This is a application to investigate the role of HIV RT fidelity in the genomic hypervariablity of HIV in response to selective pressures such as drug therapy. Presumably, it is the hypervariablity of HIV which leads to the emergence of drug-resistant strains. This genetic diversity has been hypothesized to result from error-prone replication by the viral polymerase. However, direct in vivo evidence relating the infidelity of HIV RT to genomic diversity is lacking. The investigator plans to pursue his investigation by three major objectives: First, the investigator will use random mutagenesis of the Beta-9/Beta 10 palm region of the HIV RT followed by genetic selection in a bacterial-based genetic complimentation assay to isolate HIV RT fidelity mutants. The selected mutants, and a mutant of interest already generated from his preliminary work, will then be characterized to determine the extent to which replicational accuracy has changed. Mutants which exhibit a 5-fold change in fidelity will be considered for further study. Second, the investigator will construct proviral clones containing the HIV RTs with mutations of interest and determine the degree of genetic variability in the genome of the HIV species carrying these altered RTs. Third, the investigator plans to measure the capability of HIV with mutant RT genes characterized from the previous aims to escape from the selective pressure of HIV protease inhibitors.
| Weiss, Kellie K; Bambara, Robert A; Kim, Baek (2002) Mechanistic role of residue Gln151 in error prone DNA synthesis by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). Pre-steady state kinetic study of the Q151N HIV-1 RT mutant with increased fidelity. J Biol Chem 277:22662-9 |
| Malboeuf, C M; Isaacs, S J; Tran, N H et al. (2001) Thermal effects on reverse transcription: improvement of accuracy and processivity in cDNA synthesis. Biotechniques 30:1074-8, 1080, 1082, passim |
| Diamond, T L; Kimata, J; Kim, B (2001) Identification of a simian immunodeficiency virus reverse transcriptase variant with enhanced replicational fidelity in the late stage of viral infection. J Biol Chem 276:23624-31 |
| Kim, B; Ayran, J C; Sagar, S G et al. (1999) New human immunodeficiency virus, type 1 reverse transcriptase (HIV-1 RT) mutants with increased fidelity of DNA synthesis. Accuracy, template binding, and processivity. J Biol Chem 274:27666-73 |
