Enzymatic Assay for Quantification of Deoxynucleoside Triphosphates in Human Cells Exposed to Antiretroviral 2′,3′-Dideoxynucleosides

Gao, Weiyi; Johns, D. G.; Mitsuya, Hiroaki

doi:10.1006/abio.1994.1462

Cited by 26 publications

(15 citation statements)

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“…Cold methanol extraction, which yields satisfactory recovery of nucleotides (13,37,48), failed to inactivate enzymes that degrade [ 32 P]PP i or incorporate the label into higher-molecular-weight compounds during the extraction. Persistence of active enzymes after methanol extraction has been previously reported (36).…”

Section: Discussionmentioning

confidence: 99%

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Smith¹,

Meyer²,

Asthana

et al. 2005

Antimicrob Agents Chemother

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Treatment of human immunodeficiency virus type 1 (HIV-1)-infected patients with 3-azido-3-deoxythymidine (AZT) selects for mutant forms of viral reverse transcriptase (RT) with increased ability to remove chain-terminating nucleotides from blocked DNA chains. We tested various cell extracts for the presence of endogenous acceptor substrates for this reaction. ؉ or CD8 ؉ T cells, while PP i was present at 7 to 15 M. Under these conditions, the ATP-dependent reaction predominated, and excision by the AZT-resistant mutant RT was more efficient than wild type RT. Activated CD4 ؉ or CD8 ؉ T cells contained 1.4 to 2.7 mM ATP and 55 to 79 M PP i . These cellular PP i concentrations are lower than previously reported; nonetheless, the PP i -dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PP i -dependent excision may contribute to AZT resistance in vivo, it is likely that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.Human immunodeficiency virus type 1 (HIV-1) replication is inhibited by the incorporation of chain-terminating nucleotide analogs at the 3Ј end of the nascent DNA chain during reverse transcription. Viral replication in the presence of these drugs results in selection of resistance mutations in the viral sequences encoding reverse transcriptase (RT), the replicative enzyme for HIV. Mutations at six codons in RT (M41L, D67N, K70R, L210W, T215Y or -F, and K219Q) are commonly selected by the nucleoside inhibitor 3Ј-azido-3Ј-deoxythymidine (AZT) and have been shown to confer AZT resistance through an increased ability to remove the chain terminator after it has been incorporated into DNA (1, 29, 46). These mutations are also correlated with significant resistance to other chain-terminating nucleosides (33,34,47).Excision has been shown to occur by RT-catalyzed transfer of the chain-terminating residue to a variety of acceptor substrates in vitro by a reaction that is related to pyrophosphorolysis (1,5,16,29,31,42,43). The intracellular acceptor for this reaction is unknown, but likely candidates include nucleoside triphosphates and nucleoside diphosphates as well as inorganic pyrophosphate (PP i ).In this report, we show that cell extracts contain a mixture of acceptor substrates for the excision reaction. ATP or PP i predominates depending on the cell type and its activation status. For these experiments, it was necessary to avoid errors in the measurement of PP i levels in the extracts, which can be affected by a number of factors: the breakdown of unstable intracellular compounds to form PP i during the extraction procedure, contaminating platelets, and cellular enzymes that alter PP i levels during the extraction process. Results obtained with an optimized extraction procedure disagree with widely quoted values of 130 to 150 M for intracellular PP i concentration in unstimulated lymphocytes (3); our results are more in agreement with the dat...

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Section: Discussionmentioning

confidence: 99%

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Smith¹,

Meyer²,

Asthana

et al. 2005

Antimicrob Agents Chemother

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“…We initially quantified the four dNTP pools at low hydroxyurea concentrations ('0.1 mM) in PHA-PBM cells. A sensitive enzymatic assay (with a minimum detection limit of 0.1 pmol) was chosen for dNTP determinations (12). Only the dATP pool was susceptible to depletion by hydroxyurea under these conditions: a single exposure to 0.1 mM hydroxyurea caused a rapid decrease in the dATP pool of >50% (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Sci. USA 92 (1995) where necessary, the reduction by any 2',3'-dideoxynucleoside 5'-triphosphates (ddNTPs) present in the cell extract (12).…”

Section: Methodsmentioning

confidence: 99%

Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

Gao

Johns

Chokekuchai

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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“…Indeed, recent results have shown that 3TC can still retain moderate inhibitory effects against M184V-containing viruses in primary CD4 ϩ cells, despite the high level of resistance to 3TC conferred by this substitution (51). This may be related to the fact that resting lymphocytes in vivo maintain relatively low dNTP pools, which can potentiate the effect of 3TC, since the ratio of inhibitor to substrate may be higher under these conditions than might be the case in dividing cells (18). Thus, the in vivo benefit of 3TC in the aftermath of the M184V mutation might be greater in nondividing lymphocytes than in activated lymphocytes.…”

Section: Molecular Mechanisms Responsible For Altered Interaction Of mentioning

confidence: 99%

“…In fact, the first report of a change from a methionine to valine at residue 184 (M184V) in HIV was in regard to selection for resistance in tissue culture to didanosine (ddI) (23). Later, this mutation was shown to be responsible for both high-level resistance to 3TC (6,17,18,22,57,63,69) and low-level resistance to almost all the molecules that act as nucleoside analog RT inhibitors (NRTIs) ( Table 1) (25,53,64). The appearance of the M184V substitution was found to be transiently preceded by another mutation, M184I, that also confers high-level resistance (about 1,000-fold) to 3TC (6) and occurs in patients treated with 3TC (58).…”

mentioning

confidence: 99%

Molecular Impact of the M184V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

Diallo

Götte

Wainberg

2003

Antimicrob Agents Chemother

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Enzymatic Assay for Quantification of Deoxynucleoside Triphosphates in Human Cells Exposed to Antiretroviral 2′,3′-Dideoxynucleosides

Cited by 26 publications

References 0 publications

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Intracellular Substrates for the Primer-Unblocking Reaction by Human Immunodeficiency Virus Type 1 Reverse Transcriptase: Detection and Quantitation in Extracts from Quiescent- and Activated-Lymphocyte Subpopulations

Disparate actions of hydroxyurea in potentiation of purine and pyrimidine 2',3'-dideoxynucleoside activities against replication of human immunodeficiency virus.

Molecular Impact of the M184V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

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