Manuscripts from the lab
Mammalian genomic manipulation with orthogonal Bxb1 DNA recombinase sites for the functional characterization of protein variants
ACS Synthetic Biology. 2023 Nov 3.
The Bxb1 bacteriophage serine DNA recombinase is an efficient tool for engineering recombinant DNA into the genomes of cultured cells. Generally, a single engineered “landing pad” site is introduced into the cell genome, permitting the integration of transgenic circuits or libraries of transgene variants. While sufficient for many studies, the extent of genetic manipulation possible with a single recombinase site is limiting, and insufficient for more complex cell-based assays. Here, we harnessed two orthogonal Bxb1 recombinase sites to enable alternative avenues for using mammalian synthetic biology to characterize transgenic protein variants. By designing plasmids flanked by a second pair of auxiliary recombination sites, we demonstrate that we can avoid genomic integration of undesirable bacterial DNA elements using the same starting cells engineered for whole-plasmid integration. We also created “double landing pad” cells simultaneously harboring two orthogonal Bxb1 recombinase sites at separate genomic loci, allowing complex cell-based genetic assays. Integration of a genetically encoded calcium indicator allowed for the real-time monitoring of intracellular calcium signaling dynamics, including kinetic perturbations that occur upon overexpression of the wildtype or variant version of the calcium signaling relay protein STIM1. A panel of missense mutants of the HIV-1 accessory protein Vif were paired with various paralogs within the human Apobec3 innate immune protein family to identify combinations capable or incapable of interacting within cells. These cells allow transgenic protein variant libraries to be readily paired with assay-specific protein partners or biosensors, enabling new functional readouts for large-scale genetic assays for protein function.
Expanded ACE2 dependencies of diverse SARS-like coronavirus receptor binding domains
PLoS Biology. 2022 July 27.
Viral spillover from animal reservoirs can trigger public health crises and cripple the world economy. Knowing which viruses are primed for zoonotic transmission can focus surveillance efforts and mitigation strategies for future pandemics. Successful engagement of receptor protein orthologs is necessary during cross-species transmission. The clade 1 sarbecoviruses including SARS-CoV and SARS-CoV-2 enter cells via engagement of ACE2, while the receptor for clade 2 and clade 3 remains largely uncharacterized. We developed a mixed cell pseudotyped virus infection assay to determine whether various clade 2 and 3 sarbecovirus spike proteins can enter HEK 293T cells expressing human or Rhinolophus horseshoe bat ACE2 proteins. The receptor binding domains from BtKY72 and Khosta-2 used human ACE2 for entry, while BtKY72 and Khosta-1 exhibited widespread use of diverse rhinolophid ACE2s. A lysine at ACE2 position 31 appeared to be a major determinant of the inability of these RBDs to use a certain ACE2 sequence. The ACE2 protein from R. alcyone engaged all known clade 3 and clade 1 receptor binding domains. We observed little use of Rhinolophus ACE2 orthologs by the clade 2 viruses, supporting the likely use of a separate, unknown receptor. Our results suggest that clade 3 sarbecoviruses from Africa and Europe use Rhinolophus ACE2 for entry, and their spike proteins appear primed to contribute to zoonosis under the right conditions.
Integrating thousands of PTEN variant activity and abundance measurements reveals variant subgroups and new dominant negatives in cancers.
Genome Medicine. 2021 Oct 14.
Background: PTEN is a multi-functional tumor suppressor protein regulating cell growth, immune signaling, neuronal function, and genome stability. Experimental characterization can help guide the clinical interpretation of the thousands of germline or somatic PTEN variants observed in patients. Two large-scale mutational datasets, one for PTEN variant intracellular abundance encompassing 4112 missense variants and one for lipid phosphatase activity encompassing 7244 variants, were recently published. The combined information from these datasets can reveal variant-specific phenotypes that may underlie various clinical presentations, but this has not been comprehensively examined, particularly for somatic PTEN variants observed in cancers.
Methods: Here, we add to these efforts by measuring the intracellular abundance of 764 new PTEN variants and refining abundance measurements for 3351 previously studied variants. We use this expanded and refined PTEN abundance dataset to explore the mutational patterns governing PTEN intracellular abundance, and then incorporate the phosphatase activity data to subdivide PTEN variants into four functionally distinct groups.
Results: This analysis revealed a set of highly abundant but lipid phosphatase defective variants that could act in a dominant-negative fashion to suppress PTEN activity. Two of these variants were, indeed, capable of dysregulating Akt signaling in cells harboring a WT PTEN allele. Both variants were observed in multiple breast or uterine tumors, demonstrating the disease relevance of these high abundance, inactive variants.
Conclusions: We show that multidimensional, large-scale variant functional data, when paired with public cancer genomics datasets and follow-up assays, can improve understanding of uncharacterized cancer-associated variants, and provide better insights into how they contribute to oncogenesis.
Mutants of human ACE2 differentially promote SARS-CoV and SARS-CoV-2 spike mediated infection.
