RESUMO
Influenza A virus (IAV), a major cause of human morbidity and mortality, continuously evolves in response to selective pressures. Stem-directed, broadly neutralizing antibodies (sBnAbs) targeting the influenza virus hemagglutinin (HA) are a promising therapeutic strategy, but neutralization escape mutants can develop. We used an integrated approach combining viral passaging, deep sequencing, and protein structural analyses to define escape mutations and mechanisms of neutralization escape in vitro for the F10 sBnAb. IAV was propagated with escalating concentrations of F10 over serial passages in cultured cells to select for escape mutations. Viral sequence analysis revealed three mutations in HA and one in neuraminidase (NA). Introduction of these specific mutations into IAV through reverse genetics confirmed their roles in resistance to F10. Structural analyses revealed that the selected HA mutations (S123G, N460S, and N203V) are away from the F10 epitope but may indirectly impact influenza virus receptor binding, endosomal fusion, or budding. The NA mutation E329K, which was previously identified to be associated with antibody escape, affects the active site of NA, highlighting the importance of the balance between HA and NA function for viral survival. Thus, whole-genome population sequencing enables the identification of viral resistance mutations responding to antibody-induced selective pressure.IMPORTANCE Influenza A virus is a public health threat for which currently available vaccines are not always effective. Broadly neutralizing antibodies that bind to the highly conserved stem region of the influenza virus hemagglutinin (HA) can neutralize many influenza virus strains. To understand how influenza virus can become resistant or escape such antibodies, we propagated influenza A virus in vitro with escalating concentrations of antibody and analyzed viral populations by whole-genome sequencing. We identified HA mutations near and distal to the antibody binding epitope that conferred resistance to antibody neutralization. Additionally, we identified a neuraminidase (NA) mutation that allowed the virus to grow in the presence of high concentrations of the antibody. Virus carrying dual mutations in HA and NA also grew under high antibody concentrations. We show that NA mutations mediate the escape of neutralization by antibodies against HA, highlighting the importance of a balance between HA and NA for optimal virus function.
Assuntos
Farmacorresistência Viral , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/genética , Mutação , Neuraminidase/genética , Animais , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Cães , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vacinas contra Influenza , Células Madin Darby de Rim Canino , Modelos Moleculares , Neuraminidase/química , Testes de Neutralização , Genética Reversa , Análise de Sequência de RNA , Proteínas Virais/química , Proteínas Virais/genéticaRESUMO
The fitness effects of synonymous mutations can provide insights into biological and evolutionary mechanisms. We analyzed the experimental fitness effects of all single-nucleotide mutations, including synonymous substitutions, at the beginning of the influenza A virus hemagglutinin (HA) gene. Many synonymous substitutions were deleterious both in bulk competition and for individually isolated clones. Investigating protein and RNA levels of a subset of individually expressed HA variants revealed that multiple biochemical properties contribute to the observed experimental fitness effects. Our results indicate that a structural element in the HA segment viral RNA may influence fitness. Examination of naturally evolved sequences in human hosts indicates a preference for the unfolded state of this structural element compared to that found in swine hosts. Our overall results reveal that synonymous mutations may have greater fitness consequences than indicated by simple models of sequence conservation, and we discuss the implications of this finding for commonly used evolutionary tests and analyses.
Assuntos
Aptidão Genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Vírus da Influenza A Subtipo H1N1/crescimento & desenvolvimento , Mutação Silenciosa , Substituição de Aminoácidos , Animais , Cães , Evolução Molecular , Células HEK293 , Humanos , Vírus da Influenza A Subtipo H1N1/genética , Células Madin Darby de Rim Canino , Modelos Moleculares , Filogenia , Dobramento de RNA , Suínos , Replicação ViralRESUMO
Biology has, and continues to be, shaped by evolutionary mechanisms. Within the past decade, local fitness landscapes have become experimentally tractable and are providing new perspectives on evolutionary mechanisms. Powered by next-generation sequencing, the impacts of all individual amino acid substitutions on function have been quantified for dozens of proteins. These fitness maps have been utilized to investigate the biophysical underpinnings of existing protein function as well as the appearance and enhancement of new protein functions. This review highlights emerging trends from this rapidly growing area of research, including an expanded understanding of the biophysical mechanisms underlying existing and new protein function, the roles epistasis and adaptation play in shaping evolution, and the prediction of disease-causing alleles in humans.
