In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, ep silon) on the pregenomic RNA template by the viral reverse transcriptase (R T) and is primed by the RT itself (protein priming). We have previously sho wn that the RT-E interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these r eactions remained to be identified. We now report that five cellular chaper one proteins, all known cofactors of Hsp90, were sufficient to reconstitute a duck hepatitis B virus RT active in epsilon binding and protein priming in vitro. Four proteins, Hsp90, Hsp70, Hsp40, and Hop, were required for re constitution of RT activity, and the fifth protein, p23, further enhanced t he kinetics of reconstitution. RT activation by the chaperone proteins is a dynamic process dependent on ATP hydrolysis and the Hsp90 ATPase activity. Thus, our results have defined a minimal complement of host factors necess ary and sufficient for RT activation. Furthermore, this defined in vitro re constitution system has now paved the way for future biochemical and struct ural studies to elucidate the mechanisms of RT activation and chaperone fun ctions.