Baculovirus expression of pestivirus non-structural proteins.
Bovine viral diarrhoea virus (BVDV) belongs to the pestivirus group, a genus within the Flaviviridae family. It possesses a positive-sense ssRNA genome with a single large open reading frame (ORF) encoding about 4000 amino acids. Here we report the continuation of our studies of pestivirus protein biogenesis, involving expression from the viral non-structural protein-encoding region. The 3'-terminal 60% of the BVDV ORF was cloned into a plasmid transfer vector which was then used to construct a recombinant baculovirus. Infection of Spodoptera frugiperda Sf9 cells with this recombinant virus resulted in the production of the expected mature viral proteins. Polyprotein processing by the BVDV p80 proteinase appeared to be nearly identical to that observed in authentic BVDV-infected bovine cells, and as previously shown to occur when expression of the same region was studied in a mammalian cell transient expression system. However, one viral proteolytic cleavage did not occur in the baculovirus-infected insect cells; the viral p80 proteinase failed to act at its own N terminus. This recombinant baculovirus/insect cell expression system provides an abundant source of BVDV non-structural proteins. Therefore we explored the utility of the proteins produced in this system for the detection of anti-BVDV antibodies in bovine sera. In preliminary experiments using these antigens in an ELISA we found a positive correlation between the presence of ELISA-reactive antibody and virus-neutralizing activity.
Petric
M
M
Department of Pediatrics, Johns Hopkins Hospital, Baltimore, Maryland 21205.
Yolken
R H
RH
Dubovi
E J
EJ
Wiskerchen
M
M
Collett
M S
MS
D000818:Animals; D000914:Antibodies, Viral / Q000032:analysis; D016367:Baculoviridae / Q000235:genetics*; D001912:Bovine Virus Diarrhea-Mucosal Disease / Q000382:microbiology*; D002213:Capsid / Q000096:biosynthesis* / Q000276:immunology; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay; D016366:Open Reading Frames; D010576:Pestivirus / Q000235:genetics* / Q000276:immunology; D011995:Recombination, Genetic; D014758:Viral Core Proteins / Q000096:biosynthesis* / Q000276:immunology; D017361:Viral Nonstructural Proteins
D000914:Antibodies, Viral; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins