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[This corrects the article DOI: 10.1371/journal.pgen.1005310.].
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Hepatitis C virus (HCV) encodes mechanisms to evade the multilayered antiviral actions of the host immune system. Great progress has been made in elucidating the strategies HCV employs to down-regulate interferon (IFN) production, impede IFN signaling transduction, and impair IFN-stimulated gene (ISG) expression. However, there is a limited understanding of the mechanisms governing how viral proteins counteract the antiviral functions of downstream IFN effectors due to the lack of an efficient approach to identify such interactions systematically. To study the mechanisms by which HCV antagonizes the IFN responses, we have developed a high-throughput profiling platform that enables mapping of HCV sequences critical for anti-IFN function at high resolution. Genome-wide profiling performed with a 15-nt insertion mutant library of HCV showed that mutations in the p7 region conferred high levels of IFN sensitivity, which could be alleviated by the expression of WT p7 protein. This finding suggests that p7 protein of HCV has an immune evasion function. By screening a liver-specific ISG library, we identified that IFI6-16 significantly inhibits the replication of p7 mutant viruses without affecting WT virus replication. In contrast, knockout of IFI6-16 reversed the IFN hypersensitivity of p7 mutant virus. In addition, p7 was found to be coimmunoprecipitated with IFI6-16 and to counteract the function of IFI6-16 by depolarizing the mitochondria potential. Our data suggest that p7 is a critical immune evasion protein that suppresses the antiviral IFN function by counteracting the function of IFI6-16.
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Hepacivirus/patogenicidad , Hepatitis C/inmunología , Interacciones Huésped-Patógeno/inmunología , Evasión Inmune , Interferones/inmunología , Proteínas Mitocondriales/inmunología , Proteínas Virales/inmunología , Sistemas CRISPR-Cas , Línea Celular , Perfilación de la Expresión Génica , Técnicas de Inactivación de Genes , Biblioteca de Genes , Genoma Viral , Hepacivirus/genética , Hepatitis C/virología , Humanos , Inmunidad Innata , Interferones/genética , Interferones/metabolismo , Hígado/inmunología , Hígado/metabolismo , Potencial de la Membrana Mitocondrial/inmunología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutagénesis Insercional , Transducción de Señal , Proteínas Virales/genética , Replicación ViralRESUMEN
Viruses often encode proteins with multiple functions due to their compact genomes. Existing approaches to identify functional residues largely rely on sequence conservation analysis. Inferring functional residues from sequence conservation can produce false positives, in which the conserved residues are functionally silent, or false negatives, where functional residues are not identified since they are species-specific and therefore non-conserved. Furthermore, the tedious process of constructing and analyzing individual mutations limits the number of residues that can be examined in a single study. Here, we developed a systematic approach to identify the functional residues of a viral protein by coupling experimental fitness profiling with protein stability prediction using the influenza virus polymerase PA subunit as the target protein. We identified a significant number of functional residues that were influenza type-specific and were evolutionarily non-conserved among different influenza types. Our results indicate that type-specific functional residues are prevalent and may not otherwise be identified by sequence conservation analysis alone. More importantly, this technique can be adapted to any viral (and potentially non-viral) protein where structural information is available.
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Virus de la Influenza A/genética , Virus de la Influenza B/genética , ARN Polimerasa Dependiente del ARN/genética , Proteínas Virales/genética , Secuencia de Bases , Evolución Biológica , Línea Celular , Biología Computacional , Secuencia Conservada/genética , Biblioteca de Genes , Células HEK293 , Humanos , Análisis de Secuencia de ADNRESUMEN
Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients.
