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

• Published in: Virology (New York, N.Y.) Vol. 171; no. 1; pp. 131 - 140
• Main Authors: Cohen, J., Charpilienne, A., Chilmonczyk, S., Estes, M.K.
• Format: Journal Article
• Language: English
• Published: San Diego, CA Elsevier Inc 01.07.1989; Elsevier; Published by Elsevier Inc
• Subjects: Amino Acid Sequence; baculovirus; Base Sequence; Biological and medical sciences; Blotting, Western; Cloning, Molecular; DNA-Directed RNA Polymerases - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation; Genes, Viral; Genetics; Insect Viruses - genetics; Life Sciences; Microbiology; Molecular Sequence Data; Rotavirus - enzymology; Rotavirus - genetics; Viral Proteins - genetics; Viral Proteins - immunology; Viral Structural Proteins; Virology; Virus; Bovine rotavirus; Baculoviridae; Nucleotide sequence; Duovirus; Baculovirus; Gene expression; Reoviridae; EXPRESSION DU GENE; IMMUNOLOGIE; VIROLOGIE; BIOLOGIE MOLECULAIRE
• ISSN: 0042-6822; 1096-0341
• DOI: 10.1016/0042-6822(89)90519-9

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.