Biology subjects

Phillips, A. M.

Publications and source records attributed to Phillips, A. M..

At least 19 recordsLinked to original sources

bioRxiv2026

A Selenium-Deficient Mouse Model of Mouse-Adapted SARS-CoV-2 Demonstrates Variant Emergence Observed in SARS-CoV-2 Pandemic Variants

Host selenium deficiency has been shown to generate novel genetic variants in RNA viruses. With the predicted rise of selenium deficiency globally, we sought to determine if host selenium deficiency can be a predictive factor for RNA virus variant emergence. We utilized a selenium-deficient BALB/c mouse model to investigate how host selenium status influences the emergence of viral variants in mouse-adapted SARS-CoV-2. Mice were maintained on control or selenium-deficient diets and subjected to sequential rounds of diet-matched viral passage to generate diet-specific virus populations. Deep sequencing of passaged viral populations revealed that selenium-deficient passage drove a marked increase in inter-host genomic heterogeneity and produced a distinct mutational profile relative to control passage. Eighteen mutations were identified as unique to selenium-deficient passage, including variants previously observed during natural human SARS-CoV-2 evolution. These mutations were largely maintained at sub-consensus frequencies, indicating that selenium deficiency can expand the viral quasispecies landscape that enrichs reservoirs of adaptive potential. To determine how this altered mutant spectrum affected pathogenesis, we challenged normal diet-fed adult and aged BALB/c mice with control- or selenium-deficient-passaged virus. Although overt differences in weight loss, survival, and viral burden were generally modest, selenium-deficient-passaged virus induced pronounced increases in antiviral cytokine expression. Together, these findings identify host selenium deficiency as a driver of RNA virus population diversification and show that nutritionally stressed animal models can reproducibly generate mutations observed in nature.

microbiology↗

bioRxiv2026

Biophysical trade-offs in antibody evolution are resolved by conformation-mediated epistasis

Protein evolution is constrained by multidimensional biophysical factors, in which mutations that enhance one property often compromise another. Antibodies represent an extreme case: they evolve rapidly to bind diverse antigens, yet mutations that improve affinity can disrupt folding, reduce cell-surface trafficking, or promote self-reactivity, and are typically selected against during affinity maturation. Though biophysical characterization of individual antibodies suggests that such trade-offs are pervasive, their impact on antibody evolutionary trajectories remains unclear, in part because existing high-throughput biophysical methods rely on heterologous systems that are often poorly suited for human proteins. Here, we develop a high-throughput platform to quantify multiple biophysical parameters of large libraries of full-length proteins that are natively synthesized, processed, and displayed on human cells. We apply this approach to a human antibody lineage that matures to recognize divergent SARS-CoV-2 variants by measuring the surface expression, antigen affinity, and self-reactivity for all 213 possible evolutionary intermediates between the unmutated and mature sequences. These measurements reveal that mutations differentially affect these biophysical properties - in some cases, improving one property at the expense of another. We leverage these data to compute the likelihood of all possible evolutionary paths, finding that very few paths can navigate these multidimensional requirements. The few accessible paths acquire mutations in a specific order that either circumvent trade-offs between biophysical properties or offset deleterious effects on one property with beneficial effects on another. By determining the structures of the ancestral and evolved antibodies, we find that these coordinated mutational effects arise from a conformational rearrangement that alleviates steric clashes and reshapes the biophysical landscape, enabling otherwise inaccessible mutational paths. Together, this work defines the multidimensional biophysical constraints and structural mechanisms that govern antibody evolution and establishes a general framework for mapping and predicting the biophysical effects of mutations in human proteins.

evolutionary biology↗

bioRxiv2025

Parallel but distinct adaptive routes in the budding and fission yeasts after 10,000 generations of experimental evolution

