title: Genetic selection in Escherichia coli for active human immunodeficiency virus reverse transcriptase mutants.
abstract: Most catalytically active human immunodeficiency virus (HIV) reverse transcriptase (RT) mutants characterized to date have been isolated from the virus after treatment with HIV RT inhibitors such as nucleoside analogs. However, detailed understanding of structure-function relationships, and of the roles of the several catalytic activities of HIV RT in viral replication, requires characterization of a greater diversity of mutant enzymes than has been obtained from viral variants. Coupling of a bacterial genetic selection system for functional HIV RT with random mutagenesis has yielded a large number of active mutant enzymes, most of which have not been found in viral variants. The genetic selection system, combined with biochemical characterization of active mutant proteins, affords three major benefits. First, we can increase our understanding of the roles of individual amino acids in catalysis. Second, the mutational spectrum observed among active HIV RT variants can identify amino acids that are intolerant, or relatively intolerant, of substitution. Third, this system provides us with HIV RT variants with altered biochemical properties, such as replicational fidelity and processivity. Characterization of HIV harboring these mutant RTs with defined structural and functional alterations will contribute to elucidation of the roles of each catalytic activity of HIV RT in viral replication.
authors.affiliation: Department of Pathology, School of Medicine, University of Washington, Seattle 98195, USA.
authors.forename: B
authors.initials: B
authors.lastname: Kim
mesh_terms: D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D005816:Genetic Complementation Test; D006678:HIV / Q000201:enzymology*; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012641:Selection, Genetic; D013329:Structure-Activity Relationship
chemical_list: D012194:RNA-Directed DNA Polymerase