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Journal of Virology, August 2001, p. 7410-7419, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7410-7419.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Reconstitution of a Functional Duck Hepatitis B Virus Replication Initiation Complex from Separate Reverse Transcriptase Domains Expressed in Escherichia coli

Jürgen Beck* and Michael Nassal

Department of Internal Medicine II/Molecular Biology, University Hospital Freiburg, D-79106 Freiburg, Germany

Received 23 February 2001/Accepted 14 May 2001

Hepatitis B viruses replicate through reverse transcription of an RNA intermediate, the pregenomic RNA (pgRNA). Replication is initiated de novo and requires formation of a ribonucleoprotein complex comprising the viral reverse transcriptase (P protein), an RNA stem-loop structure (varepsilon ) on the pgRNA, and cellular proteins, including the heat shock protein Hsp90, the cochaperone p23, and additional, as yet unknown, factors. Functional complexes catalyze the synthesis of a short DNA primer that is templated by varepsilon  and covalently linked to the terminal protein (TP) domain of P protein. Currently, the only system for generating such complexes in the test tube is in vitro translation of duck hepatitis B virus (DHBV) P protein in rabbit reticulocyte lysate (RRL), which also provides the necessary factors. However, its limited translation capacity precludes a closer analysis of the complex. To overcome this restriction we sought to produce larger amounts of DHBV P protein by expression in Escherichia coli, followed by complex reconstitution in RRL. Because previous attempts to generate full-length P protein in bacteria have failed we investigated whether separate expression of the TP and reverse transcriptase-RNase H (RT-RH) domains would allow higher yields and whether these domains could trans complement each other. Indeed, TP and, after minor C-terminal modifications, also RT-RH could be expressed in substantial amounts, and when added to RRL, they were capable of varepsilon -dependent DNA primer synthesis, demonstrating posttranslational activation. This reconstitution system should pave the way for a detailed understanding of the unique hepadnaviral replication initiation mechanism.

* Corresponding author. Mailing address: University Hospital Freiburg, Department of Internal Medicine II/Molecular Biology, Hugstetter Str. 55, D-79106 Freiburg, Germany. Phone: 49-761-270 3648. Fax: 49-761-270 3372. E-mail: jbeck{at}ukl.uni-freiburg.de.

Journal of Virology, August 2001, p. 7410-7419, Vol. 75, No. 16
0022-538X/01/$04.00+0   DOI: 10.1128/JVI.75.16.7410-7419.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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