Structural and functional analysis of Sendai virus nucleocapsid protein NP with monoclonal antibodies

Abstract

Monoclonal antibodies specific for Sendai virus nucleocapsid protein NP were used to map the antigenic structure of NP and to investigate the role of NP in transcription. Using nine anti-NP antibodies in competitive-binding (CB) assays, it was found that the NP molecule contained at least two topographically distinct antigenic sites. By Western blot analysis, one of the NP epitopes belonging to antigenic site I was localized to a Mr 34,000 (34K) trypsin digest fragment, and another to a Mr 48,000 (48K) fragment which remained associated with the nucleocapsid. The other antibodies which define antigenic site I did not react with either fragment; however, the results of CB would indicate that their epitopes were in a region on the tertiary structure of the NP molecule that is closely proximal to these fragments. The 48K and 34K fragments on the published NP amino acid sequence have been tentatively identified. Since the 34K and 48K fragments bind antibody, it appears that nucleocapsid-bound NP may be folded into a configuration which places at least some of these sequences on the surface of the nucleocapsid structure. Six antibodies representing both antigenic sites were purified for functional studies. All the antibodies inhibited nucleocapsid transcription in vitro to the same extent (greater than 90%); however, they differed in the amount of antibody required to produce the same effect. Within site I, antibodies producing maximum inhibition were divided into three groups: three antibodies inhibited at relatively low concentrations (0.17 microgram), one antibody inhibited at an intermediate range (0.43 microgram), and another required a 10-fold higher concentration (1.73 microgram) to produce the same effect. The antibody which detected the 48K trypsin digest fragment was the one which fell into the intermediate range for transcription inhibition, while the antibody that detected the 34K fragment was in the low range. Thus, antigenic site I, defined by CB and trypsin digestion studies, can be defined further into three subsites which appear to differ in their involvement in the transcription process. Antigenic site II was defined by a single antibody which also inhibited transcription by greater than 90%.