Identification of a Permissive Secondary Mutation That Restores the Enzymatic Activity of Oseltamivir Resistance Mutation H275Y.

Many oseltamivir resistance mutations exhibit fitness defects in the absence of drug pressure that hinders their propagation in hosts. Secondary permissive mutations can rescue fitness defects and facilitate the segregation of resistance mutations in viral populations. Previous studies have identified a panel of permissive or compensatory mutations in neuraminidase (NA) that restore the growth defect of the predominant oseltamivir resistance mutation (H275Y) in H1N1 influenza A virus. In prior work, we identified a hyperactive mutation (Y276F) that increased NA activity by approximately 70%. While Y276F had not been previously identified as a permissive mutation, we hypothesized that Y276F may counteract the defects caused by H275Y by buffering its reduced NA expression and enzyme activity. In this study, we measured the relative fitness, NA activity, and surface expression, as well as sensitivity to oseltamivir, for several oseltamivir resistance mutations, including H275Y in the wild-type and Y276F genetic background. Our results demonstrate that Y276F selectively rescues the fitness defect of H275Y by restoring its NA surface expression and enzymatic activity, elucidating the local compensatory structural impacts of Y276F on the adjacent H275Y. IMPORTANCE The potential for influenza A virus (IAV) to cause pandemics makes understanding evolutionary mechanisms that impact drug resistance critical for developing surveillance and treatment strategies. Oseltamivir is the most widely used therapeutic strategy to treat IAV infections, but mutations in IAV can lead to drug resistance. The main oseltamivir resistance mutation, H275Y, occurs in the neuraminidase (NA) protein of IAV and reduces drug binding as well as NA function. Here, we identified a new helper mutation, Y276F, that can rescue the functional defects of H275Y and contribute to the evolution of drug resistance in IAV.

The Adaptive Potential of the Middle Domain of Yeast Hsp90.

The distribution of fitness effects (DFEs) of new mutations across different environments quantifies the potential for adaptation in a given environment and its cost in others. So far, results regarding the cost of adaptation across environments have been mixed, and most studies have sampled random mutations across different genes. Here, we quantify systematically how costs of adaptation vary along a large stretch of protein sequence by studying the distribution of fitness effects of the same ≈2,300 amino-acid changing mutations obtained from deep mutational scanning of 119 amino acids in the middle domain of the heat shock protein Hsp90 in five environments. This region is known to be important for client binding, stabilization of the Hsp90 dimer, stabilization of the N-terminal-Middle and Middle-C-terminal interdomains, and regulation of ATPase-chaperone activity. Interestingly, we find that fitness correlates well across diverse stressful environments, with the exception of one environment, diamide. Consistent with this result, we find little cost of adaptation; on average only one in seven beneficial mutations is deleterious in another environment. We identify a hotspot of beneficial mutations in a region of the protein that is located within an allosteric center. The identified protein regions that are enriched in beneficial, deleterious, and costly mutations coincide with residues that are involved in the stabilization of Hsp90 interdomains and stabilization of client-binding interfaces, or residues that are involved in ATPase-chaperone activity of Hsp90. Thus, our study yields information regarding the role and adaptive potential of a protein sequence that complements and extends known structural information.

Designed Hsp90 heterodimers reveal an asymmetric ATPase-driven mechanism in vivo.

Hsp90 is a homodimeric ATPase that is essential in eukaryotes for the maturation of client proteins frequently involved in signal transduction, including many kinases and nuclear steroid hormone receptors. Competitive inhibitors of ATP binding to Hsp90 prevent client maturation and show promise as anticancer agents in clinical trials. However, the role of ATP binding and hydrolysis in each subunit of the Hsp90 dimer has been difficult to investigate because of an inability to assemble and study dimers of defined composition. We used protein engineering to generate functional Hsp90 subunits that preferentially assemble as heterodimers. We analyzed dimers wherein one subunit harbors a disruptive mutation and observed that ATP binding by both subunits is essential for function in yeast, whereas ATP hydrolysis is only required in one subunit. These findings demonstrate important functional contributions from both symmetric and asymmetric Hsp90 dimers and provide valuable reagents for future investigations of Hsp90 mechanism.