Quantitative genetics approaches designed to understand the evolution of traits have helped improve our understanding of the genetic basis of adaptation. However, they often overlook crucial aspects of adaptation, including the long-term temporal evolutionary dynamics, the predictability of evolutionary outcomes, the influence of past evolution on future evolutionary trajectories (contingency), and the diversity of molecular mechanisms underlying adaptation. Experimental evolution has been a useful tool for answering these questions, but extracting fundamental principles and predictive features of evolutionary outcomes from these datasets remains challenging due to the large number of covariates and confounding effects, such as differences in experimental setup, species lifestyle, gene content, and evolution rate. Here, we sought to circumvent these challenges by comparing distant yeast species that share several evolutionary features but differ mainly in evolutionary history and genome architecture, i.e. Saccharomyces cerevisiae and Schizosaccharomyces pombe. Thus, we evolved 10 populations of the fission yeast for 10,000 generations in the same conditions as a pre-existing budding yeast dataset (i.e. high-sugar media and hypoxic conditions), allowing us to observe repeatable evolutionary outcomes within species but diverse molecular mechanisms and targets of adaptation across species. The most frequent adapting route in these conditions involved upregulating fermentation genes and downregulating the glycolysis gene pyk1, which has not previously been observed in S. cerevisiae evolved populations or in wild Kluyveromyces lactis, but similar evolutionary paths have been observed in Schizosaccharomyces japonicus and in clinically relevant populations, such as some cancer cells. This suggests that parallelism is pervasive in the tree of life and that mechanisms of adaptation can be shared among closely related or distant species. Despite similar gene content and identical environments, recurrent adaptation across S. pombe populations involved different genes than in S. cerevisiae and was mostly detectable at the transcriptomic level. This suggests that trans-regulatory effects may play an important role in adaptation on short evolutionary timescales and that differences in evolutionary outcomes between these species may be attributed to contingency.

evolutionary biology↗

bioRxiv2024

Spatiotemporal Analysis of Lung Immune Dynamics in Lethal Coccidioides posadasii Infection

Coccidioidomycosis, or Valley Fever, is a lung disease caused by inhalation of Coccidioides fungi, prevalent in the Southwestern U.S., Mexico, and parts of Central and South America. 350,000 cases are reported annually in the U.S., although that number is expected to increase as climate change expands fungal geographic range. While 60% of infections are asymptomatic, the symptomatic 40% are often misdiagnosed due to similarities with bronchitis or pneumonia. A small subset of infection progress to severe illness, necessitating a better understanding of immune responses during lethal infection. Using single-cell RNA sequencing and spatial transcriptomics, we characterized lung responses during Coccidioides infection. We identified monocyte-derived Spp1-expressing macrophages as potential mediators of tissue remodeling and fibrosis, marked by high expression of profibrotic and proinflammatory transcripts. These macrophages showed elevated TGF-{beta} and IL-6 signaling, pathways involved in fibrosis pathogenesis. Additionally, we observed significant neutrophil infiltration and defective lymphocyte responses, indicating severe adaptive immunity dysregulation in lethal, acute infection. These findings enhance our understanding of Coccidioides infection and suggest new therapeutic targets. ImportanceCoccidioidomycosis, commonly known as Valley Fever, is a lung disease caused by the inhalation of Coccidioides fungi, which is prevalent in the Southwestern U.S., Mexico, and parts of Central and South America. With climate change potentially expanding the geographic range of this fungus, understanding the immune responses during severe infections is crucial. Our study used advanced techniques to analyze lung responses during Coccidioides infection, identifying specific immune cells that may contribute to tissue damage and fibrosis. These findings provide new insights into the disease mechanisms and suggest potential targets for therapeutic intervention, which could improve outcomes for patients suffering from severe Valley Fever.

immunology↗

bioRxiv2024

Protein sequence landscapes are not so simple: on reference-free versus reference-based inference

In a recent preprint, Park, Metzger, and Thornton reanalyze 20 empirical protein sequence-function landscapes using a "reference-free analysis" (RFA) method they recently developed. They argue that these empirical landscapes are simpler and less epistatic than earlier work suggested, and attribute the difference to limitations of the methods used in the original analyses of these landscapes, which they claim are more sensitive to measurement noise, missing data, and other artifacts. Here, we show that these claims are incorrect. Instead, we find that the RFA method introduced by Park et al. is exactly equivalent to the reference-based least-squares methods used in the original analysis of many of these empirical landscapes (and also equivalent to a Hadamard-based approach they implement). Because the reanalyzed and original landscapes are in fact identical, the different conclusions drawn by Park et al. instead reflect different interpretations of the parameters describing the inferred landscapes; we argue that these do not support the conclusion that epistasis plays only a small role in protein sequence-function landscapes.

evolutionary biology↗

bioRxiv2023

Atenolol reduces cardiac-mediated mortality in a genetic mouse model of sudden unexpected death in epilepsy

