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Journal of Virology, January 2002, p. 269-279, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.269-279.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

In Vitro Reconstitution of Functional Hepadnavirus Reverse Transcriptase with Cellular Chaperone Proteins

Jianming Hu,1* David Toft,2 Dana Anselmo,1 and Xingtai Wang1

Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts 02118,1 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 559052

Received 7 August 2001/ Accepted 5 October 2001

Initiation of reverse transcription in hepadnaviruses (hepatitis B viruses) depends on the specific binding of an RNA signal (the packaging signal, {varepsilon}) 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-{varepsilon} 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 {varepsilon} 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.

* Corresponding author. Mailing address: Department of Microbiology, Boston University School of Medicine, 715 Albany St., Boston, MA 02118-2526. Phone: (617) 638-4982. Fax: (617) 638-4286. E-mail: jmhu{at}bu.edu.

Journal of Virology, January 2002, p. 269-279, Vol. 76, No. 1
0022-538X/01/$04.00+0     DOI: 10.1128/JVI.76.1.269-279.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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