HEPADNAVIRUS P-PROTEIN UTILIZES A TYROSINE RESIDUE IN THE TP DOMAIN TO PRIME REVERSE TRANSCRIPTION

Hepadnavirus DNA minus strands are covalently linked at their 5' termi nus to the viral P gene product, which has been taken to indicate that the hepadnaviral polymerase polypeptide itself also functions as a pr otein primer for initiating reverse transcription of the RNA pregenome . The present study confirms this indication by identifying the nucleo tide-linked amino acid in the P protein sequence of the duck hepatitis B virus (DHBV). In a first set of experiments, mutational analysis of three phylogenetically conserved tyrosine residues in the DNA termina l (TP) domain indicated that of these, only tyrosine 96 was essential for both viral DNA synthesis in transfected cells and priming of DNA s ynthesis in a cell-free system. This assignment was confirmed by direc t biochemical analysis: tryptic peptides from the DHBV P protein, P-32 labelled at the priming amino acid by the initiating dGTP and additio nally labelled internally by S-35methionine, were isolated and analy zed in parallel to reference peptides synthesized chemically and P-33 labelled by a tyrosine kinase. Mobility in high-performance liquid chr omatography, as well as the release in stepwise amino acid sequencing of phospholabel and of S-35methionine, identified the priming amino acid unequivocally as the tyrosine in the sequence (91)KLSGLYQMR(99) w hich is located in the center of the TP domain. Conserved sequence mot ifs surrounding Tyr-96 allow the prediction of the priming tyrosine in other hepadnaviruses. Weak sequence similarity to picornavirus genome -linked polypeptides (VPgs) and similar gene organization suggest a co mmon origin for the mechanisms that use protein priming to initiate sy nthesis of viral DNA genomes or RNA genomes from an RNA template.