RESUMO
Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homo-polymers formation, however their antigenic structure is still incompletely known. Herein, we report that RNase-resistant intracellular NP homo-polymers have a highly ordered conformational antigenic epitope, which depends on inter-subunit interactions of monomeric NPs. Our studies have shown that in radioimmunoprecipitation (RIPA) intracellular NP polymers bind mAb N5D3 and RNase does not prevent their mAb binding. In contrast to NP polymers, NP monomeric subunits, obtained by thermo-dissociation of NP polymers, fail to bind the mAb N5D3 in RIPA. At the same time, the in vitro concentration of thermo-denatured monomeric NPs in both soluble and immobilized forms results in NP-NP association, accompanied by renaturation of the N5D3 epitope. The same results were detected by Western blotting, where the pre-denatured NP monomers were concentrated on nitrocellulose into a single 56 kDa band, which then caused NP-NP self-association as well as N5D3 epitope renaturation. Thus, the in vitro renaturation of N5D3 epitope is markedly dependent on NP monomers concentration. The results obtained suggest that in vivo formation and in vitro renaturation of the N5D3 epitope depend on inter-subunit interactions of monomeric NPs and NP-NP interactions influence the antigenic structure of the influenza virus NP polymers.
Assuntos
Antígenos Virais/química , Epitopos/química , Vírus da Influenza A Subtipo H3N8/imunologia , Nucleoproteínas/imunologia , Proteínas de Ligação a RNA/imunologia , Proteínas do Core Viral/imunologia , Animais , Anticorpos Antivirais/imunologia , Antígenos Virais/imunologia , Biopolímeros/química , Biopolímeros/imunologia , Linhagem Celular , Galinhas/virologia , Cães , Ovos/virologia , Epitopos/imunologia , Proteínas do Nucleocapsídeo , Nucleoproteínas/química , Nucleoproteínas/isolamento & purificação , Nucleoproteínas/metabolismo , Renaturação Proteica , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/isolamento & purificação , Proteínas de Ligação a RNA/metabolismo , Coelhos , Ensaio de Radioimunoprecipitação , Temperatura , Proteínas do Core Viral/química , Proteínas do Core Viral/isolamento & purificação , Proteínas do Core Viral/metabolismoRESUMO
It has been previously shown that influenza virus nucleocapsid protein (NP) forms homooligomers in vivo. Our analyses revealed that the reducing agent dithiothreitol (DTT) introduced in pulse labeling period prevented further formation of native NP-oligomers. The shortly pulse-labeled non-reduced newly synthesized NP possessed a relatively faster mobility in non-reducing PAGE and a higher resistance to protease than the reduced one. These data suggest that there is an early disulfide-dependent step in NP maturation and that the newly synthesized NP possesses the intrachain disulfide bonds. In contrast to the newly synthesized NP, the non-reduced chased NP possessed the same mobility in non-reducing PAGE and the same sensitivity to protease as the reduced NP. DTT introduced in the chase period did not prevent NP-oligomers formation and did not destabilize already formed NP-oligomers. This suggests that the chased NP monomers and NP-oligomers do not contain intrachain nor interchain disulfide bonds. It was also shown that the non-reduced newly synthesized NP could not form NP-NP complexes in vitro, and acquired such ability only after reducing. The possibility is discussed that there are several stages in the maturation of NP: the initial formation of intrachain disulfide-linked NP and conversion into disulfide-free NP, which forms non-covalently stabilized NP-oligomers. Early intrachain disulfide bonds may be necessary for the prevention of early spontaneous NP-NP association.