Expression of functional hepatitis B virus polymerase in yeast reveals it to be the sole viral protein required for correct initiation of reverse transcription.
Replication of hepatitis B viruses proceeds by reverse transcription of an RNA intermediate, a reaction catalyzed by the virus-encoded polymerase (P protein). The reaction product is a partially duplex DNA whose (-)-strand is covalently linked to the P protein. Efforts to understand the mechanism of the reaction have been severely retarded by an inability to express functional polymerase outside of viral particles. Here we report the successful expression of enzymatically active polymerase in yeast cells, by fusing the P gene to coding sequences of the retrotransposon Ty1. The enzyme initiates correctly on viral RNA in yeast cells in vivo, producing nascent DNA chains covalently linked to protein, exactly as found in virus-infected cells. Replication complexes isolated from these yeast are enzymatically active in vitro, synthesizing DNA in a reaction that is actinomycin D-resistant but sensitive to RNase pretreatment. These results indicate that P protein is the sole viral protein required for the correct priming of reverse transcription and establish a tractable system for the biochemical dissection of the reaction.
Tavis
J E
JE
Howard Hughes Medical Institute, University of California, San Francisco 94143-0502.
Ganem
D
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D004261:DNA Replication; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D003609:Dactinomycin / Q000494:pharmacology; D016368:Frameshift Mutation; D006515:Hepatitis B virus / Q000201:enzymology* / Q000235:genetics / Q000502:physiology; D010957:Plasmids; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014779:Virus Replication
D004251:DNA Transposable Elements; D004279:DNA, Viral; D012367:RNA, Viral; D011994:Recombinant Proteins; D003609:Dactinomycin; D012194:RNA-Directed DNA Polymerase