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' terminus to the viral P gene product, which has been taken to indicate that the hepadnaviral polymerase polypeptide itself also functions as a protein primer for initiating reverse transcription of the RNA pregenome. The present study confirms this indication by identifying the nucleotide-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 terminal (TP) domain indicated that of these, only tyrosine 96 was essential for both viral DNA synthesis in transfected cells and priming of DNA synthesis in a cell-free system. This assignment was confirmed by direct biochemical analysis: tryptic peptides from the DHBV P protein, 32P labelled at the priming amino acid by the initiating dGTP and additionally labelled internally by [35S]methionine, were isolated and analyzed in parallel to reference peptides synthesized chemically and 33P labelled by a tyrosine kinase. Mobility in high-performance liquid chromatography, as well as the release in stepwise amino acid sequencing of phospholabel and of [35S]methionine, identified the priming amino acid unequivocally as the tyrosine in the sequence 91KLSGLYQMK99, which is located in the center of the TP domain. Conserved sequence motifs 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 common origin for the mechanisms that use protein priming to initiate synthesis of viral DNA genomes or RNA genomes from an RNA template.
Weber
M
M
ZMBH, University of Heidelberg, Germany.
Bronsema
V
V
Bartos
H
H
Bosserhoff
A
A
Bartenschlager
R
R
Schaller
H
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004279:DNA, Viral / Q000096:biosynthesis; D003848:Deoxyguanine Nucleotides / Q000378:metabolism; D015684:Gene Products, pol / Q000378:metabolism*; D015611:Hepatitis B Virus, Duck / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry / Q000378:metabolism*; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014443:Tyrosine / Q000235:genetics*
D004279:DNA, Viral; D003848:Deoxyguanine Nucleotides; D015684:Gene Products, pol; C066027:P protein, Hepatitis B virus; D010446:Peptide Fragments; D014443:Tyrosine; C007257:2'-deoxyguanosine 5'-phosphate; D012194:RNA-Directed DNA Polymerase