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

Abstract

Single amino acid deletions in the ␤3-␤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 (⌬67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e.g. M41L, T215Y, etc.) while the deletion of Thr-69 (⌬69) is rarely found in isolates containing TAMs. Here, we show that the complex ⌬67/T69G/K70R enhances ATP-dependent phosphorolytic activity on primers terminated 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 ⌬69 (or the complex S68G/⌬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 ⌬69, ⌬67/T69G/K70R, and D67N/K70R RTs support a relevant role for Lys/Arg-70 in the excision reaction. In ⌬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 AZTtriphosphate 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 ⌬69 or S68G/⌬69/K70G, providing further biochemical evidence that explains the lack of association of ⌬69 and TAMs in HIV-1 isolates.

Delayed Emergence of HIV-1 Variants Resistant to 4′-Ethy Nyl-2-Fluoro-2′-Deoxyadenosine: Comparative Sequential Passage Study with Lamivudine, Tenofovir, Emtricitabine and BMS-986001

“…Following selection with TDF and EFdA, virtually all the resultant variants contained D67del, T69G and K70R (Table 3). Previous studies have reported that D67del alone has little effects on HIV-1's sensitivity to AZT, but the addition of T69G and K70R confers significant AZT resistance, designating these three aberrant changes as the 'delta 67 complex' [32][33][34][35]. In the present study, the delta 67 complex reduced the activity of TDF by a factor of 83 (Table 5).…”

Changes in codon-pair bias of human immunodeficiency virus type 1 have profound effects on virus replication in cell culture

“…Recombinant infectious viruses were generated as we described previously [27,28]. Briefly, 100 ng of fulllength protease or 500 ng of gag purified PCR products were cotransfected into MT-4 cells with 1 μg of protease-deleted [29] or gag-protease-deleted [30] HXB2 clone that had been previously linearized with BstE II (New England Biolabs).…”

NucAmino: a nucleotide to amino acid alignment optimized for virus gene sequences

“…The indel positional matrix had a bonus score of 6 for RT codon 69. There have been two other well-described regions with indels in these HIV-1 genes: deletions in the RT β3-β4 loop region [ 4 , 8 ] and insertions between codons 33 and 41 in protease [ 9 ]. NucAmino did not include scores for these in the indel positional matrix, because each of the RT β3-β4 loop deletions are associated with different drug-resistance interpretations and because the protease codon 33/41 insertions are not associated with drug resistance.…”