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Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening.

ABSTRACT: Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensatory effect of E214D is applicable in both seasonal influenza virus strain A/New Caledonia/20/1999 and 2009 pandemic swine influenza virus strain A/California/04/2009. The technique described here has the potential to profile a gene at the single-nucleotide level to comprehend the dynamics of mutation space and fitness and thus offers prediction power for emerging mutant species.

SUBMITTER: Wu NC

PROVIDER: S-EPMC3554097 | biostudies-literature | 2013 Jan

REPOSITORIES: biostudies-literature

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Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening.

Wu Nicholas C NC Young Arthur P AP Dandekar Sugandha S Wijersuriya Hemani H Al-Mawsawi Laith Q LQ Wu Ting-Ting TT Sun Ren R

Journal of virology 20121114 2

Compensatory mutations contribute to the appearance of the oseltamivir resistance substitution H274Y in the neuraminidase (NA) gene of H1N1 influenza viruses. Here, we describe a high-throughput screening method utilizing error-prone PCR and next-generation sequencing to comprehensively screen NA genes for H274Y compensatory mutations. We found four mutations that can either fully (R194G, E214D) or partially (L250P, F239Y) compensate for the fitness deficiency of the H274Y mutant. The compensato ...[more]

PMID: 23152521

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Project description:UNLABELLED:Influenza A virus (IAV) infections cause major morbidity and mortality, generating an urgent need for novel antiviral therapeutics. We recently established a dual myxovirus high-throughput screening protocol that combines a fully replication-competent IAV-WSN strain and a respiratory syncytial virus reporter strain for the simultaneous identification of IAV-specific, paramyxovirus-specific, and broad-spectrum inhibitors. In the present study, this protocol was applied to a screening campaign to assess a diverse chemical library with over 142,000 entries. Focusing on IAV-specific hits, we obtained a hit rate of 0.03% after cytotoxicity testing and counterscreening. Three chemically distinct hit classes with nanomolar potency and favorable cytotoxicity profiles were selected. Time-of-addition, minigenome, and viral entry studies demonstrated that these classes block hemagglutinin (HA)-mediated membrane fusion. Antiviral activity extends to an isolate from the 2009 pandemic and, in one case, another group 1 subtype. Target identification through biolayer interferometry confirmed binding of all hit compounds to HA. Resistance profiling revealed two distinct escape mechanisms: primary resistance, associated with reduced compound binding, and secondary resistance, associated with unaltered binding. Secondary resistance was mediated, unusually, through two different pairs of cooperative mutations, each combining a mutation eliminating the membrane-proximal stalk N-glycan with a membrane-distal change in HA1 or HA2. Chemical synthesis of an analog library combined with in silico docking extracted a docking pose for the hit classes. Chemical interrogation spotlights IAV HA as a major druggable target for small-molecule inhibition. Our study identifies novel chemical scaffolds with high developmental potential, outlines diverse routes of IAV escape from entry inhibition, and establishes a path toward structure-aided lead development. IMPORTANCE:This study is one of the first to apply a fully replication-competent third-generation IAV reporter strain to a large-scale high-throughput screen (HTS) drug discovery campaign, allowing multicycle infection and screening in physiologically relevant human respiratory cells. A large number of potential druggable targets was thus chemically interrogated, but mechanistic characterization, positive target identification, and resistance profiling demonstrated that three chemically promising and structurally distinct hit classes selected for further analysis all block HA-mediated membrane fusion. Viral escape from inhibition could be achieved through primary and secondary resistance mechanisms. In silico docking predicted compound binding to a microdomain located at the membrane-distal site of the prefusion HA stalk that was also previously suggested as a target site for chemically unrelated HA inhibitors. This study identifies an unexpected chemodominance of the HA stalk microdomain for small-molecule inhibitors in IAV inhibitor screening campaigns and highlights a novel mechanism of cooperative resistance to IAV entry blockers.

Project description:Neuraminidase (NA) is a pivotal surface enzyme and a key therapeutic target in combating the influenza A virus. Its evolution can lead to potential zoonotic transmission, seasonal epidemics, and the emergence of drug-resistant mutants. To gain comprehensive insights into the mutational effects and drug resistance profiles of NA, we employed a high-throughput profiling system to quantify the replication capacity of NA mutants at the single-nucleotide level in mouse lung tissues. The fitness of NA mutants is generally correlated with natural mutation occurrence and is constrained by both the requirement to maintain protein stability and NA function. Leveraging this system, we profiled the drug resistance to the three most commonly used neuraminidase inhibitors (NAIs): zanamivir, oseltamivir, and peramivir. In addition to identifying previously reported drug resistance mutations, we validated novel mutants. Notably, we identified an allosteric mutation that confers resistance to all three drugs, which may affect drug binding by interfering with the tetramerization of NA. Moreover, the fitness cost associated with drug-resistant mutations may limit their widespread dissemination. In summary, we provided a parallel characterization of NA's fitness and drug resistance landscape in an in vivo context, which may guide the rational selection of antiviral drugs for optimal therapeutic efficacy and second-generation NAI development. IMPORTANCE NA is a crucial surface antigen and drug target of influenza A virus. A comprehensive understanding of NA's mutational effect and drug resistance profiles in vivo is essential for comprehending the evolutionary constraints and making informed choices regarding drug selection to combat resistance in clinical settings. In the current study, we established an efficient deep mutational screening system in mouse lung tissues and systematically evaluated the fitness effect and drug resistance to three neuraminidase inhibitors of NA single-nucleotide mutations. The fitness of NA mutants is generally correlated with a natural mutation in the database. The fitness of NA mutants is influenced by biophysical factors such as protein stability, complex formation, and the immune response triggered by viral infection. In addition to confirming previously reported drug-resistant mutations, novel mutations were identified. Interestingly, we identified an allosteric drug-resistance mutation that is not located within the drug-binding pocket but potentially affects drug binding by interfering with NA tetramerization. The dual assessments performed in this study provide a more accurate assessment of the evolutionary potential of drug-resistant mutations and offer guidance for the rational selection of antiviral drugs.

Dataset Information

Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening.

Publications

Systematic identification of H274Y compensatory mutations in influenza A virus neuraminidase by high-throughput screening.

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