Pervasive contingency and entrenchment in a billion years of Hsp90 evolution.

Interactions among mutations within a protein have the potential to make molecular evolution contingent and irreversible, but the extent to which epistasis actually shaped historical evolutionary trajectories is unclear. To address this question, we experimentally measured how the fitness effects of historical sequence substitutions changed during the billion-year evolutionary history of the heat shock protein 90 (Hsp90) ATPase domain beginning from a deep eukaryotic ancestor to modern Saccharomyces cerevisiae We found a pervasive influence of epistasis. Of 98 derived amino acid states that evolved along this lineage, about half compromise fitness when introduced into the reconstructed ancestral Hsp90. And the vast majority of ancestral states reduce fitness when introduced into the extant S. cerevisiae Hsp90. Overall, more than 75% of historical substitutions were contingent on permissive substitutions that rendered the derived state nondeleterious, became entrenched by subsequent restrictive substitutions that made the ancestral state deleterious, or both. This epistasis was primarily caused by specific interactions among sites rather than a general effect on the protein's tolerance to mutation. Our results show that epistasis continually opened and closed windows of mutational opportunity over evolutionary timescales, producing histories and biological states that reflect the transient internal constraints imposed by the protein's fleeting sequence states.

Constrained Mutational Sampling of Amino Acids in HIV-1 Protease Evolution.

The evolution of HIV-1 protein sequences should be governed by a combination of factors including nucleotide mutational probabilities, the genetic code, and fitness. The impact of these factors on protein sequence evolution is interdependent, making it challenging to infer the individual contribution of each factor from phylogenetic analyses alone. We investigated the protein sequence evolution of HIV-1 by determining an experimental fitness landscape of all individual amino acid changes in protease. We compared our experimental results to the frequency of protease variants in a publicly available data set of 32,163 sequenced isolates from drug-naïve individuals. The most common amino acids in sequenced isolates supported robust experimental fitness, indicating that the experimental fitness landscape captured key features of selection acting on protease during viral infections of hosts. Amino acid changes requiring multiple mutations from the likely ancestor were slightly less likely to support robust experimental fitness than single mutations, consistent with the genetic code favoring chemically conservative amino acid changes. Amino acids that were common in sequenced isolates were predominantly accessible by single mutations from the likely protease ancestor. Multiple mutations commonly observed in isolates were accessible by mutational walks with highly fit single mutation intermediates. Our results indicate that the prevalence of multiple-base mutations in HIV-1 protease is strongly influenced by mutational sampling.

Mutations in Influenza A Virus Neuraminidase and Hemagglutinin Confer Resistance against a Broadly Neutralizing Hemagglutinin Stem Antibody.

Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.

CRISPR-Cas9-mediated saturated mutagenesis screen predicts clinical drug resistance with improved accuracy.

Developing tools to accurately predict the clinical prevalence of drug-resistant mutations is a key step toward generating more effective therapeutics. Here we describe a high-throughput CRISPR-Cas9-based saturated mutagenesis approach to generate comprehensive libraries of point mutations at a defined genomic location and systematically study their effect on cell growth. As proof of concept, we mutagenized a selected region within the leukemic oncogene BCR-ABL1 Using bulk competitions with a deep-sequencing readout, we analyzed hundreds of mutations under multiple drug conditions and found that the effects of mutations on growth in the presence or absence of drug were critical for predicting clinically relevant resistant mutations, many of which were cancer adaptive in the absence of drug pressure. Using this approach, we identified all clinically isolated BCR-ABL1 mutations and achieved a prediction score that correlated highly with their clinical prevalence. The strategy described here can be broadly applied to a variety of oncogenes to predict patient mutations and evaluate resistance susceptibility in the development of new therapeutics.

