title: Expression of hepatitis B virus polymerase in Ty1-his3AI retroelement of Saccharomyces cerevisiae.
abstract: Hepatitis B virus (HBV), although a DNA virus, replicates using reverse transcriptase encoded by the HBV polymerase (pol) gene. The biochemical dissection of HBV pol has been hampered by failure to liberate enzymatically active protein from nucleocapsids. Here, we have employed a yeast-based genetic approach to express the HBV reverse transcriptase. In this strategy, the reverse transcriptase of yeast retrotransposon Ty1 element is replaced with the HBV pol gene to produce the hybrid Ty1/HBV element. Additionally, the indicator gene his3AI is combined in an antisense orientation to the transcripts of the hybrid Ty1/HBVRT element. The splicing of his3AI, cDNA synthesis of the Ty1/HBVRT RNA and subsequent integration relies on the reverse transcriptase activity. The production of histidine prototrophs results from the successful reverse transcription of Ty1/HBVRThis3AI transcripts followed by either homologous recombination or integrase-mediated insertion and subsequent expression of HIS3 gene. Using this approach we successfully detected the reverse transcriptase activity of HBV in yeast strains defective in endogenous Ty1 expression. Consistent with the unique priming activity associated with HBV pol, the minus strand DNA synthesis was protein-primed. Deletion of HBV reverse transcriptase (RT) or RNase H domains resulted in a dramatic drop in histidine prototrophs. The addition of HBV encoded HBx protein in virus-like particles during in vitro RT reaction stimulated the RT reaction by severalfold. Furthermore, in the presence of 3TC, a known inhibitor of HBV reverse transcriptase, yeast His(+) growth of His protrophs was not observed. Thus, this approach, which is based on genetic selection in yeast, is safe, economic, and a reliable strategy with a potential for large scale screening of cofactors and inhibitors of HBV polymerase functions.
authors.affiliation: Department of Microbiology B172, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
authors.forename: I
authors.initials: I
authors.lastname: Qadri
authors.forename: A
authors.initials: A
authors.lastname: Siddiqui
mesh_terms: D005819:Genetic Markers; D006515:Hepatitis B virus / Q000201:enzymology*; D006639:Histidine / Q000096:biosynthesis; D006836:Hydro-Lyases / Q000235:genetics; D012194:RNA-Directed DNA Polymerase / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis*; D018626:Retroelements; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012641:Selection, Genetic
chemical_list: D005819:Genetic Markers; D011994:Recombinant Proteins; D018626:Retroelements; D006639:Histidine; D012194:RNA-Directed DNA Polymerase; D006836:Hydro-Lyases; C021434:imidazoleglycerolphosphate dehydratase