It has previously been shown that the purified influenza virus nucleoprotein (NP) forms the oligomers in vitro in NP preparations obtained from virions (Wiley et al., 1977, Virology, 79, 446-448; Ruigrok and Baudin, 1995, J. Gen. Virol., 76, 1009-1014) and infected cells (Becht and Weiss, 1991, Behring Inst Mitt., Justus-Liebig Universitat, Giessen, 89, 1-11). We have shown in this report that boiling-sensitive NP oligomers (di- and trimers) are formed in vivo in the course of intracellular influenza virus replication. They are detected by PAGE about 10 min after monomeric 56-kDa NP molecules are synthesized. NP oligomers are formed by different strains of influenza virus in different cell lines. Some influenza virus strains are characterized by complete conversion of NP monomers into oligomers and others by only partial conversion. In the Triton X-114 phase partitioning system NP oligomers show more hydrophobicity than NP monomers. NP oligomers are detected in the sedimentable and soluble fractions of both cell lysate and extracellular medium. The possibility is discussed that oligomeric NP is a native and functionally significant form of influenza virus NP.
Intracellular influenza virus nucleoprotein (NP) is characterized by a high efficiency of homopolymers 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.
FindingsIt is known that intracellular nucleoprotein (NP) is capable of self-associating to form large RNA-free homo-polymeric complexes [1,2], which are morphologically similar to the intact viral RNP [3][4][5]. We have previously shown that numerous types of RNase resistant thermo-sensitive NP polymers are detected in influenza virus infected MDCK cells [6][7][8]. After heating, NP polymers are dissociated exclusively into NP monomeric subunits. It is also known that protein-protein interactions induce conformational changes at interfaces of subunits. As a result, those polymerizing proteins may acquire new biological properties, including the exposure of new conformational epitopes [9,10]. The antigenic structure of intracellular influenza virus NP homo-polymers is still unknown.In the present study, we have analyzed the total intracellular influenza virus NP polymers and demonstrated in vivo formation and in vitro renaturation of the antigenic epitope depending on NP-NP association.
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