"Removing" questions surrounding the in vivo mechanism of HIV-1 AZT resistance
Date of Award
Doctor of Philosophy (Ph.D.)
Microbiology and Immunology
First Committee Member
Walter A. Scott, Committee Chair
Human immunodeficiency virus type 1 resistance to nucleoside reverse transcriptase inhibitors such as 3'-azido-2', 3'-dideoxythymidine (AZT) can arise through mutations in the coding region of reverse transcriptase (RT) that enhance the enzyme's ability to remove the drug after it has been incorporated. This excision activity of HIV-1 RT has been well characterized in a number of in vitro systems. However, the in vitro findings do not provide a complete picture of the in vivo significance of this resistance mechanism. Research was designed to characterize the in vivo nature of this resistance activity, thus, beginning to bridge the gap between the in vitro observations and the in vivo environment. We first 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, PPi, 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 PPi was present at 7 to 15 muM. Under these conditions, the ATP-dependent removal 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 muM PPi. In this environment, the PPi-dependent reaction predominated and excision by mutant and wild-type RT occurred with similar efficiency. While PPi-dependent excision may contribute to AZT resistance in vivo, this data suggests that selection of AZT-resistant mutants occurs primarily in an environment where the ATP-dependent reaction predominates.In order to investigate the excision of chain terminators inside cells, we designed an enzymatic assay to detect nucleotide-dependent excision products in cell extracts derived from AZT-treated, HIV-1-infected cells. This procedure allowed us to detect and quantify compounds with expected properties of AZT-containing dinucleoside polyphosphates in extracts prepared from AZT-treated cells infected with mutant viruses while none was detected in extracts from cells infected with WT virus. Thus, the enhanced ability of RT mutants to excise nucleoside reverse transcriptase inhibitors in vitro correlated with the enhanced ability of mutant viruses to make dinucleoside polyphosphates inside drug-treated cells. Ultimately, this assay can serve as a link between the in vitro data characterizing HIV-1 drug resistance and the in vivo environment and may be used to characterize the excision reaction of HIV-1 in a wide variety of in vivo settings.
Biology, Microbiology; Chemistry, Biochemistry
Smith, Anthony James, ""Removing" questions surrounding the in vivo mechanism of HIV-1 AZT resistance" (2006). Dissertations from ProQuest. 2407.