Effects of Specific Zidovudine Resistance Mutations and Substrate Structure on Nucleotide-Dependent Primer Unblocking by Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Abstract

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...

In Vitro Selection and Analysis of Human Immunodeficiency Virus Type 1 Resistant to Derivatives of β-2′,3′-Didehydro-2′,3′-Dideoxy-5-Fluorocytidine

“…This concept is supported by the observation that D-d4FC retains potency against virus containing the zidovudine resistance mutations D67N, K70R, T215Y, and K219Q, which confer excision competence (8,12). Thus, D-d4FC may be a potent inhibitor for patients with virus having zidovudine resistance mutations, although clinical studies are needed to confirm this.…”

Mechanism for nucleoside analog-mediated abrogation of HIV-1 replication: Balance between RNase H activity and nucleotide excision

“…1c) is that mutations reducing the rate of RNA degradation will increase the time available for excision of AZT or d4T from a terminated primer, leading to an increase in drug resistance. To test this prediction, we determined the antiretroviral drug sensitivity of wild-type RT, an RT mutant (AZT-R) containing a well characterized cluster of TAM (D67N, K70R, T215Y, K219Q) (6,12), and two mutants of RNase H that reduce RNase H activity, namely, D549N (15) and H539N (16,17) (Fig. 2).…”

“…Antiviral resistance to several NRTIs such as 3Ј-azido-3Ј-deoxythymidine (AZT) and 2,3-didehydro-2,3-dideoxythymidine (d4T) arises through a nucleotide excision or primer-unblocking mechanism involving hydrolytic removal of the chain-terminating nucleoside analog from the end of the primer (4,5). Mutations that confer resistance to thymidine analogs (TAM) increase the rate of nucleotide excision (6,7). Elucidating the nucleotide excision mechanism has significantly enhanced our understanding of the mechanisms that contribute to NRTI resistance; despite this advance, several aspects of the molecular mechanisms underlying the evolution of drug resistance in vivo are not well understood.…”

Trapping HIV-1 Reverse Transcriptase Before and After Translocation on DNA

“…In this mechanism, AZT-resistant HIV-1 RT unblocks AZTMP-terminated primers by a mechanism that involves nucleophilic attack by a pyrophosphate donor (2), most likely ATP (3)(4)(5)(6). We have recently proposed that several of the AZT-resistance mutations act primarily to facilitate the binding of ATP (3,4,7).…”