Downloaded from www.microbiologyresearch.org by IP: 54.162.133.179 On: Tue, 23 Feb 2016 06:00:27 Journal of General Virology (1994), 75, 1889-1899. Printed in Great Britain 1889 Structural and antigenic analysis of the nucleoprotein of bovine ephemeral fever rhabdovirus Peter J. Walker,* Yonghong Wang, Jeff A. Cowley, Sean M. McWilliam and Christophe J. N. Prehaud CSIRO Division of Tropical Animal Production, Private Bag No. 3, Indooroopilly, Queensland 4068, Australia The nucleotide sequence of the bovine ephemeral fever virus (BEFV) genome has been determined from the 3' terminus to the end of the nucleoprotein (N) gene. The 3' leader sequence comprises 50 nucleotides and shares a common terminal three nucleotides (3'-UGC-) and a downstream U-rich domain with vesicular stomatitis virus (VSV) and rabies virus. The N gene comprises 1328 nucleotides from the transcription initiation con- sensus sequence (AACAGG) to the conserved tran- scription termination-poly(A) sequence [CATG(A)7 ] and encodes a polypeptide of 431 amino acids with an estimated M r of 49159 and a pI of 5'4. The deduced amino acid sequence of the BEFV N protein is similar to those of other mammalian rhabdoviruses and is more closely related in sequence to vesiculoviruses (VSV Indiana and New Jersey, Pity, Chandipura) than to lyssaviruses (rabies and Mokola). An almost full-length clone, 1301 bp in length, of the BEFV N gene and clones derived from Y-terminal (559bp) and 3'-terminal (742 bp) fragments were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. A panel of 12 BEFV N protein-specific monoclonal antibodies was shown to react in immunoblots with fusion proteins containing the almost full-length N protein and the C- terminal fragment, but not the N-terminal fragment. Two of these antibodies also reacted with baculovirus- expressed rabies virus N protein. Polyclonal mouse ascitic fluids derived from BEF¥, rabies virus and several other related viruses were also shown to cross- react in immunoblots with purified preparations of rabies virus and BEFV N proteins. Introduction Bovine ephemeral fever virus (BEFV) is an arthropod- borne rhabdovirus which has been classified as the type species of the new genus Ephemerovirus (Wunner et al., 1994). The virus has the structural characteristics of a rhabdovirus with bullet- or cone-shaped morphology (Murphy et al., 1972), a 42S ssRNA genome (Della- Porta & Brown, 1979), a lipid envelope and five virion proteins: L (with an M r of 180K), G (81K), N (52K), M 1 (43K) and M2 (29K) (Walker et al., 1991). The G protein is a transmembrane glycoprotein which presents type- specific, neutralizing antigenic sites (Cybinski et al., 1990) and induces a protective immune response in cattle (Uren et al., 1993). The nucleoprotein (N) is phosphoryl- ated and remains associated with nucleocapsids after detergent disruption of virions in high concentrations of salt but is released by treatment with RNase A (Walker The nucleotide sequence data reported in this paper have been depositedwiththe EMBL and GenBank data librariesunderaccession number U04166. et al., 1991; Riding et al., 1993). As for other rhabdo- viruses, the N protein appears to be a major group- reactive antigen, sharing antigenic sites with the related rhabdoviruses Berrimah and Kimberley (Cybinski et al., 1990). We have reported recently that, in addition to the virion G protein, BEFV encodes a second glycoprotein (G~s) that has been detected only in infected cells (Walker et al., 1991). The function of this glycoprotein is presently unknown but it is related in structure and amino acid sequence to the virion G protein and is encoded by a gene located immediately downstream of the G gene (Walker et al., 1992). A similar arrangement of glycoprotein genes occurs in the related Adelaide River virus (ARV), suggesting that each has evolved from a common ancestral rhabdovirus in which there was a duplication of glycoprotein genes (Wang & Walker, 1993). An analysis of amino acid sequence relationships of the BEFV and ARV G and GNs proteins has indicated that, despite reported serological links to rabies virus (Calisher et al., 1989), both glycoproteins are more closely related to the virion G proteins of sigma virus and vesicular stomatitis virus (VSV) serotypes. 0001-2324 © 1994SGM