title: Activity-based selection of HIV-1 reverse transcriptase variants with decreased polymerization fidelity.
abstract: HIV-1 reverse transcriptase (HIV-1 RT) copies the RNA genome of HIV-1 into DNA, thereby committing errors at an exceptionally high frequency. Viral offspring evolve rapidly and consequently are capable of evading the immune response as well as antiviral treatment. However, error-prone viral replication could drive HIV close to extinction owing to an intolerable load of deleterious mutations. We applied a genetic selection scheme to identify variants of HIV-1 RT with a further increased error rate to study the relationship between error rate and viral replication. Using this approach, we identified 16 mutator candidates, two of which were purified and further studied in vitro. One of these variant enzymes showed a generally increased mutation frequency as compared with the reference enzyme. A single amino acid residue, R448, is probably responsible for the observed effect. Mutation of this residue, which is located within the RNase H domain of HIV-1 RT, seems to perturb the interaction with template RNA and consequently affects polymerase activity and fidelity.
authors.affiliation: Fakultät Chemie/Chemische Biologie (BCMT), Technische Universität Dortmund, Otto-Hahn-Strasse 6, D-44227 Dortmund, Germany.
authors.forename: Sascha N
authors.initials: SN
authors.lastname: Stumpp
authors.forename: Bianca
authors.initials: B
authors.lastname: Heyn
authors.forename: Susanne
authors.initials: S
authors.lastname: Brakmann
mesh_terms: D004279:DNA, Viral / Q000235:genetics; D019020:Directed Molecular Evolution; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D012367:RNA, Viral / Q000235:genetics
chemical_list: D004279:DNA, Viral; D012367:RNA, Viral; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase