Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein.
Bacterially expressed nucleocapsid (N) protein, from respiratory syncytial virus (RSV), was used to investigate RNA binding in a modified North-Western blotting protocol. The recombinant protein demonstrated no sequence specificity in binding RNA representing either the antigenomic leader sequence or the nonspecific sequence derived from a plasmid vector. When recombinant N was purified on CsCl gradients, two types of structure, both with densities indicating that they contained RNA, could be visualised by negative-stain electron microscopy. Structures similar to nucleocapsids (NC) from RSV-infected cells were observed, as were ring structures. A small fragment of the N (amino acids 1-92) was all that was required for the production of NC-like structures. Another mutant with an internal deletion could form rings but not NC-like structures. This suggests that this domain (amino acids 121-160) may be important for maintaining helical stability. Further analysis has also identified a potential site in the amino-terminus that may be involved in an interaction with the phosphoprotein. A domain model of the RSV N protein is presented which, similar to that of other paramyxoviruses, supports the idea that the amino-terminus is important for NC assembly.
Murphy
Lindsay B
LB
Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, Scotland, UK.
Loney
Colin
C
Murray
Jillian
J
Bhella
David
D
Ashton
Peter
P
Yeo
Robert P
RP
D000818:Animals; D001665:Binding Sites; D002460:Cell Line; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D012136:Respiratory Syncytial Viruses / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins