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

Zhian Zhang, John E. Tavis
2006 Journal of Biological Chemistry  
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
more » ... 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. . 2 The abbreviations used are: HBV, hepatitis B virus; DHBV, duck hepatitis B virus; pgRNA, pregenomic RNA; P, polymerase or reverse transcriptase; HIV, human immunodeficiency virus; LMH, chicken hepatoma cells; LMH-D2, LMH cells stably transfected with a DHBV dimer; HA, influenza hemagglutinin epitope tag; MAb, monoclonal antibody; nt, nucleotide(s).
doi:10.1074/jbc.m608031200 pmid:17005569 fatcat:g54jzjbmczfv5cal5dd7jioxga