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Antimicrobial Agents and Chemotherapy, May 2005, p. 1761-1769, Vol. 49, No. 5
0066-4804/05/$08.00+0     doi:10.1128/AAC.49.5.1761-1769.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Departments of Biochemistry and Molecular Biology,¹ Psychiatry, University of Miami School of Medicine, Miami, Florida²

Received 17 September 2004/ Returned for modification 19 November 2004/ Accepted 27 January 2005

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. Cell extracts incubated with HIV-1 RT and [³²P]ddAMP-terminated DNA primer/template gave rise to ³²P-labeled adenosine 2',3'-dideoxyadenosine 5',5'''–P¹,P⁴-tetraphosphate (Ap₄ddA), ddATP, Gp₄ddA, and Ap₃ddA, corresponding to the transfer of [³²P]ddAMP to ATP, PP_i, GTP, and ADP, respectively. Incubation with [³²P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous ³²P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PP_i levels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4⁺ or CD8⁺ T cells, while PP_i was present at 7 to 15 µM. 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 µM 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.

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