THE HEPATITIS-DELTA VIRUS LARGE ANTIGEN IS FARNESYLATED BOTH IN-VITROAND IN ANIMAL-CELLS

The hepatitis delta virus large antigen (IHDAg) is a virally encoded p rotein that contains a prenylation signal sequence at its carboxyl ter minus consisting of the tetrapeptide Cys-Arg-Pro-Gln. Although the pre sence of the Gln as the COOH-terminal residue generally specifies addi tion of the 15-carbon farnesyl isoprenoid, earlier reports had suggest ed that the protein is modified by the 20-carbon geranylgeranyl. The p renylation of IHDAg was examined in vitro using a fusion protein betwe en glutathione S-transferase and the COOH-terminal 117 amino acids of IHDAg (GST-IHDAg). When recombinant GST-IHDAg was incubated with bovin e brain cytosol in the presence of either farnesyl diphosphate or gera nylgeranyl diphosphate, GST-IHDAg was preferentially farnesylated. Ger anylgeranylation of the fusion protein was also observed, although at a rate considerably less than that of farnesylation. Using purified re combinant protein prenyltransferases, GST-IHDAg was found to be an exc ellent substrate (apparent K-m = 0.8 mu M) for protein farnesyltransfe rase (FTase), while modification by protein geranylgeranyltransferase I (GGTase I) was not detected. FTase was also able to catalyze geranyl geranylation of GST-IHDAg at a very low rate, suggesting that the low level of geranylgeranylation of GST-IHDAg observed in cytosolic prepar ations was mediated by FTase. Consistent with our observations on the in vitro prenylation of the GST-IHDAg fusion protein, isoprenoid analy sis of authentic IHDAg expressed in COS cells demonstrated that the pr otein was farnesylated, Geranylgeranylation of IHDAg expressed in COS cells was not observed. As prenylation of IHDAg is required for the as sembly of the hepatitis delta viral particle, these results suggest th at inhibitors of FTase may be useful therapeutic agents for treatment of delta virus infection.