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RNA viruses like SARS-CoV-2 have a high mutation rate, which contributes to their rapid evolution. The rate of mutations depends on the mutation type (e.g., A→C, A→G, etc.) and can vary between sites in the viral genome. Understanding this variation can shed light on the mutational processes at play, and is crucial for quantitative modeling of viral evolution. Using the millions of available SARS-CoV-2 full-genome sequences, we estimate rates of synonymous mutations for all 12 possible nucleotide mutation types and examine how much these rates vary between sites. We find a surprisingly high level of variability and several striking patterns: the rates of four mutation types suddenly increase at one of two gene boundaries; the rates of most mutation types strongly depend on a site's local sequence context, with up to 56-fold differences between contexts; consistent with a previous study, the rates of some mutation types are lower at sites engaged in RNA secondary structure. A simple log-linear model of these features explains ~15-60% of the fold-variation of mutation rates between sites, depending on mutation type; more complex models only modestly improve predictive power out of sample. We estimate the fitness effect of each mutation based on the number of times it actually occurs versus the number of times it is expected to occur based on the model. We identify several small regions of the genome where synonymous or noncoding mutations occur much less often than expected, indicative of strong purifying selection on the RNA sequence that is independent of protein sequence. Overall, this work expands our basic understanding of SARS-CoV-2's evolution by characterizing the virus's mutation process at the level of individual sites and uncovering several striking mutational patterns that arise from unknown mechanisms.

The cytochrome P450s enzyme family metabolizes ∼80% of small molecule drugs. Variants in cytochrome P450s can substantially alter drug metabolism, leading to improper dosing and severe adverse drug reactions. Due to low sequence conservation, predicting variant effects across cytochrome P450s is challenging. Even closely related cytochrome P450s like CYP2C9 and CYP2C19, which share 92% amino acid sequence identity, display distinct phenotypic properties. Using variant abundance by massively parallel sequencing, we measured the steady-state protein abundance of 7,660 single amino acid variants in CYP2C19 expressed in cultured human cells. Our findings confirmed critical positions and structural features essential for cytochrome P450 function, and revealed how variants at conserved positions influence abundance. We jointly analyzed 4,670 variants whose abundance was measured in both CYP2C19 and CYP2C9, finding that the homologs have different variant abundances in substrate recognition sites within the hydrophobic core. We also measured the abundance of all single and some multiple wild type amino acid exchanges between CYP2C19 and CYP2C9. While most exchanges had no effect, substitutions in substrate recognition site 4 reduced abundance in CYP2C19. Double and triple mutants showed distinct interactions, highlighting a region that points to differing thermodynamic properties between the 2 homologs. These positions are known contributors to substrate specificity, suggesting an evolutionary tradeoff between stability and enzymatic function. Finally, we analyzed 368 previously unannotated human variants, finding that 43% had decreased abundance. By comparing variant effects between these homologs, we uncovered regions underlying their functional differences, advancing our understanding of this versatile family of enzymes., Competing Interests: Conflicts of interest The authors declare no conflict of interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of the Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we used pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affected cell entry and antibody neutralization. Our experiments defined functional constraints throughout GPC. We quantified how GPC mutations affected neutralization with a panel of monoclonal antibodies. All antibodies tested were escaped by mutations that existed among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid the design of therapeutics and vaccines., Competing Interests: Declaration of interests J.D.B. is on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, and the Vaccine Company. K.G.A. is a consultant to and on the scientific advisory board of Invivyd. J.D.B. and K.H.D.C. receive royalty payments as inventors on Fred Hutch licensed patents related to viral deep mutational scanning. N.P.K. is a co-founder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. The King lab has received unrelated sponsored research agreements from Pfizer and GSK. M.M. is currently an employee of Seagen, though his contributions to this manuscript were performed when he was an employee of the Institute for Protein Design., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Human influenza virus evolves to escape neutralization by polyclonal antibodies. However, we have a limited understanding of how the antigenic effects of viral mutations vary across the human population and how this heterogeneity affects virus evolution. Here, we use deep mutational scanning to map how mutations to the hemagglutinin (HA) proteins of two H3N2 strains, A/Hong Kong/45/2019 and A/Perth/16/2009, affect neutralization by serum from individuals of a variety of ages. The effects of HA mutations on serum neutralization differ across age groups in ways that can be partially rationalized in terms of exposure histories. Mutations that were fixed in influenza variants after 2020 cause greater escape from sera from younger individuals compared with adults. Overall, these results demonstrate that influenza faces distinct antigenic selection regimes from different age groups and suggest approaches to understand how this heterogeneous selection shapes viral evolution., Competing Interests: Declaration of interests J.D.B. is on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, and the Vaccine Company. J.D.B., A.N.L., and F.C.W. receive royalty payments as inventors on Fred Hutch licensed patents related to viral deep mutational scanning. H.Y.C. reports consulting with Ellume, Pfizer, and the Bill and Melinda Gates Foundation. She has served on advisory boards for Vir, Merck, and Abbvie. She has conducted CME teaching with Medscape, Vindico, and Clinical Care Options. She has received research funding from Gates Ventures and support and reagents from Ellume and Cepheid outside of the submitted work. J.A.E. receives institutional funding from AstraZeneca, Merck, GlaxoSmithKline, and Pfizer. She is a consultant for Abbvie, AstraZeneca, GlaxoSmithKline, Meissa Vaccines, Moderna, Pfizer, and SanofiPasteur. A.L.G. reports contract testing from Abbott, Cepheid, Novavax, Pfizer, Janssen, and Hologic and research support from Gilead outside of the submitted work., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Lassa virus is estimated to cause thousands of human deaths per year, primarily due to spillovers from its natural host, Mastomys rodents. Efforts to create vaccines and antibody therapeutics must account for the evolutionary variability of Lassa virus's glycoprotein complex (GPC), which mediates viral entry into cells and is the target of neutralizing antibodies. To map the evolutionary space accessible to GPC, we use pseudovirus deep mutational scanning to measure how nearly all GPC amino-acid mutations affect cell entry and antibody neutralization. Our experiments define functional constraints throughout GPC. We quantify how GPC mutations affect neutralization by a panel of monoclonal antibodies and show that all antibodies are escaped by mutations that exist among natural Lassa virus lineages. Overall, our work describes a biosafety-level-2 method to elucidate the mutational space accessible to GPC and shows how prospective characterization of antigenic variation could aid design of therapeutics and vaccines., Competing Interests: Competing interests J.D.B. is on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, and the Vaccine Company. K.G.A. is a consultant to, and on the scientific advisory board of, Invivyd. J.D.B. and K.H.D.C. receive royalty payments as inventors on Fred Hutch licensed patents related to viral deep mutational scanning. N.P.K. is a co-founder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. The King lab has received unrelated sponsored research agreements from Pfizer and GSK.

