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 int ermediate RNA molecule. It is generally accepted that this replication scheme is carried out by a virally encoded, multifunctional polymeras e which has DNA-dependent DNA polymerase, reverse transcriptase, and R Nase H activities. Biochemical studies of the polymerase protein(s) ha ve been limited by the inability to purify useful quantities of functi onal enzyme from virus particles and, until recently, to express enzym atically active polymerase proteins in heterologous systems. An activi ty gel assay which detects in situ catalytic activities of DNA polymer ases 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 as say has provided the only means by which hepadnavirus polymerase prote ins have been detected in association with enzymatic activities. Since conventional methods have not allowed purification of useful quantiti es of enzymatically active polymerase protein(s), we have devised a pr otocol for purifying large quantities of duck hepatitis B virus (DHBV) core particles to near homogeneity. These immature virus particles co ntain DNA- and RNA-dependent DNA polymerase activities, as shown in th e endogenous DNA polymerase assay. We have used the activity gel assay to detect multiple DNA- and RNA-dependent DNA polymerase proteins ass ociated with these purified DHBV core particles. These enzymatically a ctive 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 detect ion of active hepadnavirus core-associated DNA polymerase proteins der ived from a natural host.