Sudden Unexpected Death in Epilepsy (SUDEP) is the leading cause of premature mortality in epilepsy. Genetic cardiac risk factors, including loss-of-function KCNH2 variants, have been linked to SUDEP. We hypothesised that seizures and LQTS interact to increase SUDEP risk. To investigate this, we crossed Kcnh2+/- and Gabrg2R43Q/+ mice that model LQTS and genetic epilepsy, respectively. Electrocorticography and electrocardiogram confirmed that Kcnh2+/- mice had a LQTS phenotype, while Gabrg2R43Q/+ mice displayed spontaneous seizures. Double mutant mice (Gabrg2R43Q/+/Kcnh2+/-) had both seizure and LQTS phenotypes that were indistinguishable from the respective single mutant mice. Survival analysis revealed that Gabrg2R43Q/+/Kcnh2+/- mice experienced a disproportionate higher rate of seizure-related death. Long-term oral administration of atenolol, a cardiac-selective {beta}-blocker, significantly improved survival in the Gabrg2R43Q/+/Kcnh2+/- mice. An additional mouse model, Hcn1M294L/+/Kcnh2+/-, based on a HCN1 developmental epileptic encephalopathy variant, also experienced a disproportionately higher rate of premature death that was rescued by atenolol. Kcnh2+/- mice also spent more time in ventricular arrhythmia during proconvulsant-induced seizures. Overall, the data implicates cardiac and loss-of-function KCNH2 variants as an important risk factor, and the potential repurposing of {beta}- blockers as a prevention strategy, for SUDEP in a subset of epilepsy patients.

neuroscience↗

bioRxiv2023

Five inhibitory receptors display distinct vesicular distributions in T cells

T cells can express multiple inhibitory receptors. Upon induction of T cell exhaustion in response to persistent antigen, prominently in the anti-tumor immune response, many are expressed simultaneously. Key inhibitory receptors are CTLA-4, PD-1, LAG3, TIM3 and TIGIT, as investigated here. These receptors are important as central therapeutic targets in cancer immunotherapy. Inhibitory receptors are not constitutively expressed on the cell surface, but substantial fractions reside in intracellular vesicular structures. It remains unresolved to which extent the subcellular localization of different inhibitory receptors is distinct. Using quantitative imaging of subcellular distributions and plasma membrane insertion as complemented by proximity proteomics and a biochemical analysis of the association of the inhibitory receptors with trafficking adaptors, the subcellular distributions of the five inhibitory receptors were discrete. The distribution of CTLA-4 was most distinct with preferential association with lysosomal-derived vesicles and the sorting nexin 1/2/5/6 transport machinery. With a lack of evidence for the existence of specific vesicle subtypes to explain divergent inhibitory receptor distributions, we suggest that such distributions are driven by divergent trafficking through an overlapping joint set of vesicular structures. This extensive characterization of the subcellular localization of five inhibitory receptors in relation to each other lays the foundation for the molecular investigation of their trafficking and its therapeutic exploitation.

immunology↗

bioRxiv2022

Hierarchical sequence-affinity landscapes shape the evolution of breadth in an anti-influenza receptor binding site antibody

Broadly neutralizing antibodies (bnAbs) that neutralize diverse variants of a particular virus are of considerable therapeutic interest1. Recent advances have enabled us to isolate and engineer these antibodies as therapeutics, but eliciting them through vaccination remains challenging, in part due to our limited understanding of how antibodies evolve breadth2. Here, we analyze the landscape by which an anti-influenza receptor binding site (RBS) bnAb, CH65, evolved broad affinity to diverse H1 influenza strains3, 4. We do this by generating an antibody library of all possible evolutionary intermediates between the unmutated common ancestor (UCA) and the affinity-matured CH65 antibody and measure the affinity of each intermediate to three distinct H1 antigens. We find that affinity to each antigen requires a specific set of mutations - distributed across the variable light and heavy chains - that interact non-additively (i.e., epistatically). These sets of mutations form a hierarchical pattern across the antigens, with increasingly divergent antigens requiring additional epistatic mutations beyond those required to bind less divergent antigens. We investigate the underlying biochemical and structural basis for these hierarchical sets of epistatic mutations and find that epistasis between heavy chain mutations and a mutation in the light chain at the VH-VL interface is essential for binding a divergent H1. Collectively, this work is the first to comprehensively characterize epistasis between heavy and light chain mutations and shows that such interactions are both strong and widespread. Together with our previous study analyzing a different class of anti-influenza antibodies5, our results implicate epistasis as a general feature of antibody sequence-affinity landscapes that can potentiate and constrain the evolution of breadth.