Human influenza virus evolves to escape neutralization by polyclonal antibodies. However, we have a limited understanding of how the antigenic effects of viral mutations vary across the human population, and how this heterogeneity affects virus evolution. Here we use deep mutational scanning to map how mutations to the hemagglutinin (HA) proteins of the A/Hong Kong/45/2019 (H3N2) and A/Perth/16/2009 (H3N2) strains affect neutralization by serum from individuals of a variety of ages. The effects of HA mutations on serum neutralization differ across age groups in ways that can be partially rationalized in terms of exposure histories. Mutations that fixed in influenza variants after 2020 cause the greatest escape from sera from younger individuals. Overall, these results demonstrate that influenza faces distinct antigenic selection regimes from different age groups, and suggest approaches to understand how this heterogeneous selection shapes viral evolution., Competing Interests: Competing Interests JDB is on the scientific advisory boards of Apriori Bio, Invivyd, Aerium Therapeutics, and the Vaccine Company. JDB, ANL, and FCW receive royalty payments as inventors on Fred Hutch licensed patents related to viral deep mutational scanning. HYC reports consulting with Ellume, Pfizer, and the Bill and Melinda Gates Foundation. She has served on advisory boards for Vir, Merck and Abbvie. She has conducted CME teaching with Medscape, Vindico, and Clinical Care Options. She has received research funding from Gates Ventures, and support and reagents from Ellume and Cepheid outside of the submitted work. JAE receives institutional funding from AstraZeneca, Merck, GlaxoSmithKline, and Pfizer. She is a consultant for Abbvie, Astrazeneca, GlaxoSmithKline, Meissa Vaccines, Moderna, Pfizer, and SanofiPasteur. ALG reports contract testing from Abbott, Cepheid, Novavax, Pfizer, Janssen, and Hologic and research support from Gilead outside of the submitted work.

