Date of Award

Winter 1994

Document Type


Degree Name

Doctor of Philosophy (PhD)


Biological Sciences


Biomedical Sciences

Committee Director

Laura K. Moen

Committee Member

Mark S. Elliot

Committee Member

Christopher J. Osgood

Committee Member

Miriam D. Rosenthal


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 If the subunits of the RT dimer are dissociated, the enzyme is no longer active; therefore, identification of subunit binding sites could lead to potential targets for antiviral therapy.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 greater effect than the L289R mutation. These results are consistent with recent 3-dimensional computer modeling studies which indicate that leucine 289 is too far from the dimer interface to be involved in subunit binding. Leucine 289 is probably very important for maintaining secondary structure and proper folding of the enzyme, but it is not directly involved in subunit interactions.


Dissertation submitted to the Faculty of Eastern Virginia Medical School and Old Dominion University in Partial Fulfillment of the Requirement for the Degree of Doctor of Philosophy in Biomedical Sciences.