evolutionary biology↗

bioRxiv2022

The landscape of antibody binding affinity in SARS-CoV-2 Omicron BA.1 evolution

The Omicron BA.1 variant of SARS-CoV-2 escapes convalescent sera and monoclonal antibodies that are effective against earlier strains of the virus. This immune evasion is largely a consequence of mutations in the BA.1 receptor binding domain (RBD), the major antigenic target of SARS-CoV-2. Previous studies have identified several key RBD mutations leading to escape from most antibodies. However, little is known about how these escape mutations interact with each other and with other mutations in the RBD. Here, we systematically map these interactions by measuring the binding affinity of all possible combinations of these 15 RBD mutations (215 = 32,768 genotypes) to four monoclonal antibodies (LY-CoV016, LY-CoV555, REGN10987, and S309) with distinct epitopes. We find that BA.1 can lose affinity to diverse antibodies by acquiring a few large-effect mutations and can reduce affinity to others through several small-effect mutations. However, our results also reveal alternative pathways to antibody escape that do not include every large-effect mutation. Moreover, epistatic interactions are shown to constrain affinity decline in S309 but only modestly shape the affinity landscapes of other antibodies. Together with previous work on the ACE2 affinity landscape, our results suggest that escape of each antibody is mediated by distinct groups of mutations, whose deleterious effects on ACE2 affinity are compensated by another distinct group of mutations (most notably Q498R and N501Y).

evolutionary biology↗

bioRxiv2022

Compensatory epistasis maintains ACE2 affinity in SARS-CoV-2 Omicron BA.1

The Omicron BA.1 variant emerged in late 2021 and quickly spread across the world. Compared to the ancestral Wuhan Hu-1 strain and other pre-Omicron SARS-CoV-2 variants, BA.1 has many mutations, a number of which are known to enable antibody escape 1-3. Many of these antibody-escape mutations individually decrease the spike receptor-binding domain (RBD) affinity for ACE2 in the background of early SARS-CoV-2 variants 4, but BA.1 still binds ACE2 with high affinity 5,6. The fitness and evolution of the BA.1 lineage is therefore driven by the combined effects of numerous mutations. Here, we systematically map the epistatic interactions between the 15 mutations in the RBD of BA.1 relative to the Wuhan Hu-1 strain. Specifically, we measure the ACE2 affinity of all possible combinations of these 15 mutations (2 15 = 32,768 genotypes), spanning all possible evolutionary intermediates from the ancestral Wuhan Hu-1 strain to BA.1. We find that immune escape mutations in BA.1 individually reduce ACE2 affinity but are compensated by epistatic interactions with other affinity-enhancing mutations, including Q498R and N501Y. Thus, the ability of BA.1 to evade immunity while maintaining ACE2 affinity is contingent on acquiring multiple interacting mutations. Our results implicate compensatory epistasis as a key factor driving substantial evolutionary change for SARS-CoV-2 and are consistent with Omicron BA.1 arising from a chronic infection.

evolutionary biology↗

bioRxiv2021

MAVS mediates a protective immune response in the brain to Rift Valley fever virus