Despite transformative advances in protein design with deep learning, the design of small-molecule-binding proteins and sensors for arbitrary ligands remains a grand challenge. Here we combine deep learning and physics-based methods to generate a family of proteins with diverse and designable pocket geometries, which we employ to computationally design binders for six chemically and structurally distinct small-molecule targets. Biophysical characterization of the designed binders revealed nanomolar to low micromolar binding affinities and atomic-level design accuracy. The bound ligands are exposed at one edge of the binding pocket, enabling the de novo design of chemically induced dimerization (CID) systems; we take advantage of this to create a biosensor with nanomolar sensitivity for cortisol. Our approach provides a general method to design proteins that bind and sense small molecules for a wide range of analytical, environmental, and biomedical applications.

Sequence-specific DNA-binding proteins (DBPs) play critical roles in biology and biotechnology, and there has been considerable interest in the engineering of DBPs with new or altered specificities for genome editing and other applications. While there has been some success in reprogramming naturally occurring DBPs using selection methods, the computational design of new DBPs that recognize arbitrary target sites remains an outstanding challenge. We describe a computational method for the design of small DBPs that recognize specific target sequences through interactions with bases in the major groove, and employ this method in conjunction with experimental screening to generate binders for 5 distinct DNA targets. These binders exhibit specificity closely matching the computational models for the target DNA sequences at as many as 6 base positions and affinities as low as 30-100 nM. The crystal structure of a designed DBP-target site complex is in close agreement with the design model, highlighting the accuracy of the design method. The designed DBPs function in both Escherichia coli and mammalian cells to repress and activate transcription of neighboring genes. Our method is a substantial step towards a general route to small and hence readily deliverable sequence-specific DBPs for gene regulation and editing., Competing Interests: Competing interests. C.G., R.P., R.M., C.N., F.D., and D.B. are co-inventors on a provisional patent application that incorporates discoveries described in this manuscript.

Deep mutational scanning (DMS) is a high-throughput experimental technique that measures the effects of thousands of mutations to a protein. These experiments can be performed on multiple homologs of a protein or on the same protein selected under multiple conditions. It is often of biological interest to identify mutations with shifted effects across homologs or conditions. However, it is challenging to determine if observed shifts arise from biological signal or experimental noise. Here, we describe a method for jointly inferring mutational effects across multiple DMS experiments while also identifying mutations that have shifted in their effects among experiments. A key aspect of our method is to regularize the inferred shifts, so that they are nonzero only when strongly supported by the data. We apply this method to DMS experiments that measure how mutations to spike proteins from SARS-CoV-2 variants (Delta, Omicron BA.1, and Omicron BA.2) affect cell entry. Most mutational effects are conserved between these spike homologs, but a fraction have markedly shifted. We experimentally validate a subset of the mutations inferred to have shifted effects, and confirm differences of > 1,000-fold in the impact of the same mutation on spike-mediated viral infection across spikes from different SARS-CoV-2 variants. Overall, our work establishes a general approach for comparing sets of DMS experiments to identify biologically important shifts in mutational effects.

The de novo design of three protein chains that associate to form a heterotrimer (but not any of the possible two-chain heterodimers) and that can drive the assembly of higher-order branching structures is an important challenge for protein design. We designed helical heterotrimers with specificity conferred by buried hydrogen bond networks and large aromatic residues to enhance shape complementary packing. We obtained ten designs for which all three chains cooperatively assembled into heterotrimers with few or no other species present. Crystal structures of a helical bundle heterotrimer and extended versions, with helical repeat proteins fused to individual subunits, showed all three chains assembling in the designed orientation. We used these heterotrimers as building blocks to construct larger cyclic oligomers, which were structurally validated by electron microscopy. Our three-way junction designs provide new routes to complex protein nanostructures and enable the scaffolding of three distinct ligands for modulation of cell signaling., (© 2022. The Author(s).)

