Mutational analysis of the reverse transcriptase and ribonuclease H domains of the human foamy virus

Donat Kögel, Mordechal Aboud, Rolf M. Flügel

Research output: Contribution to journalArticlepeer-review

10 Scopus citations

Abstract

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 Ni2+ 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* 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* activity indicating that the activities are genetically separable. The combined data reveal that the HFV DNA polymerase and RNase H activities are interdependent.

Original languageEnglish
Pages (from-to)2621-2625
Number of pages5
JournalNucleic Acids Research
Volume23
Issue number14
DOIs
StatePublished - 25 Jul 1995

ASJC Scopus subject areas

  • Genetics

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