Detection of DNA polymerase activities associated with purified duck hepatitis B virus core particles by using an activity gel assay.
Replication of hepadnaviruses involves reverse transcription of an intermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymerase which has DNA-dependent DNA polymerase, reverse transcriptase, and RNase H activities. Biochemical studies of the polymerase protein(s) have been limited by the inability to purify useful quantities of functional enzyme from virus particles and, until recently, to express enzymatically active polymerase proteins in heterologous systems. An activity gel assay which detects in situ catalytic activities of DNA polymerases after electrophoresis in partially denaturing polyacrylamide gels was used by M.R. Bavand and O. Laub (J. Virol. 62:626-628, 1988) to show the presence of DNA- and RNA-dependent DNA polymerase activities associated with hepatitis B virus particles produced in vitro. This assay has provided the only means by which hepadnavirus polymerase proteins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantities of enzymatically active polymerase protein(s), we have devised a protocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles contain DNA- and RNA-dependent DNA polymerase activities, as shown in the endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins associated with these purified DHBV core particles. These enzymatically active proteins appear larger than, approximately the same size as, and smaller than an unmodified DHBV polymerase protein predicted from the polymerase open reading frame. This is the first report of the detection of active hepadnavirus core-associated DNA polymerase proteins derived from a natural host.
Oberhaus
S M
SM
Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill 27599.
Newbold
J E
JE
D000818:Animals; D015139:Blotting, Southern; D004259:DNA-Directed DNA Polymerase / Q000032:analysis*; D003609:Dactinomycin / Q000494:pharmacology; D004372:Ducks; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015611:Hepatitis B Virus, Duck / Q000201:enzymology* / Q000648:ultrastructure
D003609:Dactinomycin; D004259:DNA-Directed DNA Polymerase