PLoS Pathogens. 2021 July 16
SARS-CoV and SARS-CoV-2 encode spike proteins that bind human ACE2 on the cell surface to enter target cells during infection. A small fraction of humans encode variants of ACE2, thus altering the biochemical properties at the protein interaction interface. These and other ACE2 coding mutants can reveal how the spike proteins of each virus may differentially engage the ACE2 protein surface during infection. We created an engineered HEK 293T cell line for facile stable transgenic modification, and expressed the major human ACE2 allele or 28 of its missense mutants, 24 of which are possible through single nucleotide changes from the human reference sequence. Infection with SARS-CoV or SARS-CoV-2 spike pseudotyped lentiviruses revealed that high ACE2 cell-surface expression could mask the effects of impaired binding during infection. Drastically reducing ACE2 cell surface expression revealed a range of infection efficiencies across the panel of mutants. Our infection results revealed a non-linear relationship between soluble SARS-CoV-2 RBD binding to ACE2 and pseudovirus infection, supporting a major role for binding avidity during entry. While ACE2 mutants D355N, R357A, and R357T abrogated entry by both SARS-CoV and SARS-CoV-2 spike proteins, the Y41A mutant inhibited SARS-CoV entry much more than SARS-CoV-2, suggesting differential utilization of the ACE2 side-chains within the largely overlapping interaction surfaces utilized by the two CoV spike proteins. These effects correlated well with cytopathic effects observed during SARS-CoV-2 replication in ACE2-mutant cells. The panel of ACE2 mutants also revealed altered ACE2 surface dependencies by the N501Y spike variant, including a near-complete utilization of the K353D ACE2 variant, despite decreased infection mediated by the parental SARS-CoV-2 spike. Our results clarify the relationship between ACE2 abundance, binding, and infection, for various SARS-like coronavirus spike proteins and their mutants, and inform our understanding for how changes to ACE2 sequence may correspond with different susceptibilities to infection.
An improved platform for functional assessment of large protein libraries in mammalian cells.
Nucleic Acids Res. 2019 Oct 15.
Determining the pathogenicity of genetic variants is a critical challenge, and functional assessment is often the only option. Experimentally characterizing millions of possible missense variants in thousands of clinically important genes requires generalizable, scalable assays. We describe variant abundance by massively parallel sequencing (VAMP-seq), which measures the effects of thousands of missense variants of a protein on intracellular abundance simultaneously. We apply VAMP-seq to quantify the abundance of 7,801 single-amino-acid variants of PTEN and TPMT, proteins in which functional variants are clinically actionable. We identify 1,138 PTEN and 777 TPMT variants that result in low protein abundance, and may be pathogenic or alter drug metabolism, respectively. We observe selection for low-abundance PTEN variants in cancer, and show that p.Pro38Ser, which accounts for ~10% of PTEN missense variants in melanoma, functions via a dominant-negative mechanism. Finally, we demonstrate that VAMP-seq is applicable to other genes, highlighting its generalizability.
As coauthor (or before independence)
Evidence of antigenic drift in the fusion machinery core of SARS-CoV-2 spike
bioRxiv. 2023 Sep 28.
Antigenic drift of SARS-CoV-2 is typically defined by mutations in the N-terminal domain and receptor binding domain of spike protein. In contrast, whether antigenic drift occurs in the S2 domain remains largely elusive. Here, we perform a deep mutational scanning experiment to identify S2 mutations that affect binding of SARS-CoV-2 spike to three S2 apex public antibodies. Our results indicate that spatially diverse mutations, including D950N and Q954H, which are observed in Delta and Omicron variants, respectively, weaken the binding of spike to these antibodies. Although S2 apex antibodies are known to be non-neutralizing, we show that they confer partial protection in vivo. We further demonstrate that such in vivo protection activity is diminished by the natural mutation D950N. Overall, this study indicates that the S2 domain of SARS-CoV-2 spike can undergo antigenic drift, which represents a potential challenge for the development of more universal coronavirus vaccines.
High-throughput functional mapping of variants in an arrhythmia gene, KCNE1, reveals novel biology
bioRxiv. 2023 April 29.
Background: KCNE1 encodes a 129-residue cardiac potassium channel (IKs) subunit. KCNE1 variants are associated with long QT syndrome and atrial fibrillation. However, most variants have insufficient evidence of clinical consequences and thus limited clinical utility. Variant effect mapping couples saturation mutagenesis with high-throughput sequencing to ascertain the function of thousands of protein-coding variants. Results: An assay of KCNE1 variant cell-surface expression (2,554/2,709 possible single amino acid variants) identified 502 loss-of-surface expression variants and an assay for KCNE1 function (2,539 variants) identified 588 loss-of-function variants. The latter half of the protein is dispensable for protein trafficking but essential for channel function. 22 of the 30 KCNE1 residues (73%) highly intolerant of variation were in predicted close contact with binding partners KCNQ1 or calmodulin. Our data were validated against gold standard electrophysiological data (ρ=-0.65), population and patient cohorts (32/38 concordant variants), and computational metrics (ρ=-0.55). After assay calibration, our data provide moderate-strength evidence for the ACMG/AMP functional criteria for benign and pathogenic variants. Conclusions: Comprehensive variant effect maps of KCNE1 can help provide insight into IKs channel biology and reclassify variants of uncertain significance.
Opto-MASS: a high-throughput engineering platform for genetically encoded fluorescent sensors enabling all-optical in vivo detection of monoamines and opioids
ACS Sensors. 2023 Nov 13.
Fluorescent sensor proteins are instrumental for detecting biological signals in vivo with high temporal accuracy and cell-type specificity. However, engineering sensors with physiological ligand sensitivity and selectivity is difficult because they need to be optimized through individual mutagenesis in vitro to assess their performance. The vast mutational landscape proteins constitute an obstacle that slows down sensor development. This is particularly true for sensors that require mammalian host systems to be screened. Here, we developed a novel high-throughput engineering platform that functionally tests thousands of variants simultaneously in mammalian cells and thus allows the screening of large variant numbers. We showcase the capabilities of our platform, called Optogenetic Microwell Array Screening System (Opto-MASS), by engineering novel monoamine and neuropeptide in vivo capable sensors with distinct physiological roles at high-throughput.
Rapid characterization of the functional and pharmacological consequences of Cantú syndrome KATP channel mutations in intact cells
Journal of Pharmacology and Experimental Therapeutics. 2023 Aug 1.
