Nucleotide sequence of bovine rotavirus gene 1 and expression of the gene product in Baculovirus()

The nucleotide sequence of the gene that encodes for the structural viral protein VP1 of bovine rotavirus (RF strain) has been determined. The sequence data indicate that segment 1 contains 3302 by and is A+T rich (65.3%). The positive strand of segment 1 contains a single open reading frame that extends 1088 codons and possesses 5′- and 3′-terminal untranslated regions of 18 and 20 bp, respectively. The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 124–847, very close to the apparent molecular weight of VP1 (M.W. 125,000). The deduced amino acid sequence presents significant similarities with RNA-dependent RNA polymerase of several RNA viruses. VP1 was also synthesized in baculovirus using two transfer vecors: pAC461 and pVL941. Following infection of Sf9 cells with a recombinant baculovirus, a full-length nonfusion protein was synthesised which shares properties with authentic VP1 made in monkey kidney cells. The level of VP1 synthesis was about 10-fold higher when the baculovirus recombinant was derived from the pVL941 transfer vector. In that case, VP1 was expressed in yields approximately equivalent to 10% of the cellular protein. The recombinant protein was immunoprecipitated by hyperimmune serum raised against purified rotavirus. It also was immunogenic; a hyperimmune serum made in guinea pigs reacted with VP1 using immunoprecipitation and Western blot. This serum did not possess neutralization activity.