Our goal is to prevent resistance to drugs directed against HIV RT (human immunodeficiency virus reverse transcriptase). We have recently established that HIV RT can substitute for DNA polymerase I in promoting growth of E. coli. Furthermore, HIV RT-dependent growth of E. coli is sensitive to AZT, a clinically important inhibitor of the RT. We propose to exploit this bacterial complementation system to: (1) Screen nucleoside analogs for their effectiveness in inhibiting HIV RT; (2) identify nonmutable (essential) amino acid residues in HIV RT to aid in drug design; and (3) detect resistant mutants in treated patients, prior to the onset of overt drug resistance. A bacterial system to screen large numbers of potential inhibitors of HIV RT has advantages with respect to efficiency, cost and safety. We will validate this approach by analyzing the effects of known inhibitors of HIV RT, and by determining if HIV RT mutations that confer resistance in humans render bacterial growth resistant to inhibition by different nucleoside analogs. Rapid emergence of resistant HIV mutants limits the efficacy of anti-HIV RT drugs. We will use random sequence mutagenesis, together with the bacterial complementation system, to identify amino acids in HIV RT that are nonmutable (essential). Nonmutable residues are potential targets for chemotherapeutic agents that inhibit the enzyme and cannot be evaded by viral mutagenesis. For early detection of resistant HIV mutants in patients undergoing anti- HIV RT chemotherapy, we will clone HIV RT from blood and analyze resistance in our bacterial complementation system.
| Kamath-Loeb, A S; Hizi, A; Kasai, H et al. (1997) Incorporation of the guanosine triphosphate analogs 8-oxo-dGTP and 8-NH2-dGTP by reverse transcriptases and mammalian DNA polymerases. J Biol Chem 272:5892-8 |
| Suzuki, M; Christians, F C; Kim, B et al. (1996) Tolerance of different proteins for amino acid diversity. Mol Divers 2:111-8 |
