Nucleic Acids Research Advance Access originally published online on September 5, 2007
Nucleic Acids Research 2007 35(18):6124-6136; doi:10.1093/nar/gkm628
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Nucleic Acids Research, 2007, Vol. 35, No. 18 6124-6136
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acid Enzymes |
Chaperone activation of the hepadnaviral reverse transcriptase for template RNA binding is established by the Hsp70 and stimulated by the Hsp90 system
1University Hospital Freiburg, Internal Medicine II/Molecular Biology, D-79106 Freiburg and 2Department of Chemistry, Technical University Munich, Lichtenbergstrasse 4, D-85747 Garching, Germany
*To whom correspondence should be addressed. Tel: +49 761 2703507; Fax: +49 761 2703507; Email: michael.nassal{at}uniklinik-freiburg.de
Received May 1, 2007. Revised July 31, 2007. Accepted July 31, 2007.
Hepadnaviruses are DNA viruses that replicate by protein-primed reverse transcription, employing a specialized reverse transcriptase (RT), P protein. DNA synthesis from the pregenomic RNA is initiated by binding of P to the 
signal. Using as template and a Tyr-residue for initiation, the RT synthesizes a DNA oligo (priming) as primer for full-length DNA. Priming strictly requires prior RT activation by chaperones. Active P– complexes have been reconstituted in vitro, but whether in addition to the heat-shock protein 70 (Hsp70) system the Hsp90 system is essential has been controversial. Here we quantitatively compared Hsp70 versus Hsp70 plus Hsp90 RT activation, and corroborated that the Hsp70 system alone is sufficient; however, Hsp90 as well the Hsp70 nucleotide exchange factor Bag-1 markedly stimulated activation by increasing the steady-state concentration of the activated metastable RT form P*, though by different mechanisms. Hsp90 inhibition in intact cells by geldanamycin analogs blocked hepadnavirus replication, however not completely and only at severely cytotoxic inhibitor concentrations. While compatible with a basal level of Hsp90 independent in vivo replication, unambiguous statements are precluded by the simultaneous massive upregulation of Hsp70 and Hsp90.
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