Gain-of-function (GOF) of KATP channels, resulting from mutations in either KCNJ8 (encoding Kir6.1) or ABCC9 (encoding SUR2), cause Cantú syndrome (CS), a channelopathy characterized by excess hair growth, coarse facial appearance, cardiomegaly, and lymphedema. Here, we established a pipeline for rapid analysis of CS mutation consequences in Landing pad HEK 293 cell lines stably expressing wild type and mutant human Kir6.1 and SUR2B. Thallium-influx and cell membrane potential, reported by fluorescent Tl-sensitive Fluozin-2 and voltage-sensitive DiBAC4(3) dyes, respectively, were used to assess channel activity. In the Tl-influx assay, CS-associated Kir6.1 mutations increased sensitivity to the KATP channel activator, pinacidil, but there was strikingly little effect of pinacidil for any SUR2B mutations, reflecting unexpected differences in the molecular mechanisms of Kir6.1 versus SUR2B mutations. Compared to the Tl-influx assay, the DiBAC4(3) assay presents more significant signal changes in response to subtle KATP channel activity changes, and all CS mutants (both Kir6.1 and SUR2B), but not WT channels, caused marked hyperpolarization, demonstrating that all mutants were activated under ambient conditions in intact cells. Most SUR2 CS mutations were markedly inhibited by <100nM glibenclamide, but sensitivity to inhibition by glibenclamide, repaglinide, and PNU37883A, was markedly reduced for Kir6.1 CS mutations. Understanding functional consequences of mutations can help with disease diagnosis and treatment. The analysis pipeline we have developed has the potential to rapidly identify mutational consequences, aiding future CS diagnosis, drug discovery, and individualization of treatment. Significance Statement We have developed new fluorescence-based assays of channel activities and drug sensitivities of Cantú syndrome (CS) mutations in human Kir6.1/SUR2B-dependent KATP channels, showing that Kir6.1 mutations increase sensitivity to activation by potassium channel openers, while SUR2B mutations markedly reduce KCO sensitivity. However, both Kir6.1 and SUR2B CS mutations are both more hyperpolarized than WT cells under basal conditions, confirming pathophysiologically relevant gain-of-function, validating DiBAC4(3) fluorescence to characterize hyperpolarization induced by KATP channel activity under basal, non KCO-activated conditions.
High-throughput identification of prefusion-stabilizing mutations in SARS-CoV-2 spike
Nat Commun. 2023 April 10.
Designing prefusion-stabilized SARS-CoV-2 spike is critical for the effectiveness of COVID-19 vaccines. All COVID-19 vaccines in the US encode spike with K986P/V987P mutations to stabilize its prefusion conformation. However, contemporary methods on engineering prefusion-stabilized spike immunogens involve tedious experimental work and heavily rely on structural information. Here, we established a systematic and unbiased method of identifying mutations that concomitantly improve expression and stabilize the prefusion conformation of the SARS-CoV-2 spike. Our method integrated a fluorescence-based fusion assay, mammalian cell display technology, and deep mutational scanning. As a proof-of-concept, this method was applied to a region in the S2 domain that includes the first heptad repeat and central helix. Our results revealed that besides K986P and V987P, several mutations simultaneously improved expression and significantly lowered the fusogenicity of the spike. As prefusion stabilization is a common challenge for viral immunogen design, this work will help accelerate vaccine development against different viruses.
Probing the biophysical constraints of SARS-CoV-2 spike N-terminal domain using deep mutational scanning
Sci Adv. 2022 Nov 23.
Increasing the expression level of the SARS-CoV-2 spike (S) protein has been critical for COVID-19 vaccine development. While previous efforts largely focused on engineering the receptor-binding domain (RBD) and the S2 subunit, the N-terminal domain (NTD) has been long overlooked due to the limited understanding of its biophysical constraints. In this study, the effects of thousands of NTD single mutations on S protein expression were quantified by deep mutational scanning. Our results revealed that in terms of S protein expression, the mutational tolerability of NTD residues was inversely correlated with their proximity to the RBD and S2. We also identified NTD mutations at the interdomain interface that increased S protein expression without altering its antigenicity. Overall, this study not only advances the understanding of the biophysical constraints of the NTD, but also provides invaluable insights into S-based immunogen design.
Receptor binding domain (RBD) antibodies contribute more to SARS-CoV-2 neutralization when target cells express high levels of ACE2
Viruses. 2022 Sep 16.
Neutralization assays are experimental surrogates for the effectiveness of infection- or vaccine-elicited polyclonal antibodies and therapeutic monoclonal antibodies targeting SARS-CoV-2. However, the measured neutralization can depend on details of the experimental assay. Here we systematically assess how ACE2 expression in target cells affects neutralization by antibodies to different spike epitopes in lentivirus pseudovirus neutralization assays. For high ACE2-expressing target cells, receptor binding domain (RBD) antibodies account for nearly all neutralizing activity in polyclonal human sera. But for lower ACE2-expressing target cells, antibodies targeting regions outside the RBD make a larger (although still modest) contribution to serum neutralization. These serum-level results are mirrored for monoclonal antibodies: N-terminal domain (NTD) antibodies and RBD antibodies that do not compete for ACE2 binding incompletely neutralize on high ACE2-expressing target cells, but completely neutralize on cells with lower ACE2 expression. Our results show that ACE2 expression level in the target cells is an important experimental variable, and that high ACE2 expression emphasizes the role of a subset of RBD-directed antibodies.
Shifting mutational constraints in the SARS-CoV-2 receptor-binding domain during viral evolution
Science. 2022 Jun 28.
