Expression of hepatitis B virus polymerase in Ty1-his3AI retroelement of Saccharomyces cerevisiae

Hepatitis B virus (HBV), although a DNA virus, replicates using reverse tra nscriptase encoded by the HBV polymerase (pol) gene, The biochemical dissec tion of HBV pol has been hampered by failure to liberate enzymatically acti ve protein from nucleocapsids. Here, we have employed a yeast-based genetic approach to express the HBV reverse transcriptase. In this strategy, the r everse transcriptase of yeast retrotransposon Ty1 element is replaced with the HBV pol gene to produce the hybrid Ty1/HBV element. Additionally, the i ndicator gene his3AI is combined in an antisense orientation to the transcr ipts of the hybrid Ty1/HBVRT element. The splicing of his3AI; cDNA synthesi s of the Ty1/HBVRT RNA and subsequent integration relies on the reverse tra nscriptase activity. The production of histidine prototrophs results from t he successful reverse transcription of Ty1/HBVRThis3AI transcripts followed by either homologous recombination or integrase-mediated insertion and sub sequent expression of HIS3 gene. Using this approach we successfully detect ed the reverse transcriptase activity of HBV in yeast strains defective in endogenous Ty1 expression. Consistent with the unique priming activity asso ciated with HBV pol, the minus strand DNA synthesis was protein-primed. Del etion of HBV reverse transcriptase (RT) or RNase H domains resulted in a dr amatic drop in histidine prototrophs, The addition of HBV encoded HBx prote in in virus-like particles during in vitro RT reaction stimulated the RT re action by severalfold. Furthermore, in the presence of 3TC, a known inhibit or of HBV reverse transcriptase, yeast His(+) growth of His protrophs was n ot observed. Thus, this approach, which is based on genetic selection in ye ast, is safe, economic, and a reliable strategy with a potential for large scale screening of cofactors and inhibitors of HBV polymerase functions.