A MUTATIONAL STUDY OF AMINO ACID RESIDUES INVOLVED IN PROTEIN STABILITY AND DIMERIZATION OF THE HIV-1 REVERSE TRANSCRIPTASE

John A. Wrobel, Esa Bloedon, Michael J. Conrad, Clyde A. Hutchison III

Department of Microbiology and Immunology
The University of North Carolina, Chapel Hill, NC

The HIV-1 reverse transcriptase (RT), a heterodimeric protein consisting of a 66 kD subunit and a 51 kD subunit, catalyzes the replication of the HIV-1 genome.  Previously, we developed an HIV-1 RT expression system in E. coli .  Our HIV-1 RT expression vector contains the entire HIV-1 pro and pol  genes, which code for the protease, the RT (with both DNA polymerase and ribonuclease H activities), and the integrase.  When expressed in E. coli, the pro-pol gene products are translated initially as a 120 kD polypeptide that is processed by the viral protease to produce the mature protease (11 kD), RT (66 kD and 51 kD heterodimer), and integrase (34 kD).  The wild-type RT expressed in our system is catalytically active.  Using oligonucleotide-directed mutagenesis, we have made 450 different single amino substitutions in our recombinant HIV-1 RT.  After expression in E. coli, each mutant was assayed for RNA-dependent DNA polymerase activity to test for enzyme function and Western blot analysis to estimate the stability of each mutant protein by measuring the processing of the RT into its mature heterodimeric form by the viral protease.  The resulting phenotype data provided a "genetic" means to identify amino acid side chains that are important for enzyme function or protein stability.  Currently we are combining our biological phenotype data with crystallographic data from several HIV-1 RT crystal structures to define the role of residues involved in protein stability in more detail.  Our analysis has identified several amino acid side chains involved in either hydrogen bonding or hydrophobic stabilizing interactions. Some of these stabilizing interactions in the HIV-1 RT are structurally conserved in MMLV RT crystal structure.  In another study we are using "lysine" scanning mutagenesis to identify amino acid residues important for dimerization at the subunit interface.