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Farmacorresistencia Viral , Perfilación de la Expresión Génica , Aptitud Genética , Hepacivirus/fisiología , Hepatitis C , Imidazoles/farmacología , Replicación Viral , Carbamatos , Línea Celular , Farmacorresistencia Viral/efectos de los fármacos , Farmacorresistencia Viral/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatitis C/patología , Humanos , Pirrolidinas , Valina/análogos & derivados , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
UNLABELLED: Viral proteins often display several functions which require multiple assays to dissect their genetic basis. Here, we describe a systematic approach to screen for loss-of-function mutations that confer a fitness disadvantage under a specified growth condition. Our methodology was achieved by genetically monitoring a mutant library under two growth conditions, with and without interferon, by deep sequencing. We employed a molecular tagging technique to distinguish true mutations from sequencing error. This approach enabled us to identify mutations that were negatively selected against, in addition to those that were positively selected for. Using this technique, we identified loss-of-function mutations in the influenza A virus NS segment that were sensitive to type I interferon in a high-throughput fashion. Mechanistic characterization further showed that a single substitution, D92Y, resulted in the inability of NS to inhibit RIG-I ubiquitination. The approach described in this study can be applied under any specified condition for any virus that can be genetically manipulated. IMPORTANCE: Traditional genetics focuses on a single genotype-phenotype relationship, whereas high-throughput genetics permits phenotypic characterization of numerous mutants in parallel. High-throughput genetics often involves monitoring of a mutant library with deep sequencing. However, deep sequencing suffers from a high error rate (â¼0.1 to 1%), which is usually higher than the occurrence frequency for individual point mutations within a mutant library. Therefore, only mutations that confer a fitness advantage can be identified with confidence due to an enrichment in the occurrence frequency. In contrast, it is impossible to identify deleterious mutations using most next-generation sequencing techniques. In this study, we have applied a molecular tagging technique to distinguish true mutations from sequencing errors. It enabled us to identify mutations that underwent negative selection, in addition to mutations that experienced positive selection. This study provides a proof of concept by screening for loss-of-function mutations on the influenza A virus NS segment that are involved in its anti-interferon activity.
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Virus de la Influenza A/inmunología , Virus de la Influenza A/fisiología , Interferón Tipo I/antagonistas & inhibidores , Mutación , Proteínas no Estructurales Virales/deficiencia , Proteínas no Estructurales Virales/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Virus de la Influenza A/genética , Virus de la Influenza A/crecimiento & desarrollo , Biología Molecular/métodos , ARN Viral/genética , Virología/métodosRESUMEN
BACKGROUND: The HIV-1 pandemic is not the result of a static pathogen but a large genetically diverse and dynamic viral population. The virus is characterized by a highly mutable genome rendering efforts to design a universal vaccine a significant challenge and drives the emergence of drug resistant variants upon antiviral pressure. Gaining a comprehensive understanding of the mutational tolerance of each HIV-1 genomic position is therefore of critical importance. RESULTS: Here we combine high-density mutagenesis with the power of next-generation sequencing to gauge the replication capacity and therefore mutational tolerability of single point mutations across the entire HIV-1 genome. We were able to achieve the evaluation of point mutational effects on viral replicative capacity for 5,553 individual HIV-1 nucleotide positions - representing 57% of the viral genome. Replicative capacity was assessed at 3,943 nucleotide positions for a single alternate base change, 1,459 nucleotide positions for two alternate base changes, and 151 nucleotide positions for all three possible alternate base changes. This resulted in the study of how a total of 7,314 individual point mutations impact HIV-1 replication on a single experimental platform. We further utilize the dataset for a focused structural analysis on a capsid inhibitor binding pocket. CONCLUSION: The approach presented here can be applied to any pathogen that can be genetically manipulated in a laboratory setting. Furthermore, the methodology can be utilized under externally applied selection conditions, such as drug or immune pressure, to identify genetic elements that contribute to drug or host interactions, and therefore mutational routes of pathogen resistance and escape.
