Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus.
Human foamy or spuma virus (HFV) codes for a distinct set of pol gen products. To determine the minimal requirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribo-nuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesis. The mutant gene products were bacterially expressed, purified by Ni2+ chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by the situ RT, RNase H and RNase H assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-His. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while DNA polymerase activity increased 2-fold. A deletion of 11 amino acid residues in the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant in the C-terminal RH domain showed no RNase H but retained RNase H activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.
Kögel
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Abteilung Retrovirale Genexpression, Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Aboud
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Flügel
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RM
D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D015751:Genes, pol; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016133:Polymerase Chain Reaction; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D016914:Ribonuclease H / Q000235:genetics*; D016324:Sequence Tagged Sites; D016092:Spumavirus / Q000201:enzymology* / Q000235:genetics*
D017931:DNA Primers; D004279:DNA, Viral; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H