PRIMING OF DUCK HEPATITIS-B VIRUS REVERSE TRANSCRIPTION IN-VITRO - PREMATURE TERMINATION OF PRIMER DNA INDUCED BY THE 5'-TRIPHOSPHATE OF FIALURIDINE

Hepadnaviruses employ a unique mechanism for the initiation of RNA-dir ected DNA synthesis. Initially, four bases (5'-GTAA-3') are added to a tyrosine residue of the viral polymerase by reverse transcription of a bulge sequence in epsilon, a stem-loop structure which functions as the packaging signal for pregenomic RNA. This protein-DNA complex acts as the primer for minus-strand elongation from the 3' sequence, DR1. To understand this process in greater detail, we investigated whether the protein-mediated priming of viral DNA synthesis is affected by nuc leotide analogs. By using cell-free expression of duck hepatitis B vir us (DHBV) reverse transcriptase (G.-H. Wang and C. Seeger, Cell 71:663 -670, 1992), the 5'-triphosphate of the thymidine analog fialuridine ( FIAU) was shown to inhibit the incorporation of radiolabeled TMP into primer DNA in a dose-dependent manner. Inhibition by the 5'-triphospha te of FIAU (FIAU-TP) was nearly complete at a concentration of 10 mu M . The dideoxynucleotide analogs ddGTP, ddTTP, and 3'-azidodeoxythymidi ne triphosphate, known inhibitors of DHBV endogenous DNA polymerase, d id not affect substantially the synthesis of primer DNA. Alternate sub strate analysis suggested that FIAU is incorporated efficiently into n ascent primer DNA as an analog of thymidine. Using site-directed mutag enesis to construct a mutant RNA template yielding a primer with the s equence 5'-GTAC-3', we demonstrated that FIAU-TP inhibited the incorpo ration of TMP, had no effect on that of dAMP, and decreased markedly t he incorporation of dCMP. These results show that the synthesis of ful l-length DHBV primer DNA is inhibited by FIAU-TP but not by the dideox ynucleotide analogs that we tested. The significance of these findings as they relate to HBV DNA replication is discussed.