Designing entirely new protein structures remains challenging because we do not fully understand the biophysical determinants of folding stability. Yet, some protein folds are easier to design than others. Previous work identified the 43-residue ɑββɑ fold as especially challenging: The best designs had only a 2% success rate, compared to 39 to 87% success for other simple folds [G. J. Rocklin et al., Science 357, 168-175 (2017)]. This suggested the ɑββɑ fold would be a useful model system for gaining a deeper understanding of folding stability determinants and for testing new protein design methods. Here, we designed over 10,000 new ɑββɑ proteins and found over 3,000 of them to fold into stable structures using a high-throughput protease-based assay. NMR, hydrogen-deuterium exchange, circular dichroism, deep mutational scanning, and scrambled sequence control experiments indicated that our stable designs fold into their designed ɑββɑ structures with exceptional stability for their small size. Our large dataset enabled us to quantify the influence of universal stability determinants including nonpolar burial, helix capping, and buried unsatisfied polar atoms, as well as stability determinants unique to the ɑββɑ topology. Our work demonstrates how large-scale design and test cycles can solve challenging design problems while illuminating the biophysical determinants of folding.

Antibodies, and antibody derivatives such as nanobodies, contain immunoglobulin-like (Ig) β-sandwich scaffolds which anchor the hypervariable antigen-binding loops and constitute the largest growing class of drugs. Current engineering strategies for this class of compounds rely on naturally existing Ig frameworks, which can be hard to modify and have limitations in manufacturability, designability and range of action. Here, we develop design rules for the central feature of the Ig fold architecture-the non-local cross-β structure connecting the two β-sheets-and use these to design highly stable Ig domains de novo, confirm their structures through X-ray crystallography, and show they can correctly scaffold functional loops. Our approach opens the door to the design of antibody-like scaffolds with tailored structures and superior biophysical properties., (© 2022. The Author(s).)

Engineered proteins generally must possess a stable structure in order to achieve their designed function. Stable designs, however, are astronomically rare within the space of all possible amino acid sequences. As a consequence, many designs must be tested computationally and experimentally in order to find stable ones, which is expensive in terms of time and resources. Here we use a high-throughput, low-fidelity assay to experimentally evaluate the stability of approximately 200,000 novel proteins. These include a wide range of sequence perturbations, providing a baseline for future work in the field. We build a neural network model that predicts protein stability given only sequences of amino acids, and compare its performance to the assayed values. We also report another network model that is able to generate the amino acid sequences of novel stable proteins given requested secondary sequences. Finally, we show that the predictive model-despite weaknesses including a noisy data set-can be used to substantially increase the stability of both expert-designed and model-generated proteins., Competing Interests: JMS and AZ are employed by Two Six Technologies, which has filed a patent on a portion of the technology described in this manuscript. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Predicting protein stability is a challenge due to the many competing thermodynamic effects. Through de novo protein design, one begins with a target structure and searches for a sequence that will fold into it. Previous work by Rocklin et al. introduced a data set of more than 16,000 miniproteins spanning four structural topologies with information on stability. These structures were characterized with a set of 46 structural descriptors, with no explicit inclusion of configurational entropy ( S cnf ). Our work focused on creating a set of 17 descriptors intended to capture variations in S cnf and its comparison to an extended set of 113 structural and energy model features that extend the Rocklin et al. feature set (R). The S cnf descriptors statistically discriminate between stable and unstable distributions within topologies and best describe EEHEE topology stability (where E = β sheet and H = α helix). Between 50 and 80% of the variation in each S cnf descriptor is described by linear combinations of R features. Despite containing useful information about minipeptide stability, providing S cnf features as inputs to machine learning models does not improve overall performance when predicting protein stability, as the R features sufficiently capture the implicit variations.

The FastDesign protocol in the molecular modeling program Rosetta iterates between sequence optimization and structure refinement to stabilize de novo designed protein structures and complexes. FastDesign has been used previously to design novel protein folds and assemblies with important applications in research and medicine. To promote sampling of alternative conformations and sequences, FastDesign includes stages where the energy landscape is smoothened by reducing repulsive forces. Here, we discover that this process disfavors larger amino acids in the protein core because the protein compresses in the early stages of refinement. By testing alternative ramping strategies for the repulsive weight, we arrive at a scheme that produces lower energy designs with more native-like sequence composition in the protein core. We further validate the protocol by designing and experimentally characterizing over 4000 proteins and show that the new protocol produces higher stability proteins., (© 2020 Wiley Periodicals LLC.)

