RESUMO
Influenza viruses use distinct antibody escape mechanisms depending on the overall complexity of the antibody response that is encountered. When grown in the presence of a hemagglutinin (HA) monoclonal antibody, influenza viruses typically acquire a single HA mutation that reduces the binding of that specific monoclonal antibody. In contrast, when confronted with mixtures of HA monoclonal antibodies or polyclonal sera that have antibodies that bind several HA epitopes, influenza viruses acquire mutations that increase HA binding to host cells. Recent data from our laboratory and others suggest that some humans possess antibodies that are narrowly focused on HA epitopes that were present in influenza virus strains that they were likely exposed to in childhood. Here, we completed a series of experiments to determine if humans with narrowly focused HA antibody responses are able to select for influenza virus antigenic escape variants in ovo We identified three human donors that possessed HA antibody responses that were heavily focused on a single HA antigenic site. Sera from all three of these donors selected single HA escape mutations during in ovo passage experiments, similar to what has been previously reported for single monoclonal antibodies. These single HA mutations directly reduced binding of serum antibodies used for selection. We propose that new antigenic variants of influenza viruses might originate in individuals who produce antibodies that are narrowly focused on HA epitopes that were present in viral strains that they encountered in childhood.IMPORTANCE Influenza vaccine strains must be updated frequently since circulating viral strains continuously change in antigenically important epitopes. Our previous studies have demonstrated that some individuals possess antibody responses that are narrowly focused on epitopes that were present in viral strains that they encountered during childhood. Here, we show that influenza viruses rapidly escape this type of polyclonal antibody response when grown in ovo by acquiring single mutations that directly prevent antibody binding. These studies improve our understanding of how influenza viruses evolve when confronted with narrowly focused polyclonal human antibodies.
Assuntos
Antígenos Virais/imunologia , Epitopos/imunologia , Glicoproteínas de Hemaglutininação de Vírus da Influenza/imunologia , Evasão da Resposta Imune/genética , Vírus da Influenza A Subtipo H1N1/imunologia , Influenza Humana/imunologia , Mutação , Animais , Anticorpos Neutralizantes/biossíntese , Anticorpos Neutralizantes/química , Anticorpos Antivirais/biossíntese , Anticorpos Antivirais/química , Variação Antigênica , Antígenos Virais/genética , Embrião de Galinha , Epitopos/química , Epitopos/genética , Expressão Gênica , Testes de Inibição da Hemaglutinação , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Soros Imunes/química , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/genética , Influenza Humana/virologia , Modelos Moleculares , Testes de Neutralização , Zigoto/imunologia , Zigoto/virologiaRESUMO
AID/APOBEC family enzymes are best known for deaminating cytosine bases to uracil in single-stranded DNA, with characteristic sequence preferences that can produce mutational signatures in targets such as retroviral and cancer cell genomes. These deaminases have also been proposed to function in DNA demethylation via deamination of either 5-methylcytosine (mC) or TET-oxidized mC bases (ox-mCs), which include 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. One specific family member, APOBEC3A (A3A), has been shown to readily deaminate mC, raising the prospect of broader activity on ox-mCs. To investigate this claim, we developed a novel assay that allows for parallel profiling of activity on all modified cytosines. Our steady-state kinetic analysis reveals that A3A discriminates against all ox-mCs by >3700-fold, arguing that ox-mC deamination does not contribute substantially to demethylation. A3A is, by contrast, highly proficient at C/mC deamination. Under conditions of excess enzyme, C/mC bases can be deaminated to completion in long DNA segments, regardless of sequence context. Interestingly, under limiting A3A, the sequence preferences observed with targeting unmodified cytosine are further exaggerated when deaminating mC. Our study informs how methylation, oxidation, and deamination can interplay in the genome and suggests A3A's potential utility as a biotechnological tool to discriminate between cytosine modification states.
Assuntos
Citidina Desaminase/metabolismo , Citosina/química , DNA/química , DNA/metabolismo , Proteínas/metabolismo , 5-Metilcitosina/química , Sequência de Bases , Metilação de DNA , Humanos , Técnicas In Vitro , Cinética , Modelos Biológicos , Oxirredução , Proteínas Recombinantes/metabolismo , Especificidade por SubstratoRESUMO
A diazo compound is shown to convert carboxylic acids to esters efficiently in an aqueous environment. The basicity of the diazo compound is critical: low basicity does not lead to a reaction but high basicity leads to hydrolysis. This reactivity extends to carboxylic acid groups in a protein. The ensuing esters are hydrolyzed by human cellular esterases to regenerate protein carboxyl groups. This new mode of chemical modification could enable the key advantages of prodrugs to be translated from small-molecules to proteins.