A functional hepatitis B virus X protein produced in insect cells

Abstract

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.