The Hepatitis B virus P22 protein is a nonstructural protein that is the precursor of the 17-kDa secreted e antigen (HBeAg). The mature HBeAg is obtained after the removal of the C-terminal region of P22, a process which involves a proprotein convertase. Our studies show first that the protease could cleave P22 at the C-terminal side of Arg 167 or Arg 154 and second, that the maturation process can be either done in one step or in two steps with the generation of a processing intermediate (P20). Our data also demonstrate that the removal of the P22 C terminus, which occurs mainly in the transGolgi network, can also be achieved after exocytosis. Keeping in mind this characteristic and the amino acid sequence of the cleavage sites, we concluded that furin is involved in the maturation of the HBeAg. In addition, we show that in our experimental system, the HBeAg is a 164-amino acid protein and not a 159-amino acid protein as previously reported.

A procedure has been devised for the purification of intact DNA polymerase a from early embryos of Drosophila melanogaster. The purified enzyme consists of at least three polypeptides with Mrs of 182,000, 60,000, and 50,000. These are related antigenically to the a (Mr 148,000), .l (Mr 58,000), and Y (Mr 46,000) subunits, respectively, of the DNA polymerase described previously [Banks, G. R., Boezi, J. A. & Lehman, I. R. (1979)J. Biol. Chem. 254, 9886-9892]. The-a subunit (Mr 182,000) has a molecular weight indistinguishable from that observed in extracts of freshly harvested embryos and presumably present in vivo. As in the previous preparation, the a subunit is required for DNA polymerase activity and is very likely the catalytic subunit of the enzyme. The ratio of primase to polymerase remains constant throughout the purification. Thus, the primase is very likely an integral component of the Drosophila DNA polymerase a. The purified DNA polymerase-primase contains no detectable endoor exodeoxyribonuclease and has pH, MgCl2, (NH4)2SO4, and NaCI optima identical to those reported previously. In contrast, the Km for dTTP is 3.7 ,uM as compared with 17.5 ,uM for the previous enzyme. Sensitivities to aphidicolin and N-ethylmaleiimide and resistance to dideoxy TTP are unchanged.DNA polymerase a as purified from a variety of eukaryotes consists of two or more polypeptides. Several polymerases (1-7) have been isolated as multisubunit proteins consisting of a catalytic core with an approximate Mr of 150,000 in association with three or four smaller subunits whose Mrs range from 40,000 to 60,000. However, other polymerase preparations are lacking a high molecular weight subunit (8,9). Our recent studies (10) and those of others (11)(12)(13)(14) have shown that a DNA primase activity is associated with the a polymerase and may be part of the multisubunit molecule.Uncontrolled proteolysis during purification has prevented the unambiguous determination of the subunit structure of DNA polymerase a and the structure-function relationships of its individual polypeptides. That proteolysis may yield an active but highly degraded form of the enzyme has been clearly demonstrated both in Drosophila melanogaster embryos and in calf thymus (4,(15)(16)(17) were prepared for use as described (19). Leupeptin was purchased from the Peptide Institute (Minoh-shi, Japan). Aphidicolin was prepared as a stock solution at 1 mg/ ml in 40% ethanol/10% dimethyl sulfoxide. N-Ethylmaleiimide (Schwarz/Mann) was dissolved in water immediately before use.Antisera. Antisera directed against Drosophila DNA polymerase a and its isolated a and J subunits were prepared 'as described (18). Purified IgG was prepared by ammonium sulfate precipitation and DEAE-cellulose chromatography (20).Enzymes. Escherichia coli DNA polymerase I and E. coli RNA polymerase were the generous gifts of J. Kelly and J. Kaguni, respectively, of this department. Pancreatic DNase I was purchased from Worthington.Nucleic acids. Calf thymus DNA (grade A, Calbiochem) wa...

The hepatitis B virus (HBV) P gene which encodes the reverse transcriptase and other proteins required for replication is expressed on the bicistronic mRNA pregenome which also encodes the capsid protein in its first cistron. Recent results have suggested that the hepadnaviral P gene is translated by internal entry of ribosomes upstream from the P gene, in the overlapping C gene. Using a reporter gene fused to the HBV C or P gene, we demonstrate that the C sequence does not allow internal initiation of translation. Alternatively, our results support a model in which the HBV P gene is translated by ribosomes which scan from the capped extremity of the bicistronic mRNA pregenome. The mechanism by which the ribosomes scan past four AUGs before they initiate translation at the P AUG was analyzed. Our results show that these AUGs are skipped via two mechanisms: leaky scanning on AUGs in a weak or suboptimal initiation context and translation of an out-of-C-frame minicistron followed by reinitiation at P AUG. The minicistron translation allows ribosomes to bypass an AUG in a favorable context that would otherwise be used as a start codon for translation of a truncated capsid protein. Our results suggest that this elaborated scanning mechanism permits the coordinate expression of the HBV C and P genes on the viral bicistronic mRNA pregenome.

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