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Análisis Mutacional de ADN/métodos , Genoma Viral , VIH-1/genética , Mutación Puntual , VIH-1/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Biología Molecular/métodos , Mutagénesis , Virología/métodos , Replicación ViralRESUMEN
OBJECTIVES: Daclatasvir is a highly potent inhibitor of hepatitis C virus. We estimated the active tissue concentration of daclatasvir in vivo. METHODS: We developed a mathematical model incorporating pharmacokinetic/pharmacodynamic and viral dynamics. By fitting the model to clinical data reported previously, we estimated the ratio between plasma drug concentration and active tissue concentration in vivo. RESULTS: The modelling results show that the active tissue concentration of daclatasvir is â¼9% of the concentration measured in plasma (95% CI 1%-29%). CONCLUSIONS: Using plasma concentrations as surrogates for clinical recommendations may lead to substantial underestimation of the risk of resistance.
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Antivirales/farmacocinética , Hepacivirus/efectos de los fármacos , Imidazoles/farmacocinética , Plasma/química , Antivirales/administración & dosificación , Carbamatos , Ensayos Clínicos como Asunto , Hepacivirus/aislamiento & purificación , Humanos , Imidazoles/administración & dosificación , Modelos Teóricos , Pirrolidinas , Valina/análogos & derivados , Carga ViralRESUMEN
In a single round: By combining the high-efficiency enrichment through the continuous-flow magnetic separation (CFMS) technique with the analytical power of next-generation sequencing, the generation of antibody mimetics with a single round of mRNA display is made possible. This approach eliminates iterative selection cycles and provides a path to fully automated ligand generation (see picture).
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Materiales Biomiméticos/metabolismo , ARN Mensajero/química , Secuencia de Aminoácidos , Anticuerpos/química , Anticuerpos/metabolismo , Materiales Biomiméticos/química , Ensayo de Inmunoadsorción Enzimática , Fibronectinas/química , Fibronectinas/metabolismo , Biblioteca de Genes , Humanos , Fragmentos Fc de Inmunoglobulinas/química , Fragmentos Fc de Inmunoglobulinas/metabolismo , Separación Inmunomagnética , Ligandos , Proteínas de Unión a Maltosa/química , Proteínas de Unión a Maltosa/metabolismo , ARN Mensajero/aislamiento & purificaciónRESUMEN
We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (AAHI-SC2) delivered by a nanostructured lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression and an N antigen modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the AAHI-SC2 vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to confer protease resistance. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen that included the AAHI-SC2 vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AAHI-SC2 prime followed by an AdS+N boost particularly enhanced CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving AAHI-SC2 homologous or heterologous vaccination were found to be highly neutralizing for all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strains. The findings here, taken in consideration with the availability of both vaccines in thermostable formulations, support the testing of heterologous vaccination by an AAHI-SC2 > AdS+N regimen in animal models of SARS-CoV-2 infection to assess its potential to provide increased protection against emerging SARS-CoV-2 variants particularly in regions of the world where the need for cold-chain storage has limited the distribution of other vaccines.
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COVID-19 , Vacunas Virales , Animales , Anticuerpos Neutralizantes , Antígenos Heterófilos , COVID-19/prevención & control , Vacunas contra la COVID-19 , ADN , Humanos , Ratones , SARS-CoV-2 , Vacunación , Vacunas Sintéticas , Vacunas de ARNmRESUMEN
The increasing prevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identify a set of antibodies against SARS-CoV-2 spike (S) proteins and characterize the structures of nAbs that recognize epitopes in the S1 subunit of the S glycoprotein. These structural studies reveal distinct binding modes for several antibodies, including the targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interact with angiotensin-converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. Further, we engineer a potent ACE2-blocking nAb to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is an approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.
