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Journal of Virology, December 2004, p. 13122-13131, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.13122-13131.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Requirement of Heat Shock Protein 90 for Human Hepatitis B Virus Reverse Transcriptase Function

Jianming Hu,1* Dafna Flores,1 David Toft,2 Xingtai Wang,1 and David Nguyen1

Department of Microbiology, Boston University School of Medicine, Boston, Massachusetts,1 Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota2

Received 13 May 2004/ Accepted 10 August 2004

The initiation of reverse transcription and nucleocapsid assembly in hepatitis B virus (HBV) depends on the specific recognition of an RNA signal (the packaging signal, {varepsilon}) on the pregenomic RNA (pgRNA) by the viral reverse transcriptase (RT). RT-{varepsilon} interaction in the duck hepatitis B virus (DHBV) was recently shown to require the molecular chaperone complex, the heat shock protein 90 (Hsp90). However, the requirement for RT-{varepsilon} interaction in the human HBV has remained unknown due to the inability to obtain a purified RT protein active in specific {varepsilon} binding. We now report that Hsp90 is also required for HBV RT-{varepsilon} interaction. Inhibition of Hsp90 led to diminished HBV pgRNA packaging into nucleocapsids in cells, which depends on RT-{varepsilon} interaction. Furthermore, using truncated HBV RT proteins purified from bacteria and five purified Hsp90 chaperone factors, we have developed an in vitro RT-{varepsilon} binding assay. Our results demonstrate that Hsp90, in a dynamic process that was dependent on ATP hydrolysis, facilitated RT-{varepsilon} interaction in HBV, as in DHBV. Specific {varepsilon} binding required sequences from both the amino-terminal terminal protein and the carboxy-terminal RT domain. Only the cognate HBV {varepsilon}, but not the DHBV {varepsilon}, could bind the HBV RT proteins. Furthermore, the internal bulge, but not the apical loop, of {varepsilon} was required for RT binding. The establishment of a defined in vitro reconstitution system has now paved the way for future biochemical and structural studies to elucidate the mechanisms of RT-{varepsilon} interaction and chaperone activation.

* Corresponding author. Present address: Department of Microbiology and Immunology—H107, The Pennsylvania State University, 500 University Dr., Hershey, PA 17033. Phone: (717) 531-6523. Fax: (717) 531-6522. E-mail: juh13{at}psu.edu.

Journal of Virology, December 2004, p. 13122-13131, Vol. 78, No. 23
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.23.13122-13131.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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