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...