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The SARS-CoV-2 variants replacing the first wave strain pose an increased threat by their potential ability to escape pre-existing humoral protection. An angiotensin converting enzyme 2 (ACE2) decoy that competes with endogenous ACE2 for binding of the SARS-CoV-2 spike receptor binding domain (S RBD) and inhibits infection may offer a therapeutic option with sustained efficacy against variants. Here, we used Molecular Dynamics (MD) simulation to predict ACE2 sequence substitutions that might increase its affinity for S RBD and screened candidate ACE2 decoys in vitro. The lead ACE2(T27Y/H34A)-IgG1FC fusion protein with enhanced S RBD affinity shows greater live SARS-CoV-2 virus neutralization capability than wild type ACE2. MD simulation was used to predict the effects of S RBD variant mutations on decoy affinity that was then confirmed by testing of an ACE2 Triple Decoy that included an additional enzyme activity-deactivating H374N substitution against mutated S RBD. The ACE2 Triple Decoy maintains high affinity for mutated S RBD, displays enhanced affinity for S RBD N501Y or L452R, and has the highest affinity for S RBD with both E484K and N501Y mutations, making it a viable therapeutic option for the prevention or treatment of SARS-CoV-2 infection with a high likelihood of efficacy against variants.
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Sustitución de Aminoácidos , Enzima Convertidora de Angiotensina 2/química , Enzima Convertidora de Angiotensina 2/metabolismo , Antivirales/farmacología , COVID-19/metabolismo , Descubrimiento de Drogas/métodos , Simulación de Dinámica Molecular , SARS-CoV-2/metabolismo , Transducción de Señal/efectos de los fármacos , Secuencia de Aminoácidos , COVID-19/virología , Humanos , Mutación , Unión Proteica/efectos de los fármacos , Dominios Proteicos/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Internalización del Virus/efectos de los fármacosRESUMEN
Protein libraries based on natural scaffolds enable the generation of novel molecular tools and potential therapeutics by directed evolution. Here, we report the design and construction of a high complexity library (30 x 10(13) sequences) based on the 10th fibronectin type III domain of human fibronectin (10FnIII). We examined the bacterial expression characteristics and stability of this library using a green fluorescent protein (GFP)-reporter screen, SDS-PAGE analysis, and chemical denaturation, respectively. The high throughput GFP reporter screen demonstrates that a large fraction of our library expresses significant levels of soluble protein in bacteria. However, SDS-PAGE analysis of expression cultures indicates the ratio of soluble to insoluble protein expressed varies greatly for randomly chosen library members. We also tested the stabilities of several representative variants by guanidinium chloride denaturation. All variants tested displayed cooperative unfolding transitions similar to wild-type, and two exhibited free energies of unfolding equal to wild-type 10FnIII. This work demonstrates the utility of GFP-based screening as a tool for analysis of high-complexity protein libraries. Our results indicate that a vast amount of protein sequence space surrounding the 10FnIII scaffold is accessible for the generation of novel functions by directed as well as natural evolution.
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Fibronectinas/química , Ingeniería de Proteínas , Técnicas Químicas Combinatorias , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Fibronectinas/biosíntesis , Fibronectinas/genética , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Guanidina/química , Humanos , Oligonucleótidos/química , Desnaturalización Proteica , Pliegue de Proteína , SolubilidadAsunto(s)
Interleucina-6/antagonistas & inhibidores , ARN Mensajero/metabolismo , Secuencia de Aminoácidos , Anticuerpos/inmunología , Línea Celular Tumoral , Evolución Molecular Dirigida , Humanos , Separación Inmunomagnética , Interleucina-6/genética , Interleucina-6/metabolismo , Ligandos , Microfluídica , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Unión ProteicaRESUMEN
The effect of a mutation on protein stability is traditionally measured by genetic construction, expression, purification, and physical analysis using low-throughput methods. This process is tedious and limits the number of mutants able to be examined in a single study. In contrast, functional fitness effects can be measured in a high-throughput manner by various deep mutational scanning tools. Using protein GB 1, we have recently demonstrated the feasibility of estimating the mutational stability effect ( ΔΔG) of single-substitution based on the functional fitness profile of all double-substitutions. The principle is to identify genetic backgrounds that have an exhausted stability margin. The functional effect of an additional substitution on these genetic backgrounds can then be used to compute the mutational ΔΔG based on the biophysical relationship between functional fitness and thermodynamic stability. However, to identify such genetic backgrounds, the approach described in our previous study required a benchmark dataset, which is a set of known mutational ΔΔG. In this study, a benchmark-independent approach is developed. The genetic backgrounds of interest are identified using k-means clustering with the integration of structural information. We further demonstrated that a reasonable approximation of ΔΔG can also be obtained without taking structural information into account. In summary, this study describes a novel method for computing ΔΔG from double-substitution functional fitness profiles alone, without relying on any known mutational ΔΔG as a benchmark.