Molecular Determinants of Epistasis in HIV-1 Protease: Elucidating the Interdependence of L89V and L90M Mutations in Resistance.

Protease inhibitors have the highest potency among antiviral therapies against HIV-1 infections, yet the virus can evolve resistance. Darunavir (DRV), currently the most potent Food and Drug Administration-approved protease inhibitor, retains potency against single-site mutations. However, complex combinations of mutations can confer resistance to DRV. While the interdependence between mutations within HIV-1 protease is key for inhibitor potency, the molecular mechanisms that underlie this control remain largely unknown. In this study, we investigated the interdependence between the L89V and L90M mutations and their effects on DRV binding. These two mutations have been reported to be positively correlated with one another in HIV-1 patient-derived protease isolates, with the presence of one mutation making the probability of the occurrence of the second mutation more likely. The focus of our investigation is a patient-derived isolate, with 24 mutations that we call "KY"; this variant includes the L89V and L90M mutations. Three additional KY variants with back-mutations, KY(V89L), KY(M90L), and the KY(V89L/M90L) double mutation, were used to experimentally assess the individual and combined effects of these mutations on DRV inhibition and substrate processing. The enzymatic assays revealed that the KY(V89L) variant, with methionine at residue 90, is highly resistant, but its catalytic function is compromised. When a leucine to valine mutation at residue 89 is present simultaneously with the L90M mutation, a rescue of catalytic efficiency is observed. Molecular dynamics simulations of these DRV-bound protease variants reveal how the L90M mutation induces structural changes throughout the enzyme that undermine the binding interactions.

Influenza virus drug resistance: a time-sampled population genetics perspective.

The challenge of distinguishing genetic drift from selection remains a central focus of population genetics. Time-sampled data may provide a powerful tool for distinguishing these processes, and we here propose approximate Bayesian, maximum likelihood, and analytical methods for the inference of demography and selection from time course data. Utilizing these novel statistical and computational tools, we evaluate whole-genome datasets of an influenza A H1N1 strain in the presence and absence of oseltamivir (an inhibitor of neuraminidase) collected at thirteen time points. Results reveal a striking consistency amongst the three estimation procedures developed, showing strongly increased selection pressure in the presence of drug treatment. Importantly, these approaches re-identify the known oseltamivir resistance site, successfully validating the approaches used. Enticingly, a number of previously unknown variants have also been identified as being positively selected. Results are interpreted in the light of Fisher's Geometric Model, allowing for a quantification of the increased distance to optimum exerted by the presence of drug, and theoretical predictions regarding the distribution of beneficial fitness effects of contending mutations are empirically tested. Further, given the fit to expectations of the Geometric Model, results suggest the ability to predict certain aspects of viral evolution in response to changing host environments and novel selective pressures.

A systematic survey of an intragenic epistatic landscape.

Mutations are the source of evolutionary variation. The interactions of multiple mutations can have important effects on fitness and evolutionary trajectories. We have recently described the distribution of fitness effects of all single mutations for a nine-amino-acid region of yeast Hsp90 (Hsp82) implicated in substrate binding. Here, we report and discuss the distribution of intragenic epistatic effects within this region in seven Hsp90 point mutant backgrounds of neutral to slightly deleterious effect, resulting in an analysis of more than 1,000 double mutants. We find negative epistasis between substitutions to be common, and positive epistasis to be rare--resulting in a pattern that indicates a drastic change in the distribution of fitness effects one step away from the wild type. This can be well explained by a concave relationship between phenotype and genotype (i.e., a concave shape of the local fitness landscape), suggesting mutational robustness intrinsic to the local sequence space. Structural analyses indicate that, in this region, epistatic effects are most pronounced when a solvent-inaccessible position is involved in the interaction. In contrast, all 18 observations of positive epistasis involved at least one mutation at a solvent-exposed position. By combining the analysis of evolutionary and biophysical properties of an epistatic landscape, these results contribute to a more detailed understanding of the complexity of protein evolution.