Rift Valley fever virus (RVFV) is a highly pathogenic mosquito-borne virus capable of causing hepatitis, encephalitis, blindness, hemorrhagic syndrome, and death in humans and livestock. Upon aerosol infection with RVFV, the brain is a major site of viral replication and tissue damage, yet pathogenesis in this organ has been understudied. Here, we investigated the immune response in the brain of RVFV infected mice. In response to infection, microglia initiate robust transcriptional upregulation of antiviral immune genes, as well as increased levels of activation markers and cytokine secretion that is dependent on mitochondrial antiviral-signaling protein (MAVS) and independent of toll-like receptors 3 and 7. In vivo, Mavs-/- mice displayed enhanced susceptibility to RVFV as determined by increased brain viral burden and higher mortality. Single-cell RNA sequence analysis identified microglia-specific defects in type I interferon and interferon responsive gene expression in Mavs-/- mice, as well as dysregulated lymphocyte infiltration. The results of this study provide a crucial step towards understanding the precise molecular mechanisms by which RVFV infection is controlled in the brain and will help inform the development of vaccines and antiviral therapies that are effective in preventing encephalitis. Author SummaryRift Valley fever virus causes severe disease in humans and livestock and in some cases can be fatal. There is concern about the use of Rift Valley fever virus as a bioweapon since it can be transmitted through the air, and there are no vaccines or antiviral treatments. Airborne transmission of the virus causes severe inflammation of the brain, yet little is known about the immune response against the virus in this organ. Here, we investigated the immune response in the brain to Rift Valley fever virus following intranasal infection. We determined that microglia, the resident immune cells of the brain, initiate a robust response to Rift Valley fever virus infection and identified a key immune pathway that is critical for the ability of microglia to respond to infection. When this immune pathway is rendered non-functional, mice have a dysregulated response to infection in the brain. This study provides insight into how the immune response can control Rift Valley fever virus infection of the brain.

immunology↗

bioRxiv2021

Dynamics and variability in the pleiotropic effects of adaptation in laboratory budding yeast populations

Evolutionary adaptation to a constant environment is driven by the accumulation of mutations which can have a range of unrealized pleiotropic effects in other environments. These pleiotropic consequences of adaptation can influence the emergence of specialists or generalists, and are critical for evolution in temporally or spatially fluctuating environments. While many experiments have examined the pleiotropic effects of adaptation at a snapshot in time, very few have observed the dynamics by which these effects emerge and evolve. Here, we propagated hundreds of diploid and haploid laboratory budding yeast populations in each of three environments, and then assayed their fitness in multiple environments over 1000 generations of evolution. We find that replicate populations evolved in the same condition share common patterns of pleiotropic effects across other environments, which emerge within the first several hundred generations of evolution. However, we also find dynamic and environment-specific variability within these trends: variability in pleiotropic effects tends to increase over time, with the extent of variability depending on the evolution environment. These results suggest shifting and overlapping contributions of chance and contingency to the pleiotropic effects of adaptation, which could influence evolutionary trajectories in complex environments that fluctuate across space and time.

evolutionary biology↗

bioRxiv2021

Binding affinity landscapes constrain the evolution of broadly neutralizing anti-influenza antibodies

Over the past two decades, several broadly neutralizing antibodies (bnAbs) that confer protection against diverse influenza strains have been isolated1,2. Structural and biochemical characterization of these bnAbs has provided molecular insight into how they bind distinct antigens1. However, our understanding of the evolutionary pathways leading to bnAbs, and thus how best to elicit them, remains limited. Here, we measure equilibrium dissociation constants of combinatorially complete mutational libraries for two naturally isolated influenza bnAbs3-5 (CR-9114, 16 mutations; CR-6261, 11 mutations), reconstructing all possible intermediates back to the unmutated germline sequences. We find that these two libraries exhibit strikingly different patterns of breadth: while many variants of CR-6261 display moderate affinity to diverse antigens, those of CR-9114 display appreciable affinity only in specific, nested combinations. By examining the extensive pairwise and higher-order epistasis between mutations, we find key sites with strong synergistic interactions that are highly similar across antigens for CR-6261 and different for CR-9114. Together, these features of the binding affinity landscapes strongly favor sequential acquisition of affinity to diverse antigens for CR-9114, while the acquisition of breadth to more similar antigens for CR-6261 is less constrained. These results, if generalizable to other bnAbs, may explain the molecular basis for the widespread observation that sequential exposure favors greater breadth6-8, and such mechanistic insight will be essential for predicting and eliciting broadly protective immune responses.

