title: Efficient Hsp90-independent in vitro activation by Hsc70 and Hsp40 of duck hepatitis B virus reverse transcriptase, an assumed Hsp90 client protein.
abstract: Hsp90 is a specialized chaperone that controls the activity of many key regulator proteins such as steroid hormone receptors (SHRs). Hormone binding, and therefore SHR activation, requires Hsp90, which is loaded onto the receptors by a series of events involving Hsp70, Hsp40, Hop, and p23. The reverse transcriptase (RT) of hepatitis B viruses, small DNA-containing viruses that replicate via an RNA intermediate, has been reported to depend similarly on Hsp90 for enzymatic activity. Using an in vitro reconstitution system consisting of recombinant duck hepatitis B virus RT, purified chaperones, and the authentic RNA template Depsilon, we demonstrate here that this RT can be activated efficiently by just Hsp40 and Hsc70 plus energy, without the need for Hsp90 or other cofactors. The reaction appears to proceed selectively with the Hdj1 variant of Hsp40 but not Hdj2 or its yeast homolog Ydj1. The primary reaction product is a metastable, RNA binding-competent intermediate that decays quickly in the absence of its cognate RNA but, in its presence, accumulates in an initiation-competent form over several hours. Because deletion of the RNase H domain rendered the protein partly chaperone-independent, the chaperones may be needed indirectly to relieve occlusion of the RNA binding site by this domain. Our results do not exclude that other factors contribute to RT activation in vivo, but they challenge a fundamental SHR-like dependence on Hsp90. Thus Hsc70, mostly known for its role in general protein folding, is able to effect activation of a highly specialized target protein.
authors.affiliation: Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, Hugstetter Strasse 55, Freiburg D-79106, Germany.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Beck
authors.forename: Michael
authors.initials: M
authors.lastname: Nassal
mesh_terms: D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000378:metabolism; D002417:Cattle; D003001:Cloning, Molecular; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000378:metabolism*; D015611:Hepatitis B Virus, Duck / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D008954:Models, Biological; D008956:Models, Chemical; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins; D016914:Ribonuclease H / Q000737:chemistry; D029701:Saccharomyces cerevisiae Proteins; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; C107481:DNAJA1 protein, human; C494647:DNAJB1 protein, human; D050883:HSC70 Heat-Shock Proteins; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C494371:HSPA8 protein, human; D006360:Heat-Shock Proteins; C494373:Hspa8 protein, rat; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; C070319:YDJ1 protein, S cerevisiae; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H