Prokudina Elena N, Semenova N P, Chumakov V M, Rudneva I A
The D.I. Ivanovsky Institute of Virology, Gamaleya Str. 16, 123098, Moscow, Russia.
Virus Res. 2004 Feb;99(2):169-75. doi: 10.1016/j.virusres.2003.11.008.
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.
先前已表明，流感病毒核衣壳蛋白（NP）在体内形成同源寡聚体。我们的分析显示，在脉冲标记期引入的还原剂二硫苏糖醇（DTT）可阻止天然NP寡聚体的进一步形成。在非还原聚丙烯酰胺凝胶电泳（PAGE）中，经短暂脉冲标记的未还原新合成NP比还原后的NP具有相对更快的迁移率，并且对蛋白酶具有更高的抗性。这些数据表明，NP成熟过程中存在一个早期的二硫键依赖性步骤，并且新合成的NP具有链内二硫键。与新合成的NP相反，在非还原PAGE中，未还原的追踪期NP与还原后的NP具有相同的迁移率，并且对蛋白酶具有相同的敏感性。在追踪期引入的DTT不会阻止NP寡聚体的形成，也不会破坏已形成的NP寡聚体的稳定性。这表明追踪期的NP单体和NP寡聚体既不包含链内也不包含链间二硫键。还表明，未还原的新合成NP在体外不能形成NP-NP复合物，只有在还原后才获得这种能力。文中讨论了NP成熟可能存在几个阶段的可能性：链内二硫键连接的NP的初始形成以及转化为无二硫键的NP，后者形成非共价稳定的NP寡聚体。早期的链内二硫键对于防止早期自发的NP-NP缔合可能是必要的。
