Reverse transcriptase activity of hepatitis B virus polymerase in eukaryotic cell extracts in vitro.
In hepadnaviruses, reverse transcription is primed by the viral reverse transcriptase (RT) and requires the specific interaction between the RT and the viral RNA encapsidation signal termed e. To study the activity of the RT in vitro, the current procedure uses in vitro translated duck hepatitis B virus polymerase, but not the hepatitis B virus polymerase itself, in the rabbit reticulocyte lysate expression system. Here, the hepatitis B virus (HBV) polymerase has been successfully expressed in a translational extract that was obtained from monolayer human hepatocyte cells HuH-7. The translated polypeptide retained the RNA-directed polymerase (reverse transcriptase) activity on the viral RNA template containing the e signal. We suggest that the reverse transcription event of the viral RNA coding for the polymerase and containing an e structure is concomitant to the translation of the viral polymerase to the messenger RNA. In contrast to the duck polymerase, only a fraction of the reverse transcribed complementary DNA (cDNA) was covalently bound to the HBV polymerase in this system. When the e signal was missing on the mRNA, the translated full-length HBV polymerase could not reverse transcribe the viral RNA template. A truncated HBV polymerase that was lacking the YMDD catalytic active site for the initiation of reverse transcription was unable to reverse transcribe the viral mRNA template containing the e signal. The reverse transcription activity could also be partially inhibited by employing nucleoside analogues, such as 2'-3'-dideoxy-3'-thiacytidine (3TC; lamivudine) in the expression system. The procedure described here provides a method for the in vitro screening of new anti-HBV compounds directed against wild- type and mutants of this crucial viral protein, the HBV polymerase, without the use of animals (ducks) or animal extracts (rabbit reticulocyte lysate).
Helpatitis, Lausanne, Switzerland. daniel.favre@bluewin.ch
Daniel
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Favre
D000818:Animals; D002460:Cell Line; D002645:Chickens; D006224:Cricetinae; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D005057:Eukaryotic Cells; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D006515:Hepatitis B virus / Q000378:metabolism*; D022781:Hepatocytes; D006801:Humans; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D017361:Viral Nonstructural Proteins; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase