RESUMO
Amino acid positions recognized by monoclonal antibodies (MAbs) in the influenza A virus nucleoprotein (NP) have been reported. As these residues were scattered in the three-dimensional (3D) structure of NP, no patterns of the architecture of antibody-binding sites could be inferred. Here, we used site-specific mutagenesis and ELISA to screen the amino acids surrounding position 470 recognized by the MAb 3/1 as a linear epitope. Ten amino acid residues involved in the reaction of NP with the MAb 3/1 and the MAb 469/6 were identified. Our data are the first to outline a compact site recognized by MAbs in the 3D structure of the influenza virus NP.
Assuntos
Sítios de Ligação de Anticorpos/genética , Vírus da Influenza A/genética , Vírus da Influenza A/imunologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/genética , Proteínas do Core Viral/imunologia , Análise Mutacional de DNA , Vírus da Influenza A/química , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas do Nucleocapsídeo , Conformação Proteica , Proteínas de Ligação a RNA/química , Proteínas do Core Viral/químicaRESUMO
Abstract Influenza virus nucleoprotein (NP) binds to the viral genome RNA and forms the internal ribonucleoprotein complex of the virus particle. Avian and human influenza virus NP have characteristic differences at several amino acid positions. It is not known whether any of these differences can be recognized by antibodies. In the present study five monoclonal antibodies (MAbs) were produced against NP of A/Duck/Novosibirsk/56/05 (H5N1) influenza virus. Two MAbs discerned human and avian influenza strains on ELISA testing. The NP expressed in a prokaryotic system was used for the analysis of site-specific mutants carrying amino acid substitutions in the relevant positions. Amino acid residues in positions 100 and 101 were shown to be recognized by the MAbs. The residue in position 100 is host-specific, and its recognition by the MAb 2E6 may be useful for the differentiation of human and avian viruses. The data are discussed in view of the effects of amino acid substitutions in influenza virus NP affecting both host range and antibody-binding specificity.
Assuntos
Anticorpos Antivirais/imunologia , Epitopos/imunologia , Virus da Influenza A Subtipo H5N1/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/imunologia , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Patos , Epitopos/genética , Humanos , Virus da Influenza A Subtipo H5N1/isolamento & purificação , Influenza Aviária/virologia , Influenza Humana/virologia , Proteínas Mutantes/genética , Proteínas Mutantes/imunologia , Proteínas do Nucleocapsídeo , Ligação ProteicaRESUMO
The locations of amino acid positions relevant to antigenic variation in the nucleoprotein (NP) of influenza virus are not conclusively known. We analysed the antigenic structure of influenza A virus NP by introducing site-specific mutations at amino acid positions presumed to be relevant for the differentiation of strain differences by anti-NP monoclonal antibodies. Mutant proteins were expressed in a prokaryotic system and analysed by performing ELISA with monoclonal antibodies. Four amino acid residues were found to determine four different antibody-binding sites. When mapped in a 3D X-ray model of NP, the four antigenically relevant amino acid positions were found to be located in separate physical sites of the NP molecule.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Epitopos de Linfócito B/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/imunologia , Substituição de Aminoácidos/genética , Animais , Sítios de Ligação de Anticorpos , Ensaio de Imunoadsorção Enzimática , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas do Nucleocapsídeo , Estrutura Terciária de ProteínaRESUMO
To elucidate the structure of the antigenic sites of avian H5 influenza virus haemagglutinin (HA) we analysed escape mutants of a mouse-adapted variant of the H5N2 strain A/Mallard/Pennsylvania/10218/84. A panel of five anti-H5 monoclonal antibodies (mAbs) was used to select 16 escape mutants. The mutants were tested by ELISA and haemagglutination inhibition with this panel of anti-H5 mAbs and the HA genes of the mutants were sequenced. The sequencing demonstrated that the amino acid changes were grouped in two antigenic sites. One corresponded to site A in the H3 HA. The other contained areas that are separated in the amino acid sequence but are topographically close in the three-dimensional structure and partially overlap in the reactions with mAbs. This site corresponds in part to site B in the H3 structure; it also includes a region not involved in site B that partially overlaps site Sa in the H1 HA and an antigenic area in H2 HA. Mutants with the amino acid change K152N, as well as those with the change D126N, showed reduced lethality in mice. The substitution D126N, creating a new glycosylation site, was accompanied by an increase in the sensitivity of the mutants to normal mouse serum inhibitors. Several amino acid changes in the H5 escape mutants occurred at the positions of reported changes in H2 drift variants. This coincidence suggests that the antigenic sites described and analysed here may be important for drift variation if H5 influenza virus ever appears as a pathogen circulating in humans.