Detection of an RNase H activity associated with hepadnaviruses.
Replication of the hepadnavirus DNA genome is accomplished via reverse transcription of an intermediate, pregenomic RNA molecule. This process is likely to be carried out by a virally encoded, multifunctional polymerase which possesses DNA- and RNA-dependent DNA polymerase and RNase H activities. However, the nature of the product(s) of the polymerase gene predicted to mediate these functions is unclear. Biochemical studies of the polymerase protein(s) have been limited by its apparent low abundance in virus particles and, until recently, the inability to express active polymerase protein(s) heterologously. We have used activity gel assays to detect DNA- and RNA-dependent DNA polymerase activities associated with highly purified duck hepatitis B virus (DHBV) core particles (S. M. Oberhaus and J. E. Newbold, J. Virol. 67:6558-6566, 1993). Now we report that the same approach identifies a 35-kDa RNase H activity in association with highly purified DHBV core particles and crude preparations of virions from DHBV-infected ducks and woodchuck hepatitis virus-infected woodchucks. This is the first report of the detection of an hepadnavirus-associated RNase H activity. Its apparent size is smaller than any of the DNA polymerase activities that we detected previously and significantly smaller than the full-length protein predicted from the polymerase open reading frame (p85 for DHBV). These data suggest that the viral polymerase and RNase H activities are separable and that these enzymes may coordinate their activities in vivo by forming a complex.
Oberhaus
S M
SM
Department of Microbiology and Immunology, School of Medicine, University of North Carolina at Chapel Hill 27599-7290, USA.
Newbold
J E
JE
D000818:Animals; D015139:Blotting, Southern; D004279:DNA, Viral / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004372:Ducks; D005777:Geese; D015613:Hepadnaviridae / Q000201:enzymology* / Q000302:isolation & purification; D018347:Hepadnaviridae Infections / Q000097:blood / Q000821:virology; D015611:Hepatitis B Virus, Duck / Q000201:enzymology* / Q000302:isolation & purification; D007700:Kinetics; D008099:Liver / Q000821:virology; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism*; D016914:Ribonuclease H / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D014766:Viremia / Q000097:blood / Q000821:virology
D004279:DNA, Viral; D008345:Manganese; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase; D016914:Ribonuclease H; D008274:Magnesium