Differential Removal of Thymidine Nucleotide Analogues from Blocked DNA Chains by Human Immunodeficiency Virus Reverse Transcriptase in the Presence of Physiological Concentrations of 2′-Deoxynucleoside Triphosphates

Abstract

Removal of 2,3-didehydro-3-deoxythymidine-5-monophosphate (d4TMP) from a blocked DNA chain can occur through transfer of the chain-terminating residue to a nucleotide acceptor by human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). ATP-dependent removal of either d4TMP or 3-azido-3-deoxythymidine-5-monophosphate (AZTMP) is increased in AZT resistant HIV-1 RT (containing D67N/K70R/ T215F/K219Q mutations). Removal of d4TMP is strongly inhibited by the next complementary deoxynucleoside triphosphate (50% inhibitory concentration [IC 50 ] of ϳ0.5 M), whereas removal of AZTMP is much less sensitive to this inhibition (IC 50 of >100 M). This could explain the lack of cross-resistance by AZT-resistant HIV-1 to d4T in phenotypic drug susceptibility assays.Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) and other retroviral RTs lack 3Ј-5Ј exonuclease activity (2, 30) but can remove 3Ј-terminal chain-terminating residues from blocked DNA chains through a nucleotide-dependent mechanism leading to production of dinucleoside polyphosphates (23, 24) or through pyrophosphorolysis (the reversal of polymerization) (1,4,13,29). We have recently shown (23) that HIV-1 RT containing the 3Ј-azido-3Ј-deoxythymidine (AZT) resistance mutations D67N, K70R, T215F, and K219Q (67/70/215/219 mutant RT) removes AZT-5Ј-monophosphate (AZTMP) from blocked primer-templates through the nucleotide-dependent mechanism more efficiently than does wildtype (WT) RT. The mutant enzyme also removes 2Ј,3Ј-dideoxyadenosine-5Ј-monophosphate (ddAMP) from blocked DNA chains more efficiently than does WT RT. Removal of ddAMP is strongly suppressed by physiological concentrations of deoxynucleoside triphosphates (dNTPs), whereas removal of AZTMP is much less sensitive to this inhibition (23).The chain terminator 2Ј,3Ј-didehydro-3Ј-deoxythymidine-5Ј-triphosphate (d4TTP) is efficiently incorporated into growing DNA chains by HIV-1 RT (39). Resistance to d4T can arise in cell culture through a valine-to-threonine mutation at position 75 (19, 21, 32); however, this mutation is rarely observed in HIV-1 from d4T-treated individuals (6,9,17,22,27,35). Instead, mutations associated with AZT resistance, including M41L, D67N, K70R, L210W, and T215Y/F, are frequently selected (6,8,21,22,27,32,35). The selection of AZT resistance mutations by d4T in the absence of AZT is unexpected, since phenotypic assays show little, if any, cross-resistance between these drugs (20,22). Nonetheless, clinical studies have shown that prior exposure to AZT reduces the efficacy of subsequent treatment with d4T (17), and the presence of AZT resistance mutations is correlated with reduced suppression of viral load in response to d4T-containing therapies (15, 25). These results suggest that the phenotypic assays do not fully reflect the in vivo sensitivity of HIV-1 replication to d4T.In an effort to understand the biochemical basis for the lack of cross-resistance by AZT-resistant mutants to d4T, we have investigated the ability of WT and 67/70/215/219 m...

Primer Unblocking and Rescue of DNA Synthesis by Azidothymidine (AZT)-resistant HIV-1 Reverse Transcriptase

“…We then compared the initiation and elongation steps of (Ϫ) strand DNA synthesis. The results we obtained using a DNA template and a DNA primer are consistent with previous studies (21)(22)(23)36). They show that the mutant RT harboring mutations D67N, K70R, T215F, and K219K can efficiently repair an AZT-terminated DNA primer in the presence of 3.5 mM ATP.…”

“…2B). Because ATP lysis was much slower than nucleotide addition to the 3Ј-end of an unblocked primer (30), our results suggest that ATP lysis was partially inhibited by the next incoming nucleotide (22,36). The rescue of DNA synthesis was observed with the wt RT in the presence of ATP and with resistant RT in its absence, but these reactions were too weak to allow quantitative analysis (Fig.…”

“…All ATP lysis experiments using AZT-resistant RT published to date were performed using DNA/DNA primer-template complexes (21)(22)(23)36). However, the efficiency of this reaction, like other properties of HIV-1 RT, might vary considerably during the different steps of the provirus synthesis.…”

“…Because ATP lysis is sensitive to the presence of the next incoming nucleotide (22,36), two series of experiments were performed. In the first one, the excision reaction was performed in the absence of any nucleoside triphosphate, and we monitored the appearance of a product one nucleotide shorter than the initial primer.…”

N348I in HIV-1 Reverse Transcriptase Can Counteract the Nevirapine-mediated Bias toward RNase H Cleavage during Plus-strand Initiation

“…In this instance, a pyrophosphate donor, such as ATP, can excise the nucleotide analogue, providing an important mechanism for resistance. Mutations in HIV-1 RT that confer resistance to the NRTI, 3Ј-azidothymidine, increase rates of excision (15)(16)(17)(18)(19)(20)(21). More recently, it has been shown that mutations in the connection and RNase H domains of HIV-1 RT confer resistance to NRTIs and NNRTIs (9,10,(22)(23)(24)(25)(26).…”