Transient disulfide bonds formation in conformational maturation of influenza virus nucleocapsid protein (NP).
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.
Prokudina
Elena N
EN
The D.I. Ivanovsky Institute of Virology, Gamaleya Str. 16, 123098, Moscow, Russia. prokudinaen@mail.ru
Semenova
N P
NP
Chumakov
V M
VM
Rudneva
I A
IA
D000818:Animals; D002460:Cell Line; D004220:Disulfides / Q000737:chemistry; D004229:Dithiothreitol / Q000378:metabolism; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D009980:Influenza A virus / Q000737:chemistry* / Q000254:growth & development; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins / Q000737:chemistry* / Q000302:isolation & purification; D010084:Oxidation-Reduction; D011485:Protein Binding; D011487:Protein Conformation; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins / Q000737:chemistry* / Q000302:isolation & purification
D004220:Disulfides; C048842:NP protein, Influenza A virus; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D016601:RNA-Binding Proteins; D014758:Viral Core Proteins; D004229:Dithiothreitol