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

Abstract

ABSTRACTTreatment 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 to32P-labeled adenosine 2′,3′-dideoxyadenosine 5′,5′′′−P1,P4-tetraphosphate (Ap4ddA), ddATP, Gp4ddA, and Ap3ddA, corresponding to the transfer of [32P]ddAMP to ATP, PPi, GTP, and ADP, respectively. Incubation with [32P]AZT monophosphate (AZTMP)-terminated primer/template gave rise to the analogous32P-labeled AZT derivatives. Based on the rates of formation of the specific excision products, ATP and PPilevels were determined: ATP was present at 1.3 to 2.2 mM in H9 cells, macrophages, and unstimulated CD4+or CD8+T cells, while PPiwas 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 PPi. These cellular PPiconcentrations are lower than previously reported; nonetheless, the PPi-dependent reaction predominated in extracts from activated T cells, and excision by mutant and wild-type RT occurred with similar efficiency. While PPi-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.