SARS-CoV-2 has evolved variants with substitutions in the spike receptor-binding domain (RBD) that impact its affinity for ACE2 receptor and recognition by antibodies. These substitutions could also shape future evolution by modulating the effects of mutations at other sites-a phenomenon called epistasis. To investigate this possibility, we performed deep mutational scans to measure the effects on ACE2 binding of all single amino-acid mutations in the Wuhan-Hu-1, Alpha, Beta, Delta, and Eta variant RBDs. Some substitutions, most prominently N501Y, cause epistatic shifts in the effects of mutations at other sites. These epistatic shifts shape subsequent evolutionary change, for example enabling many of the antibody-escape substitutions in the Omicron RBD. These epistatic shifts occur despite high conservation of the overall RBD structure. Our data shed light on RBD sequence-function relationships and facilitate interpretation of ongoing SARS-CoV-2 evolution.
Probing ion channel functional architecture and domain recombination compatibility by massively parallel domain insertion profiling
Nat Comm. 2021 Dec 8.
Protein domains are the basic units of protein structure and function. Comparative analysis of genomes and proteomes showed that domain recombination is a main driver of multidomain protein functional diversification and some of the constraining genomic mechanisms are known. Much less is known about biophysical mechanisms that determine whether protein domains can be combined into viable protein folds. Here, we use massively parallel insertional mutagenesis to determine compatibility of over 300,000 domain recombination variants of the Inward Rectifier K+ channel Kir2.1 with channel surface expression. Our data suggest that genomic and biophysical mechanisms acted in concert to favor gain of large, structured domain at protein termini during ion channel evolution. We use machine learning to build a quantitative biophysical model of domain compatibility in Kir2.1 that allows us to derive rudimentary rules for designing domain insertion variants that fold and traffic to the cell surface. Positional Kir2.1 responses to motif insertion clusters into distinct groups that correspond to contiguous structural regions of the channel with distinct biophysical properties tuned towards providing either folding stability or gating transitions. This suggests that insertional profiling is a high-throughput method to annotate function of ion channel structural regions.
Extracellular vesicles carry SARS-CoV-2 spike protein and serve as decoys for neutralizing antibodies.
Journal of Extracellular Vesicles. 2021 June 18
In late 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. SARS-CoV-2 and the disease it causes, coronavirus disease 2019 (COVID-19), spread rapidly and became a global pandemic in early 2020. SARS-CoV-2 spike protein is responsible for viral entry and binds to angiotensin converting enzyme 2 (ACE2) on host cells, making it a major target of the immune system – particularly neutralizing antibodies (nAbs) that are induced by infection or vaccines. Extracellular vesicles (EVs) are small membraned particles constitutively released by cells, including virally-infected cells. EVs and viruses enclosed within lipid membranes share some characteristics: they are small, sub-micron particles and they overlap in cellular biogenesis and egress routes. Given their shared characteristics, we hypothesized that EVs released from spike-expressing cells could carry spike and serve as decoys for anti-spike nAbs, promoting viral infection. Here, using mass spectrometry and nanoscale flow cytometry (NFC) approaches, we demonstrate that SARS-CoV-2 spike protein can be incorporated into EVs. Furthermore, we show that spike-carrying EVs act as decoy targets for convalescent patient serum-derived nAbs, reducing their effectiveness in blocking viral entry. These findings have important implications for the pathogenesis of SARS-CoV-2 infection in vivo and highlight the complex interplay between viruses, extracellular vesicles, and the immune system that occurs during viral infections.
Multiplexed measurement of variant abundance and activity reveals VKOR topology, active site and human variant impact.
Elife. 2020 Sep 1
Vitamin K epoxide reductase (VKOR) drives the vitamin K cycle, activating vitamin K-dependent blood clotting factors. VKOR is also the target of the widely used anticoagulant drug, warfarin. Despite VKOR’s pivotal role in coagulation, its structure and active site remain poorly understood. In addition, VKOR variants can cause vitamin K-dependent clotting factor deficiency or alter warfarin response. Here, we used multiplexed, sequencing-based assays to measure the effects of 2,695 VKOR missense variants on abundance and 697 variants on activity in cultured human cells. The large-scale functional data, along with an evolutionary coupling analysis, supports a four transmembrane domain topology, with variants in transmembrane domains exhibiting strongly deleterious effects on abundance and activity. Functionally constrained regions of the protein define the active site, and we find that, of four conserved cysteines putatively critical for function, only three are absolutely required. Finally, 25% of human VKOR missense variants show reduced abundance or activity, possibly conferring warfarin sensitivity or causing disease.
MHC class II transactivator CIITA induces cell resistance to Ebola virus and SARS-like coronaviruses.
Science. 2020 Aug 27
Recent outbreaks of Ebola virus (EBOV) and SARS-CoV-2 have exposed our limited therapeutic options and poor understanding of cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the MHC class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein (EboGP). We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and identify an additional function of these proteins beyond their canonical roles in antigen presentation.
High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KV11.1.
Heart Rhythm. 2020 June 6.
KCHN2 encodes the KV11.1 potassium channel responsible for IKr, a major repolarization current during the cardiomyocyte action potential. Variants in KCNH2 that lead to decreased IKr have been associated with long QT syndrome type 2 (LQT2). The mechanism of LQT2 is most often induced loss of KV11.1 trafficking to the cell surface. Accurately discriminating between variants with normal and abnormal trafficking would aid in understanding the deleterious nature of these variants; however, the volume of reported nonsynonymous KCNH2 variants precludes the use of conventional methods for functional study.
