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

Zhian Zhang,John E Tavis

doi:10.1074/jbc.m608031200

Abstract

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.

Highlights

Hepadnaviruses are small DNA-containing viruses that replicate by reverse transcription and infect primates, rodents, and birds
In vitro-translated P derivatives were diluted into IPP150, and equal aliquots of the translation mixture were immunoprecipitated with saturating amounts of Monoclonal antibody 11 (mAb 11), mAb 3F10, or anti-␤-galactosidase (Anti-␤-gal)
MgCl2 and 5 ␮Ci of the ability to modify the C terminus of P without ablating its [␣-32P]dGTP (Ͼ3,000 Ci/mmol; Amersham Biosciences) were ability to prime DNA synthesis and created two P derivatives added to the translation reactions, and the samples were incu- carrying alterations at their C termini to permit their electrobated at 37 °C for 30 min

Summary

Introduction

Hepadnaviruses are small DNA-containing viruses that replicate by reverse transcription and infect primates, rodents, and birds (reviewed in Ref. 1). These data indicate that P mutants with lesions that inactivate the reverse transcriptase active site or ablate the hydroxyl on Tyr-96 that forms the covalent linkage with DNA cannot act as dominant negative inhibitors of the wild-type enzyme, nor can they complement each other and prime DNA synthesis in trans.

Results

Conclusion