A functional hepatitis B virus X protein produced in insect cells.
We have constructed a recombinant baculovirus from Autographa californica nuclear polyhedrosis virus, called AcX, that expresses the gene encoding the hepatitis B virus X protein in infected Spodoptera frugiperda (Sf21AE) insect cells. A 16.5-kDa monomer and a 33-kDa dimer of the X protein were detected in extracts from AcX-infected cells on immunoblots using a polyclonal anti-X antibody. The biological activity of the insect cell-produced X protein was assayed by fusing AcX-infected Sf21AE cells with African green monkey kidney (CV-1) cells and then transfecting the fused cells with the reporter plasmid pSV2cat. The expression of the cat gene in CV-1:Sf21AE(AcX) fusions was seven times higher than that derived from CV-1:Sf21AE(AcMNPV) fusions, indicating that the insect cell-produced X protein was functional. The transactivation function of the insect cell-produced X protein was also verified by scrape-loading nuclear extracts of AcX-infected Sf21AE cells into pSV2cat-transfected CV-1 cells. Treatment of the AcX-infected cell nuclear extracts with anti-X antisera prior to scrape-loading eliminated the transactivating activity of the extracts. We conclude that the insect cell-produced X protein was functionally identical to that generated in mammalian cells.
Spandau
D F
DF
Department of Pathology, Indiana University School of Medicine, Indianapolis 46202.
Wang
H G
HG
Fraser
M J
MJ
Lee
C H
CH
D000818:Animals; D016367:Baculoviridae / Q000235:genetics / Q000276:immunology; D002460:Cell Line; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics / Q000378:metabolism; D004742:Enhancer Elements, Genetic / Q000235:genetics; D000882:Haplorhini; D006515:Hepatitis B virus / Q000235:genetics* / Q000276:immunology; D009036:Moths; D010957:Plasmids / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D013539:Simian virus 40 / Q000235:genetics; D015534:Trans-Activators / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D015533:Transcriptional Activation / Q000235:genetics*; D054334:Viral Regulatory and Accessory Proteins
D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D054334:Viral Regulatory and Accessory Proteins; C057628:hepatitis B virus X protein; D015500:Chloramphenicol O-Acetyltransferase