Effects of the Δ67 Complex of Mutations in Human Immunodeficiency Virus Type 1 Reverse Transcriptase on Nucleoside Analog Excision

Abstract

), contains numerous amino acid substitutions and a deletion of codon 67, which we have designated the ⌬67 complex of mutations. We have expressed and purified HIV-1 RT containing these mutations. We compared the polymerase and pyrophosphorolysis (excision) activity of an RT with the ⌬67 complex of mutations to wild-type RT and the two other AZT-resistant variants described above. All of the AZT-resistant variants we tested excise AZTMP and 9-[2-(R)-(phosphonomethoxy)propyl]adenine (PMPA [tenofovir]) from the end of a primer more efficiently than wild-type RT. Although the variant RTs excised d4TMP less efficiently than AZTMP and PMPA, they were able to excise d4TMP more efficiently than wild-type RT. HIV-1 RT containing the ⌬67 complex of mutations was not able to excise as broad a range of NRTIs as the fingers insertion variant SSGR/T215Y, but it was able to polymerize efficiently with low concentrations of deoxynucleoside triphosphates and seems to be able to excise AZTMP and PMPA at lower ATP concentrations than AZT-R or SSGR/T215Y, suggesting that a virus containing the ⌬67 complex of mutations would replicate reasonably well in quiescent cells, even in the presence of AZT.Drug resistance remains a major problem in the treatment of human immunodeficiency virus type 1 (HIV-1) infections. Nucleoside analog reverse transcriptase inhibitors (NRTIs) are widely used in combination therapy; mutations in the HIV-1 reverse transcriptase (RT) reduce the effectiveness of these inhibitors. Resistant RT variants have an increased ability to discriminate between the normal deoxynucleoside triphosphate (dNTP) and the NRTI. This discrimination can occur at the level of incorporation; i.e., the RT variant does not incorporate the NRTI as efficiently as the wild-type HIV-1 RT. Alternatively, the discrimination can occur after the incorporation of the NRTI by increasing the removal (excision) of the NRTI blocking the 3Ј end of the primer. Excision occurs by pyrophosphorolysis by means of either pyrophosphate itself or a pyrophosphate donor such as ATP. Pyrophosphorolysis is, mechanistically, the reverse of polymerization. During the chemical step of polymerization, the 3Ј end of the primer is located in what has been designated as the priming or P site, and the incoming dNTP is bound in the nucleotide binding or N site (3)(4)(5). This is the ternary complex (RT plus templateprimer [T/P] plus dNTP). For excision to be able to remove an NRTI blocking the 3Ј end of the primer, the end of the primer must be the N site in a binary complex (RT plus T/P). A pyrophosphate donor, PP i or ATP, reacts with the NRTI at the 3Ј end of the primer, yielding an unblocked primer and an NRTI triphosphate or a dinucleoside tetraphosphate, depending on which pyrophosphate donor is used (24). In vitro, PP i is an effective pyrophosphate donor, leading to the removal of 3Ј-azido-3Ј-deoxythymidine 5Ј-monophosphate (AZTMP) from the end of a primer (1, 3-5, 21, 24-28, 30). However, the RT variants that are associated with resistance to 3Ј-azido-3Ј-deo...

Relative Fitness and Replication Capacity of a Multinucleoside Analogue-Resistant Clinical Human Immunodeficiency Virus Type 1 Isolate with a Deletion of Codon 69 in the Reverse Transcriptase Coding Region

“…The molecular mechanism underlying resistance to RT inhibitors mediated by ⌬69 remains to be elucidated. In the presence of accompanying TAMs, recombinant HIV-1 RT containing ⌬67 showed significant nucleoside analogue excision activity on primers terminated with zidovudine, stavudine, and tenofovir (2). However, the contribution of nucleotide selectivity mechanisms affecting discrimination against triphosphorylated derivatives of the inhibitor cannot be ruled out.…”

Insertions in the Human Immunodeficiency Virus Type 1 Protease and Reverse Transcriptase Genes: Clinical Impact and Molecular Mechanisms

“…Susceptibility testing showed these patient-derived deletion strains had reduced susceptibilities to three to six NRTIs (26,39,51,58,66). Reports conflict on whether the deletion alone confers reduced susceptibility to NRTIs (26, 51, 66); however, this point is of minor significance since all deletion strains carry additional drug resistance mutations that engage in complex interactions that affect susceptibility to both NRTIs and NNRTIs (5,27,66).…”

The L74V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase Counteracts Enhanced Excision of Zidovudine Monophosphate Associated with Thymidine Analog Resistance Mutations

“…The rate of ATP-mediated rescue of an AZT-blocked DNA primer can be affected by a number of factors, including the rate of ternary complex formation (which requires positioning of AZT in the priming or P site of the enzyme and binding of the next incoming deoxynucleoside triphosphate at the nucleotide binding or N site), affinity of the enzyme for ATP, and positioning of the ␥ phosphate of ATP with respect to the phosphodiester bond between the terminal AZT-MP and the penultimate nucleoside monophosphate (4,5,30).…”