HIV-1 replication is inhibited by the incorporation of chain-terminating nucleotides at the 3 end of the growing DNA chain. Here we show a nucleotide-dependent reaction catalyzed by HIV-1 reverse transcriptase that can efficiently remove the chain-terminating residue, yielding an extendible primer terminus. Radioactively labeled 3-terminal residue from the primer can be transferred into a product that is resistant to calf intestinal alkaline phosphatase and sensitive to cleavage by snake venom phosphodiesterase. The products formed from different nucleotide substrates have unique electrophoretic migrations and have been identified as dinucleoside tri-or tetraphosphates. The reaction is inhibited by dNTPs that are complementary to the next position on the template (K i Ϸ 5 M), suggesting competition between dinucleoside polyphosphate synthesis and DNA polymerization. Dinucleoside polyphosphate synthesis was inhibited by an HIV-1 specific non-nucleoside inhibitor and was absent in mutant HIV-1 reverse transcriptase deficient in polymerase activity, indicating that this activity requires a functional polymerase active site. We suggest that dinucleoside polyphosphate synthesis occurs by transfer of the 3 nucleotide from the primer to the pyrophosphate moiety in the nucleoside dior triphosphate substrate through a mechanism analogous to pyrophosphorolysis. Unlike pyrophosphorolysis, however, the reaction is nucleotide-dependent, is resistant to pyrophosphatase, and produces dinucleoside polyphosphates. Because it occurs at physiological concentrations of ribonucleoside triphosphates, this reaction may determine the in vivo activity of many nucleoside antiretroviral drugs.

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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...

Phosphonoformate (foscarnet) is a pyrophosphate (PP i ) analogue and a potent inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), acting through the PP i binding site on the enzyme. HIV-1 RT can unblock a chain-terminated DNA primer by phosphorolytic transfer of the terminal residue to an acceptor substrate (PP i or a nucleotide such as ATP) which also interacts with the PP i binding site. Primer-unblocking activity is increased in mutants of HIV-1 that are resistant to the chain-terminating nucleoside inhibitor 3-azido-3-deoxythymidine (AZT). We have compared the primer-unblocking activity for HIV-1 RT containing various foscarnet resistance mutations (K65R, W88G, W88S, E89K, S117T, Q161L, M164I, and the double mutant Q161L/H208Y) alone or in combination with AZT resistance mutations. The level of primer-unblocking activity varied over a 150-fold range for these enzymes and was inversely correlated with foscarnet resistance and directly correlated with AZT resistance. Based on published crystal structures of HIV-1 RT, many of the foscarnet resistance mutations affect residues that do not make direct contact with the catalytic residues of RT, the incoming deoxynucleoside triphosphate (dNTP), or the primer-template. These mutations may confer foscarnet resistance and reduce primer unblocking by indirectly decreasing the binding and retention of foscarnet, PP i , and ATP. Alternatively, the binding position or orientation of PP i , ATP, or the primer-template may be changed in the mutant enzyme complex so that molecular interactions required for the unblocking reaction are impaired while dNTP binding and incorporation are not.Phosphonoformate (foscarnet) inhibits a wide variety of DNA and RNA polymerases including human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (40, 43). Foscarnet competes with pyrophosphate (PP i ) for binding in PP i exchange reactions, suggesting that foscarnet interacts with the site on the enzyme where PP i is formed (7, 38). On structural grounds, the site of interaction for PP i (and, presumably, for foscarnet) should lie within the deoxynucleoside triphosphate (dNTP) binding site since PP i is formed from the ␤ and ␥ phosphates of the dNTP substrate, but foscarnet inhibition of DNA synthesis in vitro is noncompetitive with dNTP substrates (58), indicating that foscarnet and dNTP bind to distinguishable forms of RT. In addition, median-effect analysis comparing foscarnet and 3Ј-azido-3Ј-deoxythymidine (AZT) triphosphate (AZTTP) alone or as mixtures has shown that inhibition by these compounds is mutually exclusive (10, 22, 50), implying that binding of either inhibitor prevents binding of the other to the same enzyme molecule.In intact cells, the inhibition of HIV-1 by foscarnet and AZT is synergistic (10,22). The mechanism of this synergy is unclear, but it has recently been demonstrated that AZT monophosphate (AZTMP) incorporation can be reversed by the primer-unblocking activity of RT, in which the chain-terminating residue is trans...

Nucleotide-dependent unblocking of chain-terminated DNA by human immunodeficiency virus type 1 reverse transcriptase (RT) is enhanced by the presence of mutations associated with 3-azido-3-deoxythymidine (AZT) resistance. The increase in unblocking activity was greater for mutant combinations associated with higher levels of in vivo AZT resistance. The difference between mutant and wild-type activity was further enhanced by introduction of a methyl group into the nucleotide substrate and was decreased for a nonaromatic substrate, suggesting that -interactions between RT and an aromatic structure may be facilitated by these mutations.Many nucleoside analogues, including 3Ј-azido-3Ј-deoxythymidine (AZT), inhibit human immunodeficiency virus type 1 (HIV-1) replication. The phosphorylated forms of these compounds are incorporated during DNA synthesis by the HIV-1 reverse transcriptase (RT), resulting in chain termination and inhibition of viral replication (7,9,12,22,31,32). Mutations at codons 41, 67, 70, 210, 215, and 219 in the HIV-1 RT gene result in resistance of HIV-1 to AZT in cell culture assays (29). Substitutions of phenylalanine and tyrosine for threonine at position 215 (T215F and T215Y) are the predominant mutations observed in vivo and are considered the most important for the resistance phenotype (16,17,25).The inhibitory effect of incorporating a chain-terminating nucleotide analogue can be partially relieved by a reaction catalyzed by RT in which the terminating nucleotide is removed from the 3Ј end of a DNA chain by transfer to a nucleotide di-or triphosphate, producing an unblocked DNA chain and dinucleoside polyphosphate with the chain terminator linked to the nucleotide acceptor through a tri-or tetraphosphate chain (19,21). HIV-1 RT can also transfer the chain-terminating residue to pyrophosphate (PP i ), regenerating the triphosphate form of the chain terminator (1, 5, 10, 23). These observations have suggested that enhanced removal of 3Ј-azido-3Ј-deoxythymidine-5Ј-monophosphate (AZTMP) is a possible mechanism for AZT resistance, and an increase in the removal reaction has been reported for RT containing various AZT resistance mutations (1,2,18,19). The biochemical contribution of each of these mutations in the removal reaction remains unclear. Boyer et al. (2) have modeled the amino acid substitutions associated with AZT resistance, as well as ATP or PP i , into the three-dimensional structure of HIV-1 RT and concluded that several of these amino acid substitutions could affect the binding of ATP but are unlikely to affect binding of PP i .In this report we describe further investigation into the contributions made by specific mutations in HIV-1 RT to its removal activity and the effects of changes in the structure of the nucleotide substrate on the wild-type (WT) and mutant activities. The mutations associated with AZT resistance identify a region of HIV-1 RT that may interact with the nucleoside moiety of a transition intermediate to facilitate the formation of the dinucleoside tetraphospha...