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Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mutación , Streptococcus/química , Streptococcus/genética , Sustitución de Aminoácidos , Modelos Moleculares , Estabilidad Proteica , Estructura Terciaria de Proteína , TermodinámicaRESUMEN
The structure of fitness landscapes is critical for understanding adaptive protein evolution. Previous empirical studies on fitness landscapes were confined to either the neighborhood around the wild type sequence, involving mostly single and double mutants, or a combinatorially complete subgraph involving only two amino acids at each site. In reality, the dimensionality of protein sequence space is higher (20(L)) and there may be higher-order interactions among more than two sites. Here we experimentally characterized the fitness landscape of four sites in protein GB1, containing 20(4) = 160,000 variants. We found that while reciprocal sign epistasis blocked many direct paths of adaptation, such evolutionary traps could be circumvented by indirect paths through genotype space involving gain and subsequent loss of mutations. These indirect paths alleviate the constraint on adaptive protein evolution, suggesting that the heretofore neglected dimensions of sequence space may change our views on how proteins evolve.
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Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Evolución Molecular , MutaciónRESUMEN
The classical picture of H-bonds has evolved considerably. In contrast to earlier expectations, C-H...O H-bonds are now known to be prevalent in both small organic and large biological systems. However, there are few reports on the energetic contribution of C-H...O H-bonds in protein or polypeptide systems and we do not know whether such interactions are stabilizing. Here we investigate C-H...O H-bonding interactions between Phe and Glu side chains by determining their effects on the helicity of model alpha-helical peptides using a combination of CD and NMR spectroscopy. The results suggest that Glu/Phe C-H...O H-bonding interactions stabilize helical structure, but only in the orientation Glu --> Phe (N --> C). Each Glu --> Phe (N --> C) interaction can contribute approximately -0.5 kcal mol(-1) to the stability of helical peptide. In the reverse orientation, Phe --> Glu (N --> C) appears to contribute negligibly. pH titrations provide further evidence for the existence of C-H...O H-bonds. The C-H...O H-bonding interactions in these peptides are insensitive to the screening effect of added neutral salt. Our results provide quantitative energetic information on C-H...O H-bonds that should be useful for empirical force-field calibration.
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Ácido Glutámico/química , Péptidos/química , Fenilalanina/química , Secuencia de Aminoácidos , Dicroismo Circular , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Sales (Química)RESUMEN
BACKGROUND: Nonadditivity in fitness effects from two or more mutations, termed epistasis, can result in compensation of deleterious mutations or negation of beneficial mutations. Recent evidence shows the importance of epistasis in individual evolutionary pathways. However, an unresolved question in molecular evolution is how often and how significantly fitness effects change in alternative genetic backgrounds. RESULTS: To answer this question, we quantified the effects of all single mutations and double mutations between all positions in the IgG-binding domain of protein G (GB1). By observing the first two steps of all possible evolutionary pathways using this fitness profile, we were able to characterize the extent and magnitude of pairwise epistasis throughout an entire protein molecule. Furthermore, we developed a novel approach to quantitatively determine the effects of single mutations on structural stability (ΔΔGU). This enabled determination of the importance of stability effects in functional epistasis. CONCLUSIONS: Our results illustrate common biophysical mechanisms for occurrences of positive and negative epistasis. Our results show pervasive positive epistasis within a conformationally dynamic network of residues. The stability analysis shows that significant negative epistasis, which is more common than positive epistasis, mostly occurs between combinations of destabilizing mutations. Furthermore, we show that although significant positive epistasis is rare, many deleterious mutations are beneficial in at least one alternative mutational background. The distribution of conditionally beneficial mutations throughout the domain demonstrates that the functional portion of sequence space can be significantly expanded by epistasis.
