants of Interest" (VOIs) or "Variants of Concern" (VOCs), as classified by the United States Centers for Disease Control (CDC) (3). The distinction between VOIs and the higher alert VOCs is whether a negative clinical impact is suspected or confirmed. VBMs are variants that would be classified as VOCs if not for low prevalence.Early statistical and algorithmic identification of the key Spike amino acid changes contributing to future putative VBM/VOI/VOCs are of clear benefit to public health strategy. Such predictions could enhance the identification of vulnerabilities for antibody-based therapeutics, vaccines, and diagnostics. Predicting future successful mutations would extend the time available to develop proactive responses at earlier stages of spread. It would also complement existing forecasting efforts which seek to predict overall SARS-CoV-2 incidence, hospitalizations, and death over time (4-6). Focus on the success of individual mutations rather than genomic variants also facilitates longer-term forecasting. The
SARS-CoV-2 has evolved variants with substitutions in the spike receptor-binding domain (RBD) that impact its affinity for ACE2 receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites--a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single amino-acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently N501Y, cause epistatic shifts in the effects of mutations at other sites, thereby shaping subsequent evolutionary change. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.
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