evolutionary biology↗

bioRxiv2021

Development of Potent and Effective Synthetic SARS-CoV-2 Neutralizing Nanobodies

The respiratory virus responsible for Coronavirus disease 2019 (COVID-19), Severe acute respiratory syndrome coronavirus 2 (SARS-2), has impacted nearly every aspect of life worldwide, claiming the lives of over 2.5 million people globally, at the time of this publication. Neutralizing nanobodies (VHH) represent a promising therapeutic intervention strategy to address the current SARS-2 pandemic and provide a powerful toolkit to address future virus outbreaks. Using a synthetic, high-diversity VHH bacteriophage library, several potent neutralizing VHH antibodies were identified and evaluated for their capacity to tightly bind to the SARS-2 receptor-binding domain (RBD), to prevent binding of SARS-2 spike (S) to the cellular receptor Angiotensin-converting enzyme 2 (ACE2), and to neutralize viral infection. Preliminary preclinical evaluation of multiple nanobody candidates demonstrate that they are prophylactically and therapeutically effective in vivo against wildtype SARS-2. The identified and characterized nanobodies described herein represent viable candidates for further preclinical evaluation and another tool to add to our therapeutic arsenal to address the COVID-19 pandemic. Author SummaryTo fully address the on-going pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-2), it will be important to have both vaccines and therapeutic strategies to prevent and mitigate the effects of SARS-2. In this study, we describe the identification and characterization of potently neutralizing humanized single domain heavy chain (VHH) antibodies that have binding affinity for both the original Wuhan strain and widely circulating B.1.1.7/UK strain. VHH antibodies have the same therapeutic potential as conventional antibodies in half the size and with greater stability and solubility. Using a synthetic humanized high-diversity VHH phage library we identified several candidates with strong affinity for the SARS-2 spike that block the interaction of SARS-2 spike with the cellular receptor ACE2, and effectively neutralize infection with SARS-2 in vitro. By sequencing viral escape mutants generated in the presence of each VHH we mapped the binding sites of the VHH antibodies and assessed their affinity against newly emerging SARS-2 variants. Finally, we demonstrate that two of these VHH antibodies show prophylactic and therapeutic efficacy in vivo against challenge with SARS-2. This study establishes that screening highly diverse VHH phage libraries against viral threats can yield highly effective therapeutic agents in real time.

immunology↗

bioRxiv2021

Molecular and cell biological analysis of SwrB in Bacillus subtilis

Swarming motility is flagellar-mediated movement over a solid surface and Bacillus subtilis cells require an increase in flagellar density to swarm. SwrB is a protein of unknown function required for swarming that is necessary to increase the number of flagellar hooks but not basal bodies. Previous work suggested that SwrB activates flagellar type III secretion but the mechanism by which it might perform this function is unknown. Here we show that SwrB likely acts sub-stoichiometrically as it localizes as puncta at the membrane in numbers fewer than that of flagellar basal bodies. Moreover the action of SwrB is likely transient as puncta of SwrB were not dependent on the presence of the basal bodies and rarely co-localized with flagellar hooks. Random mutagenesis of the SwrB sequence found that a histidine within the transmembrane segment was conditionally required for activity and punctate localization. Finally, three hydrophobic residues that precede a cytoplasmic domain of poor conservation abolished SwrB activity when mutated and caused aberrant migration during electrophoresis. Our data are consistent with a model in which SwrB interacts with the flagellum, changes conformation to activate type III secretion, and departs. IMPORTANCEType III secretion systems (T3SS) are elaborate nanomachines that form the core of the bacterial flagellum and injectisome of pathogens. The machines not only secrete proteins like virulence factors but also secrete the structural components for their own assembly. Moroever, proper construction requires complex regulation to ensure that the parts are roughly secreted in the order in which they are assembled. Here we explore a poorly understood activator the flagellar T3SS activation in Bacillus subtilis called SwrB. To aid mechanistic understanding, we determine the rules for subcellular punctate localization, the topology with respect to the membrane, and critical residues required for SwrB function.

microbiology↗

bioRxiv2021

The Host Cell's Endoplasmic Reticulum Proteostasis Network Profoundly Shapes the Protein Sequence Space Accessible to HIV Envelope