Eliciting broadly neutralizing antibodies (bnAbs) targeting envelope (Env) is a major goal of HIV vaccine development, but cross-clade breadth from immunization has only sporadically been observed. Recently, Xu et al (2018) elicited cross-reactive neutralizing antibody responses in a variety of animal models using immunogens based on the epitope of bnAb VRC34.01. The VRC34.01 antibody, which was elicited by natural human infection, targets the N terminus of the Env fusion peptide, a critical component of the virus entry machinery. Here we precisely characterize the functional epitopes of VRC34.01 and two vaccine-elicited murine antibodies by mapping all single amino-acid mutations to the BG505 Env that affect viral neutralization. While escape from VRC34.01 occurred via mutations in both fusion peptide and distal interacting sites of the Env trimer, escape from the vaccine-elicited antibodies was mediated predominantly by mutations in the fusion peptide. Cryo-electron microscopy of four vaccine-elicited antibodies in complex with Env trimer revealed focused recognition of the fusion peptide and provided a structural basis for development of neutralization breadth. Together, these functional and structural data suggest that the breadth of vaccine-elicited antibodies targeting the fusion peptide can be enhanced by specific interactions with additional portions of Env. Thus, our complete maps of viral escape both delineate pathways of resistance to these fusion peptide-directed antibodies and provide a strategy to improve the breadth or potency of future vaccine-induced antibodies against Env's fusion peptide., Competing Interests: The authors have declared that no competing interests exist.

The immediate evolutionary space accessible to HIV is largely determined by how single amino acid mutations affect fitness. These mutational effects can shift as the virus evolves. However, the prevalence of such shifts in mutational effects remains unclear. Here, we quantify the effects on viral growth of all amino acid mutations to two HIV envelope (Env) proteins that differ at [Formula: see text]100 residues. Most mutations similarly affect both Envs, but the amino acid preferences of a minority of sites have clearly shifted. These shifted sites usually prefer a specific amino acid in one Env, but tolerate many amino acids in the other. Surprisingly, shifts are only slightly enriched at sites that have substituted between the Envs-and many occur at residues that do not even contact substitutions. Therefore, long-range epistasis can unpredictably shift Env's mutational tolerance during HIV evolution, although the amino acid preferences of most sites are conserved between moderately diverged viral strains., Competing Interests: HH, AD, SH, JO, JB No competing interests declared, (© 2018, Haddox et al.)

Precisely defining how viral mutations affect HIV's sensitivity to antibodies is vital to develop and evaluate vaccines and antibody immunotherapeutics. Despite great effort, a full map of escape mutants has not been delineated for an anti-HIV antibody. We describe a massively parallel experimental approach to quantify how all single amino acid mutations to HIV Envelope (Env) affect neutralizing antibody sensitivity in the context of replication-competent virus. We apply this approach to PGT151, a broadly neutralizing antibody recognizing a combination of Env residues and glycans. We confirm sites previously defined by structural and functional studies and reveal additional sites of escape, such as positively charged mutations in the antibody-Env interface. Evaluating the effect of each amino acid at each site lends insight into biochemical mechanisms of escape throughout the epitope, highlighting roles for charge-charge repulsions. Thus, comprehensively mapping HIV antibody escape gives a quantitative, mutation-level view of Env evasion of neutralization., (Copyright © 2017 Elsevier Inc. All rights reserved.)

HIV is notorious for its capacity to evade immunity and anti-viral drugs through rapid sequence evolution. Knowledge of the functional effects of mutations to HIV is critical for understanding this evolution. HIV's most rapidly evolving protein is its envelope (Env). Here we use deep mutational scanning to experimentally estimate the effects of all amino-acid mutations to Env on viral replication in cell culture. Most mutations are under purifying selection in our experiments, although a few sites experience strong selection for mutations that enhance HIV's replication in cell culture. We compare our experimental measurements of each site's preference for each amino acid to the actual frequencies of these amino acids in naturally occurring HIV sequences. Our measured amino-acid preferences correlate with amino-acid frequencies in natural sequences for most sites. However, our measured preferences are less concordant with natural amino-acid frequencies at surface-exposed sites that are subject to pressures absent from our experiments such as antibody selection. Our data enable us to quantify the inherent mutational tolerance of each site in Env. We show that the epitopes of broadly neutralizing antibodies have a significantly reduced inherent capacity to tolerate mutations, rigorously validating a pervasive idea in the field. Overall, our results help disentangle the role of inherent functional constraints and external selection pressures in shaping Env's evolution., Competing Interests: The authors have declared that no competing interests exist.