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Proteínas Bacterianas/química , Epistasis Genética , Evolución Molecular , Modelos Genéticos , Proteínas Bacterianas/genética , Modelos Moleculares , Mutación , Estabilidad Proteica , Estructura Terciaria de ProteínaRESUMEN
Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce â¼ 2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of â¼ 0.005% to â¼ 0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population.
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VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Genoma Viral/genética , Reproducibilidad de los ResultadosRESUMEN
UNLABELLED: Pairing high-throughput sequencing technologies with high-throughput mutagenesis enables genome-wide investigations of pathogenic organisms. Knowledge of the specific functions of protein domains encoded by the genome of the hepatitis C virus (HCV), a major human pathogen that contributes to liver disease worldwide, remains limited to insight from small-scale studies. To enhance the capabilities of HCV researchers, we have obtained a high-resolution functional map of the entire viral genome by combining transposon-based insertional mutagenesis with next-generation sequencing. We generated a library of 8,398 mutagenized HCV clones, each containing one 15-nucleotide sequence inserted at a unique genomic position. We passaged this library in hepatic cells, recovered virus pools, and simultaneously assayed the abundance of mutant viruses in each pool by next-generation sequencing. To illustrate the validity of the functional profile, we compared the genetic footprints of viral proteins with previously solved protein structures. Moreover, we show the utility of these genetic footprints in the identification of candidate regions for epitope tag insertion. In a second application, we screened the genetic footprints for phenotypes that reflected defects in later steps of the viral life cycle. We confirmed that viruses with insertions in a region of the nonstructural protein NS4B had a defect in infectivity while maintaining genome replication. Overall, our genome-wide HCV mutant library and the genetic footprints obtained by high-resolution profiling represent valuable new resources for the research community that can direct the attention of investigators toward unidentified roles of individual protein domains. IMPORTANCE: Our insertional mutagenesis library provides a resource that illustrates the effects of relatively small insertions on local protein structure and HCV viability. We have also generated complementary resources, including a website (http://hangfei.bol.ucla.edu) and a panel of epitope-tagged mutant viruses that should enhance the research capabilities of investigators studying HCV. Researchers can now detect epitope-tagged viral proteins by established antibodies, which will allow biochemical studies of HCV proteins for which antibodies are not readily available. Furthermore, researchers can now quickly look up genotype-phenotype relationships and base further mechanistic studies on the residue-by-residue information from the functional profile. More broadly, this approach offers a general strategy for the systematic functional characterization of viruses on the genome scale.
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Genoma Viral , Hepacivirus/genética , Proteínas Virales/genética , Línea Celular , Mapeo Cromosómico , Clonación Molecular , Elementos Transponibles de ADN/genética , ADN Viral/genética , Biblioteca de Genes , Hepacivirus/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutagénesis Insercional , Plásmidos , Análisis de Secuencia de ADN , Transcripción Genética , Transfección , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Genetic research on influenza virus biology has been informed in large part by nucleotide variants present in seasonal or pandemic samples, or individual mutants generated in the laboratory, leaving a substantial part of the genome uncharacterized. Here, we have developed a single-nucleotide resolution genetic approach to interrogate the fitness effect of point mutations in 98% of the amino acid positions in the influenza A virus hemagglutinin (HA) gene. Our HA fitness map provides a reference to identify indispensable regions to aid in drug and vaccine design as targeting these regions will increase the genetic barrier for the emergence of escape mutations. This study offers a new platform for studying genome dynamics, structure-function relationships, virus-host interactions, and can further rational drug and vaccine design. Our approach can also be applied to any virus that can be genetically manipulated.