Duan CY, Poticha D, Stoeckli TC, Lu J, Petropoulos C, Whitcomb J, McHenry CS, Kuritzkes DR; Conference on Retroviruses and Opportunistic Infections.

Program Abstr 8th Conf Retrovir Oppor Infect Conf Retrovir Oppor Infect 8th 2001 Chic Ill. 2001 Feb 4-8; 8: 175 (abstract no. 442).

Univ of Colorado Hlth Sci Ctr, Denver.

Background: The objective of this study was to characterize RT derived from an AZT-resistant clinical isolate and to determine whether this enzyme was cross-resistant to d4T-TP in vitro. Methods: Cloned wild-type (18A) and mutant (18C) (M41L/D67N/K70R/T215Y/K219Q) RT genes from HIV-1 isolates obtained before and after AZT treatment, respectively, were expressed in E. coli and purified to homogeneity. RT activity was assayed by 3H-thymidine incorporation using an HIV RNA template with an 18-nucleotide DNA primer annealed to the primer binding site in the absence or presence of AZTTP or d4TTP. All assays were conducted in the presence or absence of 3.2 micromolar ATP or 150 micromolar pyrophosphate. Results: Specific activity of the wild-type and mutant enzymes in the absence of drug was not significantly different. The Km for dTTP was 3.00 micromolar for 18A RT and 3.64 micromolar for 18C RT (P = 0.294). 18C RT demonstrated a 16- fold increase in IC50 for AZTTP compared to the 18A enzyme (12.4 +/- 0.25 micromolar vs 0.78 +/- 0.20 micromolar; P < 0.001) and a 14-fold increase in IC50 for d4TTP compared to 18A RT (14.4 +/- 0.77 micromolar vs 1.01 +/- 0.11 micromolar; P < 0.001). Likewise, the Ki of AZTTP for 18C RT (1.35 +/- 0.24 micromolar) was significantly greater than for 18A RT (0.28 +/- 0.05 micromolar; P = 0.025). Similarly, the K(i) of d4TTP was significantly higher for 18C RT (3.03 +/- 0.61 micromolar) than for 18A RT (0.33 +/- 0.02 micromolar; P = 0.025). In the presence of ATP, IC50s of 18C to AZTTP and d4TTP were increased 4-fold and 5-fold, respectively, as compared to the IC50s in the absence of ATP. With 150 micromolar pyrophosphate in the RT assay, IC50s of 18C to AZTTP and d4TTP increased 3-fold and 1.43-fold, respectively, as compared to the IC50 s in the absence of pyrophosphate. Conclusions: Purified HIV-1 RT from an AZT-resistant isolate demonstrated reduced inhibition by AZTTP and by d4TTP compared to RT from a paired WT isolate. This finding provides additional evidence for cross-resistance between AZT and d4T and may provide a biochemical explanation for the lower efficacy of d4T in patients with prior AZT treatment.

Publication Types:

• Meeting Abstracts

Keywords:

• 3'-azido-3'-deoxythymidine 5'-triphosphate
• DNA Primers
• Diphosphates
• HIV Infections
• HIV-1
• Humans
• In Vitro
• Stavudine
• Thymine Nucleotides
• Zidovudine
• reverse transcriptase, Human immunodeficiency virus 1
• thymidine 5'-triphosphate

Other ID:

• GWAIDS0006729

UI: 102244225

From Meeting Abstracts