Analysis of the earliest steps of hepadnavirus replication: genome repair after infectious entry into hepatocytes does not depend on viral polymerase activity.
Hepadnaviruses contain a relaxed circular DNA genome (RC-DNA) with discontinuities in both strands. Upon infectious entry into a host cell, this genome is converted into a covalently closed superhelical form (CCC-DNA), which later serves as the template for transcription. Here we examined whether the viral polymerase activity is required for this repair reaction. Primary hepatocytes prepared from embryonated duck eggs were infected with the duck hepatitis B virus. Conversion of the RC-DNA into the CCC-DNA was then analyzed by a newly developed polymerase chain reaction technique. This method allows the efficient discrimination between the two DNA forms and is sensitive enough to monitor repair of the infecting viral DNA in the absence of replication and amplification. Thus, we were able to monitor this process in the presence of a potent inhibitor of the viral polymerase, the nucleoside analog 2',3'-dideoxyguanosine. The data show that inhibition of the viral polymerase activity has no influence on genome repair, suggesting that this enzymatic function is not required for conversion of the RC-DNA into the CCC-DNA. Consequently, antiviral drugs blocking the polymerase activity cannot prevent the infectious entry of the virus into a host cell.
Köck
J
J
Department of Virology, University of Ulm, Germany.
Schlicht
H J
HJ
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002455:Cell Division; D002478:Cells, Cultured; D004260:DNA Repair / Q000187:drug effects; D004279:DNA, Viral / Q000235:genetics / Q000302:isolation & purification; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D015224:Dideoxynucleosides / Q000494:pharmacology; D004372:Ducks; D004625:Embryo, Nonmammalian; D016679:Genome, Viral; D015613:Hepadnaviridae / Q000201:enzymology / Q000235:genetics / Q000502:physiology*; D008099:Liver / Q000166:cytology / Q000382:microbiology*; D008969:Molecular Sequence Data; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction / Q000379:methods
D004279:DNA, Viral; D015224:Dideoxynucleosides; D019384:Nucleic Acid Synthesis Inhibitors; D009838:Oligodeoxyribonucleotides; C050220:2',3'-dideoxyguanosine; D004259:DNA-Directed DNA Polymerase