Positive Selection Drives Preferred Segment Combinations during Influenza Virus Reassortment.

Influenza A virus (IAV) has a segmented genome that allows for the exchange of genome segments between different strains. This reassortment accelerates evolution by breaking linkage, helping IAV cross species barriers to potentially create highly virulent strains. Challenges associated with monitoring the process of reassortment in molecular detail have limited our understanding of its evolutionary implications. We applied a novel deep sequencing approach with quantitative analysis to assess the in vitro temporal evolution of genomic reassortment in IAV. The combination of H1N1 and H3N2 strains reproducibly generated a new H1N2 strain with the hemagglutinin and nucleoprotein segments originating from H1N1 and the remaining six segments from H3N2. By deep sequencing the entire viral genome, we monitored the evolution of reassortment, quantifying the relative abundance of all IAV genome segments from the two parent strains over time and measuring the selection coefficients of the reassorting segments. Additionally, we observed several mutations coemerging with reassortment that were not found during passaging of pure parental IAV strains. Our results demonstrate how reassortment of the segmented genome can accelerate viral evolution in IAV, potentially enabled by the emergence of a small number of individual mutations.

Latent effects of Hsp90 mutants revealed at reduced expression levels.

In natural systems, selection acts on both protein sequence and expression level, but it is unclear how selection integrates over these two dimensions. We recently developed the EMPIRIC approach to systematically determine the fitness effects of all possible point mutants for important regions of essential genes in yeast. Here, we systematically investigated the fitness effects of point mutations in a putative substrate binding loop of yeast Hsp90 (Hsp82) over a broad range of expression strengths. Negative epistasis between reduced expression strength and amino acid substitutions was common, and the endogenous expression strength frequently obscured mutant defects. By analyzing fitness effects at varied expression strengths, we were able to uncover all mutant effects on function. The majority of mutants caused partial functional defects, consistent with this region of Hsp90 contributing to a mutation sensitive and critical process. These results demonstrate that important functional regions of proteins can tolerate mutational defects without experimentally observable impacts on fitness.

Evolution of the influenza A virus genome during development of oseltamivir resistance in vitro.

Influenza A virus (IAV) is a major cause of morbidity and mortality throughout the world. Current antiviral therapies include oseltamivir, a neuraminidase inhibitor that prevents the release of nascent viral particles from infected cells. However, the IAV genome can evolve rapidly, and oseltamivir resistance mutations have been detected in numerous clinical samples. Using an in vitro evolution platform and whole-genome population sequencing, we investigated the population genomics of IAV during the development of oseltamivir resistance. Strain A/Brisbane/59/2007 (H1N1) was grown in Madin-Darby canine kidney cells with or without escalating concentrations of oseltamivir over serial passages. Following drug treatment, the H274Y resistance mutation fixed reproducibly within the population. The presence of the H274Y mutation in the viral population, at either a low or a high frequency, led to measurable changes in the neuraminidase inhibition assay. Surprisingly, fixation of the resistance mutation was not accompanied by alterations of viral population diversity or differentiation, and oseltamivir did not alter the selective environment. While the neighboring K248E mutation was also a target of positive selection prior to H274Y fixation, H274Y was the primary beneficial mutation in the population. In addition, once evolved, the H274Y mutation persisted after the withdrawal of the drug, even when not fixed in viral populations. We conclude that only selection of H274Y is required for oseltamivir resistance and that H274Y is not deleterious in the absence of the drug. These collective results could offer an explanation for the recent reproducible rise in oseltamivir resistance in seasonal H1N1 IAV strains in humans.

Experimental illumination of a fitness landscape.

