Expression and characterization of hepadnavirus reverse transcriptases

Jianming Hu, Christoph Seeger

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

The successful expression of the hepadnavirus reverse transcriptase in several different expression systems as described earlier has greatly facilitated biochemical and genetical analysis of this most unusual enzyme. The demonstration that the polymerase is the only vital protein needed to initiate vital DNA synthesis de novo using a novel protein-priming mechanism, has hastened efforts to identify key players in the priming reaction and has yielded important information regarding the requirement of both the polymerase and the RNA template. A tyrosine residue at the N terminus of the polymerase polypeptide has been identified as the primer for reverse transcription, and two separate domains of the enzyme have been shown to participate in binding to the ε RNA template. Detailed analyses using the in vitro-expressed enzyme have led to the surprising conclusion that the viral RNA template for initiation of DNA synthesis is the same sequence motif ε, previously identified as the RNA packaging signal, and that a strand transfer is required for the continuation of vital DNA synthesis. However, it has not yet been possible to obtain highly purified enzyme in vitro, which is a prerequisite for further detailed biochemical and structural studies. Expression levels in the reticulocyte lysate are too low to be feasible for this purpose and the yeast system is not useful for large-scale purification due to contamination by Ty proteins and the uncertainty of proteolytic processing of the Ty-polymerase fusion. The recent success in expressing high levels of HBV polymerase in insect cells may facilitate the production of large amounts of the polymerase enzyme for further biochemical and structural studies.

Original languageEnglish (US)
Pages (from-to)195-208
Number of pages14
JournalMethods in enzymology
Volume275
DOIs
StatePublished - Jan 1 1996

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology

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