The purpose of this study was to report a high-throughput, multiplexed screening method for KCNH2 genetic variants capable of measuring the cell surface abundance of hundreds of missense variants in the resulting KV11.1 channel. We developed a method to quantitate KV11.1 variant trafficking on a pilot region of 11 residues in the S5 helix. We generated trafficking scores for 220 of 231 missense variants in the pilot region. For 5 of 5 variants, high-throughput trafficking scores validated when tested in single variant flow cytometry and confocal microscopy experiments. We further explored these results with planar patch electrophysiology and found that loss-of-trafficking variants do not produce IKr. Conversely, but expectedly, some variants that traffic normally were still functionally compromised. We describe a new method for detecting KV11.1 trafficking-deficient variants in a multiplexed assay. This new method accurately generated trafficking data for variants in KV11.1 and is extendable both to all residues in KV11.1 and to other cell surface proteins.
A Premalignant Cell-Based Model for Functionalization and Classification of PTEN Variants.
Cancer Research. 2020 May 4
As sequencing becomes more economical, we are identifying sequence variations in the population faster than ever. For disease-associated genes, it is imperative that we differentiate a sequence variant as either benign or pathogenic such that the appropriate therapeutic interventions or surveillance can be implemented. PTEN (Phosphatase and TENsin homolog) is a frequently mutated tumor suppressor that has been linked to the PTEN hamartoma tumor syndrome. While the domain structure of PTEN and the functional impact of a number of its most common tumor-linked mutations have been characterized, there is a lack of information about many recently identified clinical variants. To address this challenge, we developed a cell-based assay that utilizes a premalignant phenotype of normal mammary epithelial cells lacking PTEN. We measured the ability of PTEN variants to rescue the spheroid formation phenotype of PTEN-/- MCF10A cells maintained in suspension. As proof of concept, we functionalized 47 missense variants using this assay, only 19 of which have clear classifications in ClinVar. We utilized a machine learning model trained with annotated genotypic data to classify variants as benign or pathogenic based on our functional scores. Our model predicted with high accuracy that loss of PTEN function was indicative of pathogenicity. We also determined that the pathogenicity of certain variants may have arisen from reduced stability of the protein product. Overall, this assay outperformed computational predictions, was scalable, and had a short run time, serving as an ideal alternative for annotating the clinical significance of cancer-associated PTEN variants.
Massively parallel variant characterization identifies NUDT15 alleles associated with thiopurine toxicity.
Proc. Natl. Acad. Sci. U.S.A. 2020 Feb 24
As a prototype of genomics-guided precision medicine, individualized thiopurine dosing based on pharmacogenetics is a highly effective way to mitigate hematopoietic toxicity of this class of drugs. Recently, NUDT15 deficiency was identified as a genetic cause of thiopurine toxicity, and NUDT15-informed preemptive dose reduction was quickly adopted in clinical settings. To exhaustively identify pharmacogenetic variants in this gene, we developed massively parallel NUDT15 function assays to determine the variants’ effect on protein abundance and thiopurine cytotoxicity. Of the 3,097 possible missense variants, we characterized the abundance of 2,922 variants and found 54 hotspot residues at which variants resulted in complete loss of protein stability. Analyzing 2,935 variants in the thiopurine cytotoxicity-based assay, we identified 17 additional residues where variants altered NUDT15 activity without affecting protein stability. We identified structural elements key to NUDT15 stability and/or catalytical activity with single amino acid resolution. Functional effects for NUDT15 variants accurately predicted toxicity risk alleles in patients treated with thiopurines with far superior sensitivity and specificity compared to bioinformatic prediction algorithms. In conclusion, our massively parallel variant function assays identified 1,152 deleterious NUDT15 variants, providing a comprehensive reference of variant function and vastly improving the ability to implement pharmacogenetics-guided thiopurine treatment individualization.
Deep Mutational Scan of an SCN5A Voltage Sensor.
Circ Genom Precis Med. 2020 Jan 12
Background – Variants in ion channel genes have classically been studied in low-throughput by patch clamping. Deep Mutational Scanning (DMS) is a complementary approach that can simultaneously assess function of thousands of variants. Methods – We have developed and validated a method to perform a DMS of variants in SCN5A, which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of SCN5A in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells. Results – In preliminary experiments, challenge with three drugs (veratridine, brevetoxin, and ouabain) could discriminate wildtype channels from gain and loss of function pathogenic variants. High-throughput sequencing of the pre- and post-drug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The DMS scores identified 40 putative gain of function and 33 putative loss of function variants. For 8/9 variants, patch clamping data was consistent with the scores. Conclusions – These experiments demonstrate the accuracy of a high-throughput in vitro scan of SCN5A variant function, which can be used to identify deleterious variants in SCN5A and other ion channel genes.
Multiplex assessment of protein variant abundance by massively parallel sequencing.
Nat Genet. 2018 Jun; 50(6):874-882
Multiplex genetic assays can simultaneously test thousands of genetic variants for a property of interest. However, limitations of existing multiplex assay methods in cultured mammalian cells hinder the breadth, speed and scale of these experiments. Here, we describe a series of improvements that greatly enhance the capabilities of a Bxb1 recombinase-based landing pad system for conducting different types of multiplex genetic assays in various mammalian cell lines. We incorporate the landing pad into a lentiviral vector, easing the process of generating new landing pad cell lines. We also develop several new landing pad versions, including one where the Bxb1 recombinase is expressed from the landing pad itself, improving recombination efficiency more than 2-fold and permitting rapid prototyping of transgenic constructs. Other versions incorporate positive and negative selection markers that enable drug-based enrichment of recombinant cells, enabling the use of larger libraries and reducing costs. A version with dual convergent promoters allows enrichment of recombinant cells independent of transgene expression, permitting the assessment of libraries of transgenes that perturb cell growth and survival. Lastly, we demonstrate these improvements by assessing the effects of a combinatorial library of oncogenes and tumor suppressors on cell growth. Collectively, these advancements make multiplex genetic assays in diverse cultured cell lines easier, cheaper and more effective, facilitating future studies probing how proteins impact cell function, using transgenic variant libraries tested individually or in combination.
