Abstract
Single amino acid deletions in the β3-β4 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 (Δ67 complex) are usually associated with thymidine analog resistance mutations (TAMs) (e.g. M41L, T215Y, etc.) while the deletion of Thr-69 (Δ69) is rarely found in isolates containing TAMs. Here, we show that the complex Δ67/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 Δ69 (or the complex S68G/Δ69/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 Δ69, Δ67/T69G/K70R, and D67N/K70R RTs support a relevant role for Lys/Arg-70 in the excision reaction. In Δ69 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 Δ69 or S68G/Δ69/K70G, providing further biochemical evidence that explains the lack of association of Δ69 and TAMs in HIV-1 isolates.
Highlights
K219E reduced both ATP- and pyrophosphate-mediated excision of primers terminated with thymidine analogues, only when introduced in reverse transcriptase (RT) bearing ⌬69 or S68G/⌬69/K70G, providing further biochemical evidence that explains the lack of association of ⌬69 and thymidine analog resistance mutations (TAMs) in human immunodeficiency virus type 1 (HIV-1) isolates
A significant number of mutations conferring resistance to nucleotide RT inhibitors (NRTIs) has been demonstrated to appear in the 3-4 hairpin loop of HIV-1 RT (1, 5)
Amino acid sequences around the 3-4 region of the RT show remarkable variability, with ⌬69/ ⌬70 deletions appearing frequently in combination with the Q151M complex (21, 23, 40, 41), and ⌬67 in isolates containing TAMs (22, 26, 40)
Summary
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. One of them is based on nucleotide selectivity, and is exemplified by lamivudine-resistance mutations M184V and M184I, and by the Q151M complex (A62V/ V75I/F77L/F116Y/Q151M) that confers resistance to multiple NRTIs (6, 7) These amino acid substitutions affect residues of the dNTP binding site of the RT (8), while reducing the enzyme’s ability to incorporate the triphosphorylated NRTIs. The second mechanism involves enhanced excision of the JOURNAL OF BIOLOGICAL CHEMISTRY 20615. The prevalence of 3-4 hairpin loop deletions has been estimated to be less than 0.2% among patients failing antiretroviral treatment including NRTIs (21, 22) These deletions may appear together with mutations of the Q151M complex or with TAMs. The deletion of RT codon 69 (encoding for Thr-69) has been observed in combination with one or more mutations of the Q151M complex in viral isolates resistant to multiple NRTIs (21, 23). Biochemical studies have shown that in the absence of TAMs, ⌬69 decreases ATP-mediated excision of primers terminated with AZT as well as the catalytic efficiency of incorporation of 3TC triphosphate (3TCTP) relative to dCTP by the HIV-1 RT (24)
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