Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus
Kögel, Donat; Aboud, Mordechal; Flügel, Rolf M.; Abteilung Retrovirale Genexpression, Angewandte Tumorivologie, Deutsches Krebsorschungszentrum69009 Heidelberg, Germany; Aboud Mordechal; Department of Microbiology and Immunology, Ben-Gurion University of the Negev
Журнал:
Nucleic Acids Research
Дата:
1995
Аннотация:
Human foamy or spuma virus (HFV) codes for a distinct set of pol gene products. To determine the minimalrequirements for the HFV enzymatic activities, defined residues of the reverse transcriptase (RT) and ribonuclease H (RNase H) domain of the HFV pol gene were mutated by site-specific PCR mutagenesls. The mutant gene products were bacterially expressed, purified by Ni<sup>2+</sup> chelate affinity chromatography and characterised by Western blotting. The enzymatic activities of the individual recombinant HFV pol mutant proteins were characterised by in situ RT, RNase H and RNase H<sup>*</sup> assays. Two substitution mutants reached RT activity levels higher than that of the intact recombinant HFV RT-RH-Hls. When the catalytically essential D508 was substituted by A508, 5% of RNase H activity was retained while ONA polymerase activity increased 2-fold. A deletion of 11 amlno acid residues In the hinge region completely abolished DNA polymerase while RNase H activity decreased 2-fold. A deletion mutant In the C-termlnal RH domain showed no RNase H but retained RNase H<sup>*</sup> activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.
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