Effects of insertional and point mutations on the functions of the duck hepatitis B virus polymerase.
The polymerase (P) gene of hepadnaviruses encodes a large polypeptide that appears to participate in several steps in the viral life cycle: packaging of viral RNA, providing the primer for synthesis of minus-strand DNA, synthesizing minus-strand DNA from an RNA template and plus-strand DNA from a DNA template, and degrading viral RNA in RNA-DNA hybrids. To assist in the assignment of these functions to domains of the duck hepatitis B virus polymerase protein, we have constructed a series of substitution mutations and a large insertion mutation, based in part on amino acid sequence comparisons with other proteins known to exhibit reverse transcriptase (RT) and RNase H activities. We found that changes in highly conserved sequences in putative RT and RNase H domains in the carboxy-terminal half of the protein dramatically reduced synthesis of both strands of viral DNA without major effects on RNA packaging into subviral cores. Thus we can uncouple RNA packaging and DNA synthesis but cannot separate RT and RNase H activities as has been done with human hepatitis B virus. The viability of a mutant with a large insertion (123 amino acids) upstream of the RT and RNase H domain indicates that a hinge region may separate parts of the polymerase protein implicated in priming and polymerization.
Department of Microbiology, University of California, San Francisco 94143-0502.
L J
LJ
Chang
R C
RC
Hirsch
D
D
Ganem
H E
HE
Varmus
D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004722:Endoribonucleases / Q000235:genetics*; D005814:Genes, Viral; D015611:Hepatitis B Virus, Duck / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009841:Oligonucleotides; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D016914:Ribonuclease H; D013329:Structure-Activity Relationship; D015678:Viral Structural Proteins / Q000235:genetics
D009841:Oligonucleotides; D015678:Viral Structural Proteins; D012194:RNA-Directed DNA Polymerase; D004722:Endoribonucleases; D016914:Ribonuclease H