The sequence space accessible to evolving proteins can be enhanced by cellular chaperones that assist biophysically defective clients in navigating complex folding landscapes. It is also possible, however, for proteostasis mechanisms that promote strict quality control to greatly constrain accessible protein sequence space. Unfortunately, most efforts to understand how proteostasis mechanisms influence evolution rely on artificial inhibition or genetic knockdown of specific chaperones. The few experiments that perturb quality control pathways also generally modulate the levels of only individual quality control factors. Here, we use chemical genetic strategies to tune proteostasis networks via natural stress response pathways that regulate levels of entire suites of chaperones and quality control mechanisms. Specifically, we upregulate the unfolded protein response (UPR) to test the hypothesis that the host endoplasmic reticulum (ER) proteostasis network shapes the sequence space accessible to human immunodeficiency virus-1 (HIV) envelope (Env) protein. Elucidating factors that enhance or constrain Env sequence space is critical because Env evolves extremely rapidly, yielding HIV strains with antibody and drug escape mutations. We find that UPR-mediated upregulation of ER proteostasis factors, particularly those controlled by the IRE1-XBP1s UPR arm, globally reduces Env mutational tolerance. Conserved, functionally important Env regions exhibit the largest decreases in mutational tolerance upon XBP1s activation. This phenomenon likely reflects strict quality control endowed by XBP1s-mediated remodeling of the ER proteostasis environment. Intriguingly and in contrast, specific regions of Env, including regions targeted by broadly neutralizing antibodies, display enhanced mutational tolerance when XBP1s is activated, hinting at a role for host proteostasis network hijacking in potentiating antibody escape. These observations reveal a key function for proteostasis networks in decreasing instead of expanding the sequence space accessible to client proteins, while also demonstrating that the host ER proteostasis network profoundly shapes the mutational tolerance of Env in ways that could have important consequences for HIV adaptation.

biochemistry↗

bioRxiv2021

Loss-of-function variants in the cardiac Kv11.1 channel as a genetic biomarker for SUDEP

ObjectiveTo compare the frequency and impact on channel function of KCNH2 variants in SUDEP patients with epilepsy controls comprising patients older than 50 years, a group with low SUDEP risk, and establish loss-of-function KCNH2 variants as predictive biomarkers of SUDEP risk. MethodsWe searched for KCNH2 variants with a minor allele frequency of < 5%. Functional analysis in Xenopus laevis oocytes was performed for all KCNH2 variants identified. ResultsKCNH2 variants were found in 11.1% (10/90) of SUDEP individuals compared to 6.0% (20/332) of epilepsy controls (p = 0.11). Loss-of-function KCNH2 variants, defined as causing > 20% reduction in maximal amplitude, were observed in 8.9% (8/90) SUDEP patients compared to 3.3% (11/332) epilepsy controls suggesting about three-fold enrichment (nominal p = 0.04). KCNH2 variants that did not change channel function occurred at a similar frequency in SUDEP (2.2%; 2/90) and epilepsy control (2.7%; 9/332) cohorts (p > 0.99). Rare KCNH2 variants (< 1% allele frequency) associated with greater loss of function and an [~]11-fold enrichment in the SUDEP cohort (nominal p = 0.03). In silico tools were unable to predict the impact of a variant on function highlighting the need for electrophysiological analysis. ConclusionsThese data show that loss-of-function KCNH2 variants are enriched in SUDEP patients and suggest that cardiac mechanisms contribute to SUDEP risk. We propose that genetic screening in combination with functional analysis can identify loss-of-function KCNH2 variants that could act as biomarkers of an individuals SUDEP risk.

neuroscience↗

bioRxiv2021

The Genetic Basis of Differential Autodiploidization in Evolving Yeast Populations

Spontaneous whole-genome duplication, or autodiploidization, is a common route to adaptation in experimental evolution of haploid budding yeast populations. The rate at which autodiploids fix in these populations appears to vary across strain backgrounds, but the genetic basis of these differences remains poorly characterized. Here we show that the frequency of autodiploidization differs dramatically between two closely related laboratory strains of Saccharomyces cerevisiae, BY4741 and W303. To investigate the genetic basis of this difference, we crossed these strains to generate hundreds of unique F1 segregants and tested the tendency of each segregant to autodiplodize across hundreds of generations of laboratory evolution. We find that variants in the SSD1 gene are the primary genetic determinant of differences in autodiploidization. We then used multiple laboratory and wild strains of S. cerevisiae to show that clonal populations of strains with a functional copy of SSD1 autodiploidize more frequently in evolution experiments, while knocking out this gene or replacing it with the W303 allele reduces autodiploidization propensity across all genetic backgrounds tested. These results suggest a potential strategy for modifying rates of spontaneous whole-genome duplications in laboratory evolution experiments in haploid budding yeast. They may also have relevance to other settings in which eukaryotic genome stability plays an important role, such as biomanufacturing and the treatment of pathogenic fungal diseases and cancers.

evolutionary biology↗