title: In vitro reconstitution of functional hepadnavirus reverse transcriptase with cellular chaperone proteins.
abstract: Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, epsilon) on the pregenomic RNA template by the viral reverse transcriptase (RT) and is primed by the RT itself (protein priming). We have previously shown that the RT-epsilon interaction and protein priming require the cellular heat shock protein, Hsp90. However, additional host factors required for these reactions remained to be identified. We now report that five cellular chaperone 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 reconstitution of RT activity, and the fifth protein, p23, further enhanced the 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 necessary and sufficient for RT activation. Furthermore, this defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT activation and chaperone functions.
authors.affiliation: Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118, USA. jmhu@bu.edu
authors.forename: Jianming
authors.initials: J
authors.lastname: Hu
authors.forename: David
authors.initials: D
authors.lastname: Toft
authors.forename: Dana
authors.initials: D
authors.lastname: Anselmo
authors.forename: Xingtai
authors.initials: X
authors.lastname: Wang
mesh_terms: D018833:Chaperonins / Q000737:chemistry / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D015613:Hepadnaviridae / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D014779:Virus Replication
chemical_list: D018841:HSP90 Heat-Shock Proteins; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D018833:Chaperonins