title: Investigations into the amino-terminal domain of the respiratory syncytial virus nucleocapsid protein reveal elements important for nucleocapsid formation and interaction with the phosphoprotein.
abstract: 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.
authors.affiliation: Medical Research Council Virology Unit, Church Street, Glasgow G11 5JR, Scotland, UK.
authors.forename: Lindsay B
authors.initials: LB
authors.lastname: Murphy
authors.forename: Colin
authors.initials: C
authors.lastname: Loney
authors.forename: Jillian
authors.initials: J
authors.lastname: Murray
authors.forename: David
authors.initials: D
authors.lastname: Bhella
authors.forename: Peter
authors.initials: P
authors.lastname: Ashton
authors.forename: Robert P
authors.initials: RP
authors.lastname: Yeo
mesh_terms: 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
chemical_list: D019590:Nucleocapsid Proteins; D010750:Phosphoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins