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J. Biol. Chem., Vol. 281, Issue 47, 35794-35801, November 24, 2006

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The Duck Hepatitis B Virus Reverse Transcriptase Functions as a Full-length Monomer^*

Zhian Zhang and John E. Tavis¹

From the Department of Molecular Microbiology and Immunology and Saint Louis University Liver Center, Saint Louis University School of Medicine, St. Louis, Missouri 63104

Hepadnaviral reverse transcription occurs within cytoplasmic capsid particles and is catalyzed by a virally encoded reverse transcriptase, but the primary structure and multimeric state of the polymerase during reverse transcription are poorly understood. We measured these parameters for the duck hepatitis B virus polymerase employing active enzyme translated in vitro and derived from intracellular core particles and mature virions. In vitro-translated polymerase immunoprecipitated as a monomer, and polymerase molecules with complementary defects in the enzymatic active site and tyrosine 96, which primes DNA synthesis, could not complement or inhibit each other in priming assays. Western analysis using antibodies recognizing epitopes throughout the polymerase combined with nuclease digestion of permeabilized virion-derived capsid particles revealed that only full-length polymerase molecules were in virions and that they were all covalently attached to large DNA molecules. Because DNA synthesis is primed by the polymerase itself and only one copy of the viral DNA is in each capsid, the polymerase must function as an uncleaved monomer. Therefore, a single polymerase monomer is encapsidated, primes DNA synthesis, synthesizes both DNA strands, and participates in the three-strand transfers of DNA synthesis, with all steps after DNA priming performed while the polymerase is covalently coupled to the product DNA. Because the N-terminal domain of the polymerase is displaced from the active site on the same molecule by the viral DNA during reverse transcription, P must be structurally dynamic during DNA synthesis. Therefore, non-nucleoside compounds that interfere with this change may be novel antiviral agents.

Received for publication, August 22, 2006 , and in revised form, September 26, 2006.

^* This work was supported by Grant AI38447 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, 1402 S. Grand Blvd., Saint Louis, MO 63104. Tel.: 314-977-8893; Fax: 314-977-8717; E-mail: tavisje{at}slu.edu.

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