On the design of CRISPR-based single cell molecular screens.
Nat Methods. 2018 Apr; 15(4):271-274.
Several groups recently coupled CRISPR perturbations and single-cell RNA-seq for pooled genetic screens. We demonstrate that vector designs of these studies are susceptible to ∼50% swapping of guide RNA-barcode associations because of lentiviral template switching. We optimized a published alternative, CROP-seq, in which the guide RNA also serves as the barcode, and here confirm that this strategy performs robustly and doubled the rate at which guides are assigned to cells to 94%.
A platform for functional assessment of large variant libraries in mammalian cells.
Nucleic Acids Res. 2017 Jun;15(4):e102.
Sequencing-based, massively parallel genetic assays have revolutionized our ability to quantify the relationship between many genotypes and a phenotype of interest. Unfortunately, variant library expression platforms in mammalian cells are far from ideal, hindering the study of human gene variants in their physiologically relevant cellular contexts. Here, we describe a platform for phenotyping variant libraries in transfectable mammalian cell lines in two steps. First, a landing pad cell line with a genomically integrated, Tet-inducible cassette containing a Bxb1 recombination site is created. Second, a single variant from a library of transfected, promoter-less plasmids is recombined into the landing pad in each cell. Thus, every cell in the recombined pool expresses a single variant, allowing for parallel, sequencing-based assessment of variant effect. We describe a method for incorporating a single landing pad into a defined site of a cell line of interest, and show that our approach can be used generate more than 20 000 recombinant cells in a single experiment. Finally, we use our platform in combination with a sequencing-based assay to explore the N-end rule by simultaneously measuring the effects of all possible N-terminal amino acids on protein expression.
Host and viral determinants for MxB restriction of HIV-1 infection.
Retrovirology. 2014 Oct; 11:90.
BACKGROUND: Interferon-induced cellular proteins play important roles in the host response against viral infection. The Mx family of dynamin-like GTPases, which include MxA and MxB, target a wide variety of viruses. Despite considerable evidence demonstrating the breadth of antiviral activity of MxA, human MxB was only recently discovered to specifically inhibit lentiviruses. Here we assess both host and viral determinants that underlie MxB restriction of HIV-1 infection.
RESULTS: Heterologous expression of MxB in human osteosarcoma cells potently inhibited HIV-1 infection (~12-fold), yet had little to no effect on divergent retroviruses. The anti-HIV effect manifested as a partial block in the formation of 2-long terminal repeat circle DNA and hence nuclear import, and we accordingly found evidence for an additional post-nuclear entry block. A large number of previously characterized capsid mutations, as well as mutations that abrogated integrase activity, counteracted MxB restriction. MxB expression suppressed integration into gene-enriched regions of chromosomes, similar to affects observed previously when cells were depleted for nuclear transport factors such as transportin 3. MxB activity did not require predicted GTPase active site residues or a series of unstructured loops within the stalk domain that confer functional oligomerization to related dynamin family proteins. In contrast, we observed an N-terminal stretch of residues in MxB to harbor key determinants. Protein localization conferred by a nuclear localization signal (NLS) within the N-terminal 25 residues, which was critical, was fully rescuable by a heterologous NLS. Consistent with this observation, a heterologous nuclear export sequence (NES) abolished full-length MxB activity. We additionally mapped sub-regions within amino acids 26-90 that contribute to MxB activity, finding sequences present within residues 27-50 particularly important.
CONCLUSIONS: MxB inhibits HIV-1 by interfering with minimally two steps of infection, nuclear entry and post-nuclear trafficking and/or integration, without destabilizing the inherent catalytic activity of viral preintegration complexes. Putative MxB GTPase active site residues and stalk domain Loop 4 — both previously shown to be necessary for MxA function — were dispensable for MxB antiviral activity. Instead, we highlight subcellular localization and a yet-determined function(s) present in the unique MxB N-terminal region to be required for HIV-1 restriction.
Structural insight into HIV-1 restriction by MxB.
Cell Host Microbe. 2014 Nov; 16:1-12.
* contributed equally
The myxovirus resistance (Mx) proteins are interferon-induced dynamin GTPases that can inhibit a variety of viruses. Recently, MxB, but not MxA, was shown to restrict HIV-1 by an unknown mechanism that likely occurs in close proximity to the host cell nucleus and involves the viral capsid. Here, we present the crystal structure of MxB and reveal determinants involved in HIV-1 restriction. MxB adopts an extended antiparallel dimer and dimerization, but not higher-ordered oligomerization, is critical for restriction. Although MxB is structurally similar to MxA, the orientation of individual domains differs between MxA and MxB, and their antiviral functions rely on separate determinants, indicating distinct mechanisms for virus inhibition. Additionally, MxB directly binds the HIV-1 capsid, and this interaction depends on dimerization and the N terminus of MxB as well as the assembled capsid lattice. These insights establish a framework for understanding the mechanism by which MxB restricts HIV-1.
Nucleoporin NUP153 phenylalanine-glycine motifs engage a common binding pocket within the HIV-1 capsid protein to mediate lentiviral infectivity.
PLoS Pathog. 2013 Oct;9(10):e1003693.
