442   Biochemical Evidence of Cross-Resistance to Stavudine (d4T) Triphosphate in Purified HIV-1 Reverse Transcriptase (RT) Derived from a Zidovudine (AZT)- Resistant Isolate.

C. Y. Duan*¹, D. Poticha¹, T. C. Stoeckli¹, J. Lu¹, C. Petropoulos², J. Whitcomb², C. S. McHenry¹, and D. R. Kuritzkes¹.
¹Univ. of Colorado Hlth. Sci. Ctr., Denver and²ViroLogic, S. San Francisco, CA.

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 by³H-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 mM ATP or 150mM pyrophosphate.

Results:Specific activity of the wild-type and mutant enzymes in the absence of drug was not significantly different. The K_mfor dTTP was 3.00mM for 18A RT and 3.64mM for 18C RT (P = 0.294). 18C RT demonstrated a 16-fold increase in IC₅₀for AZTTP compared to the 18A enzyme (12.4+0.25mM vs 0.78+0.20mM; P < 0.001) and a 14-fold increase in IC₅₀for d4TTP compared to 18A RT (14.4+0.77mM vs 1.01+0.11mM; P < 0.001). Likewise, the K_iof AZTTP for 18C RT (1.35+0.24mM) was significantly greater than for 18A RT (0.28+0.05mM; P = 0.025). Similarly, the K_iof d4TTP was significantly higher for 18C RT (3.03+0.61mM) than for 18A RT (0.33+0.02mM; 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 150mM pyrophosphate in the RT assay, IC₅₀s of 18C to AZTTP and d4TTP increased 3-fold and 1.43-fold, respectively, as compared to the IC₅₀s 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.