RESUMO
The replicative properties of influenza virus hemagglutinin (HA) mutants with altered receptor binding characteristics were analyzed following intranasal inoculation of mice. Among the mutants examined was a virus containing a Y98F substitution at a conserved position in the receptor binding site that leads to a 20-fold reduction in binding. This mutant can replicate as well as wild-type (WT) virus in MDCK cells and in embryonated chicken eggs but is highly attenuated in mice, exhibiting titers in lungs more than 1,000-fold lower than those of the WT. The capacity of the Y98F mutant to induce antibody responses and the structural locations of HA reversion mutations are examined.
Assuntos
Hemaglutininas Virais/genética , Hemaglutininas Virais/metabolismo , Vírus da Influenza A/genética , Vírus da Influenza A/patogenicidade , Mutação de Sentido Incorreto , Proteínas Virais/genética , Proteínas Virais/metabolismo , Substituição de Aminoácidos/genética , Animais , Anticorpos Antivirais/sangue , Sítios de Ligação , Peso Corporal , Linhagem Celular , Embrião de Galinha , Cães , Testes de Inibição da Hemaglutinação , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/imunologia , Pulmão/virologia , Camundongos , Camundongos Endogâmicos BALB C , Modelos Moleculares , Infecções por Orthomyxoviridae/prevenção & controle , Infecções por Orthomyxoviridae/virologia , Estrutura Terciária de Proteína , Virulência , Replicação Viral/fisiologiaRESUMO
A panel of eight single amino acid deletion mutants was generated within the first 24 residues of the fusion peptide domain of the of the hemagglutinin (HA) of A/Aichi/2/68 influenza A virus (H3N2 subtype). The mutant HAs were analyzed for folding, cell surface transport, cleavage activation, capacity to undergo acid-induced conformational changes, and membrane fusion activity. We found that the mutant DeltaF24, at the C-terminal end of the fusion peptide, was expressed in a non-native conformation, whereas all other deletion mutants were transported to the cell surface and could be cleaved into HA1 and HA2 to activate membrane fusion potential. Furthermore, upon acidification these cleaved HAs were able to undergo the characteristic structural rearrangements that are required for fusion. Despite this, all mutants were inhibited for fusion activity based on two separate assays. The results indicate that the mutant fusion peptide domains associate with target membranes in a non-functional fashion, and suggest that structural features along the length of the fusion peptide are likely to be relevant for optimal membrane fusion activity.
Assuntos
Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Glicoproteínas de Hemaglutininação de Vírus da Influenza/fisiologia , Vírus da Influenza A Subtipo H3N2/genética , Vírus da Influenza A Subtipo H3N2/fisiologia , Deleção de Sequência , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/fisiologia , Internalização do Vírus , Sequência de Aminoácidos , Animais , Linhagem Celular , Cricetinae , Genes Virais , Glicoproteínas de Hemaglutininação de Vírus da Influenza/química , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Proteínas Virais de Fusão/químicaRESUMO
Influenza virus entry occurs in endosomes, where acidification triggers irreversible conformational changes of the hemagglutinin glycoprotein (HA) that are required for membrane fusion. The acid-induced HA structural rearrangements have been well documented, and several models have been proposed to relate these to the process of membrane fusion. However, details regarding the role of specific residues in the initiation of structural rearrangements and membrane fusion are lacking. Here we report the results of studies on the HA of A/Aichi/2/68 virus (H3 subtype), in which mutants with changes at several ionizable residues in the vicinity of the "fusion peptide" were analyzed for their effects on the pH at which conformational changes and membrane fusion occur. A variety of phenotypes was obtained, including examples of substitutions that lead to an increase in HA stability at reduced pH. Of particular note was the observation that a histidine to tyrosine substitution at HA1 position 17 resulted in a decrease in pH at which HA structural changes and membrane fusion take place by 0.3 relative to WT. The results are discussed in relation to possible mechanisms by which HA structural rearrangements are initiated at low pH and clade-specific differences near the fusion peptide.