Subunit Interactions of Recombinant HIV-1 Reverse Transcriptase With Mutations at L289
type: CreativeWork
description: Reverse transcriptase (RT) is a dimeric enzyme required for replication of the human immunodeficiency virus (HIV). If the subunits of the RT dimer are dissociated, the enzyme is no longer active; therefore, identification of subunit binding sites could lead to potential targets for antiviral therapy. In order to identify where subunit binding of RT occurs, mutations were made at leucine (L) 289, a residue believed to be involved in dimerization through hydrophobic interactions with other leucines. L289 is the central leucine of a leucine repeat sequence which resembles a leucine zipper protein-DNA binding motif. Two mutations, leucine to arginine (L289R) and leucine to proline (L289P), were created using PCR mutagenesis. The mutations in the RT gene were verified by DNA sequencing, and cloned into a yeast expression vector using recombinant DNA techniques. The RT mutants were purified from yeast and compared to wild type RT in terms of specific activity, subunit dissociation, and subunit association using RT polymerase activity assays, fluorescence studies, and analytical gel filtration. Only the L289P mutant showed significantly less specific activity than wild type RT, and neither mutation affected RNase H activity. Both mutants dissociated into monomers slightly more easily than wild type RT, and both mutants were slower to associate to form dimers than wild type RT. L289P dimers dissociated slightly more easily than L289R dimers and were slower to reassociate. These mutations had only moderate effects on subunit interactions. If leucine 289 was part of a leucine zipper and directly involved in subunit binding, then both mutations would have had much greater effects on subunit interactions. Also, since RT subunit interactions are known to be hydrophobic in nature, the mutation to arginine, which is very hydrophilic, was expected to have a greater effect on dimerization than the proline mutation. However, the opposite was observed and the L289P mutation had a grea…
abstract: Reverse transcriptase (RT) is a dimeric enzyme required for replication of the human immunodeficiency virus (HIV). If the subunits of the RT dimer are dissociated, the enzyme is no longer active; therefore, identification of subunit binding sites could lead to potential targets for antiviral therapy. In order to identify where subunit binding of RT occurs, mutations were made at leucine (L) 289, a residue believed to be involved in dimerization through hydrophobic interactions with other leucines. L289 is the central leucine of a leucine repeat sequence which resembles a leucine zipper protein-DNA binding motif. Two mutations, leucine to arginine (L289R) and leucine to proline (L289P), were created using PCR mutagenesis. The mutations in the RT gene were verified by DNA sequencing, and cloned into a yeast expression vector using recombinant DNA techniques. The RT mutants were purified from yeast and compared to wild type RT in terms of specific activity, subunit dissociation, and subunit association using RT polymerase activity assays, fluorescence studies, and analytical gel filtration. Only the L289P mutant showed significantly less specific activity than wild type RT, and neither mutation affected RNase H activity. Both mutants dissociated into monomers slightly more easily than wild type RT, and both mutants were slower to associate to form dimers than wild type RT. L289P dimers dissociated slightly more easily than L289R dimers and were slower to reassociate. These mutations had only moderate effects on subunit interactions. If leucine 289 was part of a leucine zipper and directly involved in subunit binding, then both mutations would have had much greater effects on subunit interactions. Also, since RT subunit interactions are known to be hydrophobic in nature, the mutation to arginine, which is very hydrophilic, was expected to have a greater effect on dimerization than the proline mutation. However, the opposite was observed and the L289P mutation had a grea…
author: Smith, Jacquelyn R.
keywords: Immune deficiency, HIV, Mutations, Biochemistry, Molecular Biology
date published: 1994-01-01T08:00:00Z
date modified: 2026-07-24T03:35:30Z
provider: Old Dominion University
publisher: Networked Digital Library of Theses and Dissertations
type: WebPage
source organization: Old Dominion University