The genes of all organisms have been shaped by selective pressures. The relationship between gene sequence and fitness has tremendous implications for understanding both evolutionary processes and functional constraints on the encoded proteins. Here, we have exploited deep sequencing technology to experimentally determine the fitness of all possible individual point mutants under controlled conditions for a nine-amino acid region of Hsp90. Over the past five decades, limited glimpses into the relationship between gene sequence and function have sparked a long debate regarding the distribution, relative proportion, and evolutionary significance of deleterious, neutral, and advantageous mutations. Our systematic experimental measurement of fitness effects of Hsp90 mutants in yeast, evaluated in the light of existing population genetic theory, are remarkably consistent with a nearly neutral model of molecular evolution.

Dominant negative mutations in yeast Hsp90 reveal triage decision mechanism targeting client proteins for degradation.

Most of the fundamental processes of cells are mediated by proteins. However, the biologically-relevant mechanism of most proteins are poorly understood. Dominant negative mutations have provided a valuable tool for investigating protein mechanisms but can be difficult to isolate because of their toxic effects. We used a mutational scanning approach to identify dominant negative mutations in yeast Hsp90. Hsp90 is a chaperone that forms dynamic complexes with many co-chaperones and client proteins. In vitro analyses have elucidated some key biochemical states and structures of Hsp90, co-chaperones, and clients; however, the biological mechanism of Hsp90 remains unclear. For example, high throughput studies have found that many E3 ubiquitin ligases bind to Hsp90, but it is unclear if these are primarily clients or acting to tag other clients for degradation. We introduced a library of all point mutations in the ATPase domain of Hsp90 into yeast and noticed that 176 were more than 10-fold depleted at the earliest point that we could analyze. There were two hot spot regions of the depleted mutations that were located at the hinges of a loop that closes over ATP. We quantified the dominant negative growth effects of mutations in the hinge regions using a library of mutations driven by an inducible promoter. We analyzed individual dominant negative mutations in detail and found that addition of the E33A mutation that prevents ATP hydrolysis by Hsp90 abrogated the dominant negative phenotype. Pull-down experiments did not reveal any stable binding partners, indicating that the dominant effects were mediated by dynamic complexes. DN Hsp90 decreased the expression level of two model Hsp90 clients, glucocorticoid receptor (GR) and v-src kinase. Using MG132, we found that GR was rapidly destabilized in a proteasome-dependent fashion. These findings provide evidence that the binding of E3 ligases to Hsp90 may serve a quality control function fundamental to eukaryotes.

Contributions of Hyperactive Mutations in Mpro from SARS-CoV-2 to Drug Resistance.

The appearance and spread of mutations that cause drug resistance in rapidly evolving diseases, including infections by the SARS-CoV-2 virus, are major concerns for human health. Many drugs target enzymes, and resistance-conferring mutations impact inhibitor binding or enzyme activity. Nirmatrelvir, the most widely used inhibitor currently used to treat SARS-CoV-2 infections, targets the main protease (Mpro) preventing it from processing the viral polyprotein into active subunits. Our previous work systematically analyzed resistance mutations in Mpro that reduce binding to inhibitors; here, we investigate mutations that affect enzyme function. Hyperactive mutations that increase Mpro activity can contribute to drug resistance but have not been thoroughly studied. To explore how hyperactive mutations contribute to resistance, we comprehensively assessed how all possible individual mutations in Mpro affect enzyme function using a mutational scanning approach with a fluorescence resonance energy transfer (FRET)-based yeast readout. We identified hundreds of mutations that significantly increased the Mpro activity. Hyperactive mutations occurred both proximal and distal to the active site, consistent with protein stability and/or dynamics impacting activity. Hyperactive mutations were observed 3 times more than mutations which reduced apparent binding to nirmatrelvir in recent studies of laboratory-grown viruses selected for drug resistance. Hyperactive mutations were also about three times more prevalent than nirmatrelvir binding mutations in sequenced isolates from circulating SARS-CoV-2. Our findings indicate that hyperactive mutations are likely to contribute to the natural evolution of drug resistance in Mpro and provide a comprehensive list for future surveillance efforts.

