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

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Kögel D,Aboud M,Flügel RM

doi

10.1093/nar/23.14.2621

subject

Has Abstract

pub_date

1995-07-25 00:00:00

pages

2621-5

issue

14

eissn

0305-1048

issn

1362-4962

pii

5c0071

journal_volume

23

pub_type

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