3′-azido-3′-deoxythymidine-(5′)-tetraphospho-(5′)-adenosine, the product of ATP-Mediated excision of chain-terminating AZTMP, is a potent chain-terminating substrate for HIV-1 reverse transcriptase

The resistance of HIV-1 to 3'-azido-3'-deoxythymidine (AZT) involves phosphorolytic excision of chain-terminating AZT-5'-monophosphate (AZTMP). Both pyrophosphate (PPi) and ATP act as excision substrates in vitro, but the intracellular substrate used during replication of AZT-resistant HIV is still unknown. PPi-mediated excision produces AZT-5'-triphosphate (AZTTP), which could be immediately re-used as a substrate for viral DNA chain termination. In contrast, ATP-mediated excision produces the novel compound AZT-(5')-tetraphospho-(5')-adenosine (AZTp4A). Since little is known of the interaction of AZTp4A with HIV-1 RT, we carried out kinetic and molecular modeling studies to probe this. AZTp4A was found to be a potent inhibitor of HIV-1 RT-catalyzed DNA synthesis and of both ATP- and PPi-mediated AZTMP excision. AZTp4A is in fact an excellent chain-terminating substrate for AZT-resistant RT-catalyzed DNA synthesis, better than AZTTP (k(pol)/K-d = 6.2 and 11.9 for AZTTP and AZTp4A, respectively). The affinity of AZT-resistant HIV-1 RT for AZTp4A is at least 30000-fold greater than that for the excision substrate ATP and similar to 10-fold greater than that for AZTTP. Dissociation of newly formed AZTp4A from RT may therefore provide a significant rate-limiting step for continued HIV-1 DNA synthesis. Our studies show that the products of PPi- and ATP-mediated excision of chain-terminating AZTMP (AZTTP and AZTp4A, respectively) are both potent chain-terminating substrates for HIV-1 RT, suggesting that there is no obvious benefit to HIV using ATP instead of PPi as the excision substrate.