Lentiviruses can infect non-dividing cells, and various cellular transport proteins provide crucial functions for lentiviral nuclear entry and integration. We previously showed that the viral capsid (CA) protein mediated the dependency on cellular nucleoporin (NUP) 153 during HIV-1 infection, and now demonstrate a direct interaction between the CA N-terminal domain and the phenylalanine-glycine (FG)-repeat enriched NUP153 C-terminal domain (NUP153(C)). NUP153(C) fused to the effector domains of the rhesus Trim5α restriction factor (Trim-NUP153(C)) potently restricted HIV-1, providing an intracellular readout for the NUP153(C)-CA interaction during retroviral infection. Primate lentiviruses and equine infectious anemia virus (EIAV) bound NUP153(C) under these conditions, results that correlated with direct binding between purified proteins in vitro. These binding phenotypes moreover correlated with the requirement for endogenous NUP153 protein during virus infection. Mutagenesis experiments concordantly identified NUP153(C) and CA residues important for binding and lentiviral infectivity. Different FG motifs within NUP153(C) mediated binding to HIV-1 versus EIAV capsids. HIV-1 CA binding mapped to residues that line the common alpha helix 3/4 hydrophobic pocket that also mediates binding to the small molecule PF-3450074 (PF74) inhibitor and cleavage and polyadenylation specific factor 6 (CPSF6) protein, with Asn57 (Asp58 in EIAV) playing a particularly important role. PF74 and CPSF6 accordingly each competed with NUP153(C) for binding to the HIV-1 CA pocket, and significantly higher concentrations of PF74 were needed to inhibit HIV-1 infection in the face of Trim-NUP153(C) expression or NUP153 knockdown. Correlation between CA mutant viral cell cycle and NUP153 dependencies moreover indicates that the NUP153(C)-CA interaction underlies the ability of HIV-1 to infect non-dividing cells. Our results highlight similar mechanisms of binding for disparate host factors to the same region of HIV-1 CA during viral ingress. We conclude that a subset of lentiviral CA proteins directly engage FG-motifs present on NUP153 to affect viral nuclear import.
Differential effects of human immunodeficiency virus type 1 capsid and cellular factors nucleoporin 153 and LEDGF/p75 on the efficiency and specificity of viral DNA integration.
J Virol. 2013 Jan;87(1):648-58.
Retroviruses integrate into cellular DNA nonrandomly. Lentiviruses such as human immunodeficiency virus type 1 (HIV-1) favor the bodies of active genes and gene-enriched transcriptionally active regions of chromosomes. The interaction between lentiviral integrase and the cellular protein lens epithelium-derived growth factor (LEDGF)/p75 underlies the targeting of gene bodies, whereas recent research has highlighted roles for the HIV-1 capsid (CA) protein and cellular factors implicated in viral nuclear import, including transportin 3 (TNPO3) and nucleoporin 358 (NUP358), in the targeting of gene-dense regions of chromosomes. Here, we show that CA mutations, which include the substitution of Asp for Asn74 (N74D), significantly reduce the dependency of HIV-1 on LEDGF/p75 during infection and that this difference correlates with the efficiency of viral DNA integration. The distribution of integration sites mapped by Illumina sequencing confirms that the N74D mutation reduces integration into gene-rich regions of chromosomes and gene bodies and reveals previously unrecognized roles for NUP153 (another HIV-1 cofactor implicated in viral nuclear import) and LEDGF/p75 in the targeting of the viral preintegration complex to gene-dense regions of chromatin. A role for the CA protein in determining the dependency of HIV-1 on LEDGF/p75 during infection highlights a connection between the viral capsid and chromosomal DNA integration.
The requirement for nucleoporin NUP153 during human immunodeficiency virus type 1 infection is determined by the viral capsid.
J Virol. 2011 Aug;85(15):7818-27.
Lentiviruses likely infect nondividing cells by commandeering host nuclear transport factors to facilitate the passage of their preintegration complexes (PICs) through nuclear pore complexes (NPCs) within nuclear envelopes. Genome-wide small interfering RNA screens previously identified karyopherin β transportin-3 (TNPO3) and NPC component nucleoporin 153 (NUP153) as being important for infection by human immunodeficiency virus type 1 (HIV-1). The knockdown of either protein significantly inhibited HIV-1 infectivity, while infection by the gammaretrovirus Moloney murine leukemia virus (MLV) was unaffected. Here, we establish that primate lentiviruses are particularly sensitive to NUP153 knockdown and investigate HIV-1-encoded elements that contribute to this dependency. Mutants lacking functional Vpr or the central DNA flap remained sensitive to NUP153 depletion, while MLV/HIV-1 chimera viruses carrying MLV matrix, capsid, or integrase became less sensitive when the latter two elements were substituted. Two capsid missense mutant viruses, N74D and P90A, were largely insensitive to NUP153 depletion, as was wild-type HIV-1 when cyclophilin A was depleted simultaneously or when infection was conducted in the presence of cyclosporine A. The codepletion of NUP153 and TNPO3 yielded synergistic effects that outweighed those calculated based on individual knockdowns, indicating potential interdependent roles for these factors during HIV-1 infection. Quantitative PCR revealed normal levels of late reverse transcripts, a moderate reduction of 2-long terminal repeat (2-LTR) circles, and a relatively large reduction in integrated proviruses upon NUP153 knockdown. These results suggest that capsid, likely by the qualities of its uncoating, determines whether HIV-1 requires cellular NUP153 for PIC nuclear import.
Differential sensitivities of retroviruses to integrase strand transfer inhibitors.
J Virol. 2011 Apr;85(7):3677-82.
Integrase inhibitors are emerging anti-human immunodeficiency virus (HIV) drugs, and multiple retroviruses and transposable elements were evaluated here for susceptibilities to raltegravir (RAL) and elvitegravir (EVG). All viruses, including primate and nonprimate lentiviruses, a Betaretrovirus, a Gammaretrovirus, and the Alpharetrovirus Rous sarcoma virus (RSV), were susceptible to inhibition by RAL. EVG potently inhibited all lentiviruses and intermediately inhibited Betaretrovirus and Gammaretrovirus infections yet was basically ineffective against RSV. Substitutions based on HIV type 1 (HIV-1) resistance changes revealed that integrase residue Ser150 contributed significantly to the resistance of RSV. The drugs intermediately inhibited intracisternal A-particle retrotransposition but were inactive against Sleeping Beauty transposition and long interspersed nucleotide element 1 (LINE-1) retrotransposition.
