Differential Incorporation and Removal of Antiviral Deoxynucleotides by Human DNA Polymerase γ

Abstract

Mitochondrial toxicity can result from antiviral nucleotide analog therapy used to control human immunodeficiency virus type 1 infection. We evaluated the ability of such analogs to inhibit DNA synthesis by the human mitochondrial DNA polymerase (pol ␥) by comparing the insertion and exonucleolytic removal of six antiviral nucleotide analogs. Apparent steady-state K m and k cat values for insertion of 2,3-dideoxy-TTP (ddTTP), 3-azido-TTP (AZT-TP), 2,3-dideoxy-CTP (ddCTP), 2,3-didehydro-TTP (D4T-TP), (-)-2,3-dideoxy-3-thiacytidine (3TC-TP), and carbocyclic 2,3-didehydro-ddGTP (CBV-TP) indicated incorporation of all six analogs, albeit with varying efficiencies. Dideoxynucleotides and D4T-TP were utilized by pol ␥ in vitro as efficiently as natural deoxynucleotides, whereas AZT-TP, 3TC-TP, and CBV-TP were only moderate inhibitors of DNA chain elongation. Inefficient excision of dideoxynucleotides, D4T, AZT, and CBV from DNA predicts persistence in vivo following successful incorporation. In contrast, removal of 3-terminal 3TC residues was 50% as efficient as natural 3 termini. Finally, we observed inhibition of exonuclease activity by concentrations of AZT-monophosphate known to occur in cells. Thus, although their greatest inhibitory effects are through incorporation and chain termination, persistence of these analogs in DNA and inhibition of exonucleolytic proofreading may also contribute to mitochondrial toxicity.More than 36 million people are infected by the human immunodeficiency virus worldwide, where 5.3 million new infections occurred during 2000 (1). Although antiviral therapy effectively extends the life of individuals, the death toll continues to rise; 3 million people, the highest number since the epidemic began, died from AIDS in 2000 (1). Nucleoside analogs utilized in antiviral therapy are readily incorporated into DNA by the HIV-1 1 reverse transcriptase. Although viral replication is effectively inhibited by DNA chain terminators, cellular side effects also result. The continuous antiviral therapy required to keep the HIV infection under control has increased the chance for severe antiviral analog induced toxicity. Current antiviral nucleoside analog therapy against HIV clearly results in compromised mitochondrial function due to inhibition of the mitochondrial DNA polymerase (2, 3).AZT was the first analog to be approved for anti-HIV therapy in 1985 were the first to report mitochondrial myopathies in HIV-infected individuals undergoing AZT treatment. Control studies thereafter demonstrated that these induced myopathies, most notably visualized histologically as ragged red fibers, were indeed caused by AZT treatment and were not a consequence of the HIV infection (5). This study revealed reduced amounts of mitochondrial DNA in AZT-treated skeletal muscle (5). Further clinical evidence has demonstrated that mitochondrial myopathy slowly and cumulatively develops during AZT treatment (6).The second class of antiviral nucleoside analogs approved for HIV therapy are the dideoxynucleoside analogs ddI...

Transgenic expression of the deoxynucleotide carrier causes mitochondrial damage that is enhanced by NRTIs for AIDS

“…17 The DNC TG here offered an approach to evaluate NRTI toxicity in vivo and linked NRTI toxicity to mitochondrial import and nucleotide homeostasis. 4 For some time, our group [26][27][28] and others [29][30][31][32][33][34][35] have studied mechanisms of mitochondrial toxicity of NRTIs focusing on inhibition of DNA pol g (reviewed in Kaguni 36 ). NRTIs are firmly linked to altered mtDNA replication 10,37,38 through the DNA pol g hypothesis.…”

3′-Azido-3′-deoxythymidine (AZT) inhibits thymidine phosphorylation in isolated rat liver mitochondria: A possible mechanism of AZT hepatotoxicity

“…These toxicities are believed to be mediated through mitochondrial damage and are associated with mitochondrial DNA depletion [15][16][17]. The current prevailing hypothesis for this damage is by 3′-azido-3′-deoxythymidine-5′-triphosphate (AZTTP) inhibition of the mitochondrial DNA polymerase γ [18].…”

“…The current prevailing hypothesis for this damage is by 3′-azido-3′-deoxythymidine-5′-triphosphate (AZTTP) inhibition of the mitochondrial DNA polymerase γ [18]. However, several groups have demonstrated that relative to other nucleoside analog triphosphates, AZTTP is not a very good inhibitor of polymerase γ (50% inhibitory concentration (IC 50 ) of 100-200 μM AZTTP) [16,17,19]. Further, because 3′-azido-3′-deoxythymidine-5′-monophosphate (AZTMP) is a poor substrate for the cytosolic thymidylate kinase [20,21], levels of AZTTP high enough to inhibit polymerase γ have not been reported even in mitotic tissue [20,22].…”

Mitochondrial DNA Depletion, Oxidative Stress, and Mutation: Mechanisms 0f Dysfunction from Nucleoside Reverse Transcriptase Inhibitors

“…Human DNA pol-␥ is inhibited by 50% with 20 M AZT triphosphate in a reverse transcriptase assay (Lim and Copeland, 2001). Gray et al (1995) showed that Hela cell DNA pol-␥ incorporated 3TC triphosphate nearly as well as ddC triphosphate into DNA.…”

“…Human DNA pol-␥ removes chain terminators poorly compared with normal nucleotides. AZTmonophosphate is the most persistent chain terminator to DNA pol-␥ exonuclease activity (Lim and Copeland, 2001). It is possible that this could be significant mechanistically in the observed mitochondrial toxicity of DNA by AZT and related NRTIs.…”