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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 (
) 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
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
-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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