The requirement for cellular transportin 3 (TNPO3 or TRN-SR2) during infection maps to human immunodeficiency virus type 1 capsid and not integrase.
J Virol. 2010 Jan;84(1):397-406.
* contributed equally
Recent genome-wide screens have highlighted an important role for transportin 3 in human immunodeficiency virus type 1 (HIV-1) infection and preintegration complex (PIC) nuclear import. Moreover, HIV-1 integrase interacted with recombinant transportin 3 protein under conditions whereby Moloney murine leukemia virus (MLV) integrase failed to do so, suggesting that integrase-transportin 3 interactions might underscore active retroviral PIC nuclear import. Here we correlate infectivity defects in transportin 3 knockdown cells with in vitro protein binding affinities for an expanded set of retroviruses that include simian immunodeficiency virus (SIV), bovine immunodeficiency virus (BIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), and Rous sarcoma virus (RSV) to critically address the role of integrase-transportin 3 interactions in viral infection. Lentiviruses, with the exception of FIV, display a requirement for transportin 3 in comparison to MLV and RSV, yielding an infection-based dependency ranking of SIV > HIV-1 > BIV and EIAV > MLV, RSV, and FIV. In vitro pulldown and surface plasmon resonance assays, in contrast, define a notably different integrase-transportin 3 binding hierarchy: FIV, HIV-1, and BIV > SIV and MLV > EIAV. Our results therefore fail to support a critical role for integrase binding in dictating transportin 3 dependency during retrovirus infection. In addition to integrase, capsid has been highlighted as a retroviral nuclear import determinant. Accordingly, MLV/HIV-1 chimera viruses pinpoint the genetic determinant of sensitization to transportin 3 knockdown to the HIV-1 capsid protein. We therefore conclude that capsid, not integrase, is the dominant viral factor that dictates transportin 3 dependency during HIV-1 infection.
Polybasic KKR motif in the cytoplasmic tail of Nipah virus fusion protein modulates membrane fusion by inside-out signaling.
J Virol. 2007 May;81(9):4520-32.
The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membrane-adjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3- to 5-fold. These results were corroborated in a reverse-pseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper- or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where approximately 20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P = 0.007, r(2) = 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.
N-glycans on Nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry.
J Virol. 2006 May;80(10):4878-89.
Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusion-inhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F’s interactions with NiV-G, providing support for the attachment protein “displacement” model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this “shielding” role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.
The Impact of Genetic Variants on PTEN Molecular Functions and Cellular Phenotypes.
Cold Spring Harb Perspect Med. 2019 Aug 26. pii: a036228. doi: 10.1101/cshperspect.a036228.
Phosphatase and tensin homolog (PTEN) is a tumor suppressor that directly regulates a diverse array of cellular phenotypes, including growth, migration, morphology, and genome stability. How a single protein impacts so many important cellular processes remains a fascinating question. This question has been partially resolved by the characterization of a slew of missense variants that alter or eliminate PTEN’s various molecular functions, including its enzymatic activity, subcellular localization, and posttranslational modifications. Here, we review what is known about how PTEN variants impact molecular function and, consequently, cellular phenotype. In particular, we highlight eight informative “sentinel variants” that abrogate distinct molecular functions of PTEN. We consider two published massively parallel assays of variant effect that measured the effect of thousands of PTEN variants on protein abundance and enzymatic activity. Finally, we discuss how characterization of clinically ascertained variants, establishment of clinical sequencing databases, and massively parallel assays of variant effect yield complementary datasets for dissecting PTEN‘s role in disease.
Viral and cellular requirements for the nuclear entry of retroviral preintegration nucleoprotein complexes.
Viruses. 2013 Oct 7;5(10):2483-511. doi: 10.3390/v5102483.
Retroviruses integrate their reverse transcribed genomes into host cell chromosomes as an obligate step in virus replication. The nuclear envelope separates the chromosomes from the cell cytoplasm during interphase, and different retroviral groups deal with this physical barrier in different ways. Gammaretroviruses are dependent on the passage of target cells through mitosis, where they are believed to access chromosomes when the nuclear envelope dissolves for cell division. Contrastingly, lentiviruses such as HIV-1 infect non-dividing cells, and are believed to enter the nucleus by passing through the nuclear pore complex. While numerous virally encoded elements have been proposed to be involved in HIV-1 nuclear import, recent evidence has highlighted the importance of HIV-1 capsid. Furthermore, capsid was found to be responsible for the viral requirement of various nuclear transport proteins, including transportin 3 and nucleoporins NUP153 and NUP358, during infection. In this review, we describe our current understanding of retroviral nuclear import, with emphasis on recent developments on the role of the HIV-1 capsid protein.
Viral latency and potential eradication of HIV-1.
Expert Rev Anti Infect Ther. 2012 Aug;10(8):855-7.
* contributed equally
Although HAART can suppress plasma viral loads to undetectable levels, individuals infected with HIV-1 harbor latent reservoirs of integrated proviruses that re-emerge upon the cessation of drug treatment. The 2012 Keystone Symposium on Frontiers in HIV Pathogenesis, Therapy and Eradication highlighted the current understanding of latent infection and new methods to activate and target these reservoirs for eradication. This report focuses on a select few aspects of the discussion, including the extent that ongoing replication might contribute to the persistent viral reservoir, recent advances in activating the expression of latent proviruses, progress in developing effective animal models and potential avenues to eradicate the cells that constitute the latent reservoir.