MUTATIONAL ANALYSIS OF THE REVERSE-TRANSCRIPTASE AND RIBONUCLEASE-H DOMAINS OF THE HUMAN FOAMY VIRUS

Human foamy or spuma virus (H RT) codes for a distinct set of pol gene products, To determine the minimal requirements for the HN 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 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 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-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.