Solubility-promoting function of Hsp90 contributes to client maturation and robust cell growth.

The Hsp90 chaperone is required for the maturation of signal transduction clients, including many kinases and nuclear steroid hormone receptors. The binding and hydrolysis of ATP by Hsp90 drive conformational rearrangements in three structure domains. Two intrinsically disordered regions of Hsp90 located between these domains and at the C terminus have traditionally been considered to impart flexibility. We discovered that the charged nature of these acid-rich disordered regions imparts a solubility-promoting function to Hsp90 that is important for its cellular activity in yeast. Both the solubility-promoting function and ATPase activity must occur in the same Hsp90 molecule in order to support robust growth, suggesting that the solubility-promoting function is required during the ATP-driven client maturation process. Expression of model clients together with Hsp90 variants indicated interdependent solubilities mediated by the aggregation propensities of both the client and Hsp90. We propose a model whereby the charge-rich disordered regions of Hsp90 serve a solubility-promoting function important for complexes with aggregation-prone clients. These findings demonstrate a novel biological function of the intrinsically disordered regions in Hsp90 and provide a compelling rationale for why their charged properties are conserved throughout eukaryotic evolution.

Systematic profiling of dominant ubiquitin variants reveals key functional nodes contributing to evolutionary selection.

Dominant-negative mutations can help to investigate the biological mechanisms and to understand the selective pressures for multifunctional proteins. However, most studies have focused on recessive mutant effects that occur in the absence of a second functional gene copy, which overlooks the fact that most eukaryotic genomes contain more than one copy of many genes. We have identified dominant effects on yeast growth rate among all possible point mutations in ubiquitin expressed alongside a wild-type allele. Our results reveal more than 400 dominant-negative mutations, indicating that dominant-negative effects make a sizable contribution to selection acting on ubiquitin. Cellular and biochemical analyses of individual ubiquitin variants show that dominant-negative effects are explained by varied accumulation of polyubiquitinated cellular proteins and/or defects in conjugation of ubiquitin variants to ubiquitin ligases. Our approach to identify dominant-negative mutations is general and can be applied to other proteins of interest.

Mutational fitness landscape and drug resistance.

Robust technology has been developed to systematically quantify fitness landscapes that provide valuable opportunities to improve our understanding of drug resistance and define new avenues to develop drugs with reduced resistance susceptibility. We outline the critical importance of drug resistance studies and the potential for fitness landscape approaches to contribute to this effort. We describe the major technical advancements in mutational scanning, which is the primary approach used to quantify protein fitness landscapes. There are many complex steps to consider in planning and executing mutational scanning projects including developing a selection scheme, generating mutant libraries, tracking the frequency of variants using next-generation sequencing, and processing and interpreting the data. Key experimental parameters impacting each of these steps are discussed to aid in planning fitness landscape studies. There is a strong need for improved understanding of drug resistance, and fitness landscapes provide a promising new approach.

A complete allosteric map of a GTPase switch in its native cellular network.

Allosteric regulation is central to protein function in cellular networks. A fundamental open question is whether cellular regulation of allosteric proteins occurs only at a few defined positions or at many sites distributed throughout the structure. Here, we probe the regulation of GTPases-protein switches that control signaling through regulated conformational cycling-at residue-level resolution by deep mutagenesis in the native biological network. For the GTPase Gsp1/Ran, we find that 28% of the 4,315 assayed mutations show pronounced gain-of-function responses. Twenty of the sixty positions enriched for gain-of-function mutations are outside the canonical GTPase active site switch regions. Kinetic analysis shows that these distal sites are allosterically coupled to the active site. We conclude that the GTPase switch mechanism is broadly sensitive to cellular allosteric regulation. Our systematic discovery of new regulatory sites provides a functional map to interrogate and target GTPases controlling many essential biological processes.