Prokudina Elena N, Semenova Nataly, Chumakov Valery, Stitz Lothar
The D.I. Ivanovsky Institute of Virology, Gamaleya str, 16, Moscow, Russia.
Virol J. 2008 Feb 29;5:37. doi: 10.1186/1743-422X-5-37.
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.
细胞内流感病毒核蛋白（NP）的特点是具有高效形成同聚物的能力，然而其抗原结构仍不完全清楚。在此，我们报告耐核糖核酸酶的细胞内NP同聚物具有高度有序的构象抗原表位，这取决于单体NP的亚基间相互作用。我们的研究表明，在放射免疫沉淀（RIPA）中，细胞内NP聚合物与单克隆抗体N5D3结合，核糖核酸酶并不阻止它们与单克隆抗体的结合。与NP聚合物相反，通过NP聚合物热解离获得的NP单体亚基在RIPA中不能与单克隆抗体N5D3结合。同时，体外热变性的可溶性和固定化形式的单体NP浓度会导致NP-NP缔合，同时N5D3表位复性。蛋白质免疫印迹检测也得到了相同的结果，预变性的NP单体在硝酸纤维素膜上浓缩成一条单一的56 kDa条带，随后导致NP-NP自缔合以及N5D3表位复性。因此，N5D3表位的体外复性明显依赖于NP单体的浓度。所得结果表明，N5D3表位的体内形成和体外复性取决于单体NP的亚基间相互作用，且NP-NP相互作用影响流感病毒NP聚合物的抗原结构。
