Hepatitis B virus polymerase gene: expression of the long open reading frame using the baculovirus expression system.
A recombinant baculovirus was constructed containing a copy of the hepatitis B virus (HBV) genome which was inserted to produce an in-frame fusion of the precore (pre-C) coding region with the first 11 amino acids of the polyhedrin gene. The recombinant baculovirus expressed the 25K pre-C protein and two novel proteins, of approximately 93K and 72K. Both the 93K and 72K proteins are recognized by an anti-polymerase monoclonal antibody. Northern blot analysis of the mRNA produced during infection of Spodoptera frugiperda cells by the HBV recombinant baculovirus detected only one HBV mRNA species, suggesting that the three HBV-specific proteins expressed are translated from the same mRNA. No larger fusion proteins cross-reacting with either anti-core or polymerase antibodies were detected. These findings suggest that the two proteins encoded within the HBV polymerase gene are not produced via a core-polymerase fusion intermediate but by internal binding of ribosomes. These results are the first clear demonstration of efficient expression of two bona fide unprocessed polymerase proteins in a 1:1 ratio from an unspliced pre-C mRNA-like transcript. With the successful expression of the polymerase gene in insect cells it is now possible to produce large amounts of these proteins, allowing a more detailed structural and functional analysis of these proteins.
McGlynn
E
E
Research Department, Pharmaceuticals Division, Ciba-Geigy Limited, Basel, Switzerland.
Reutener
S
S
Matter
A
A
Wildner
G
G
Will
H
H
Lydon
N B
NB
D016367:Baculoviridae / Q000235:genetics*; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D015870:Gene Expression; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics; D016366:Open Reading Frames
D004259:DNA-Directed DNA Polymerase