Thymidine Analogue Excision and Discrimination Modulated by Mutational Complexes Including Single Amino Acid Deletions of Asp-67 or Thr-69 in HIV-1 Reverse Transcriptase

Single amino acid deletions in the beta 3-beta 4 hairpin loop of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been identified in heavily treated patients. The deletion of Asp-67 together with mutations T69G and K70R (Delta 67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e. g. M41L, T215Y, etc.) while the deletion of Thr-69 (Delta 69) is rarely found in isolates containing TAMs. Here, we show that the complex Delta 67/T69G/K70R enhances ATP-dependent phosphorolytic activity on primers terminated with 3'-azido-3'-deoxythymidine (AZT) or 2',3'-didehydro-2',3terminated with 3'-azido-3'-deoxythymidine (AZT) or 2',3'-didehydro-2',3'-dideoxythymidine (d4T) both in the presence or absence of TAMs (i.e. M41L/T215Y), while Delta 69 (or the complex S68G/Delta 69/K70G) antagonize the effects of TAMs in ATP-mediated excision. These effects are consistent with AZT susceptibility data obtained with recombinant HIV-1 bearing the relevant RTs. Molecular dynamics studies based on models of wild-type HIV-1 RT and mutant Delta 69, Delta 67/T69G/K70R, and D67N/K70R RTs support a relevant role for Lys/Arg-70 in the excision reaction. In Delta 69 RT, the side chain of Lys-70 locates away from the putative pyrophosphate binding site. Therefore, its participation in interactions required for the excision reaction is unlikely. Our theoretical studies also suggest a role for Lys-219 in thymidine analog excision/discrimination. However, pre-steady-state kinetics revealed only minor differences in selectivity of AZT-triphosphate versus dTTP between deletion-containing RTs and their homologous enzymes having the K219E mutation. K219E reduced both ATP- and pyrophosphate-mediated excision of primers terminated with thymidine analogues, only when introduced in RTs bearing Delta 69 or S68G/Delta 69/K70G, providing further biochemical evidence that explains the lack of association of Delta 69 and TAMs in HIV-1 isolates.