DNA-dependent RNA Synthesis Research Articles

Next-Generation Sequencing Methods to Determine the Accuracy of Retroviral Reverse Transcriptases: Advantages and Limitations

Retroviruses, like other RNA viruses, mutate at very high rates and exist as genetically heterogeneous populations. The error-prone activity of viral reverse transcriptase (RT) is largely responsible for the observed variability, most notably in HIV-1. In addition, RTs are widely used in biotechnology to detect RNAs and to clone expressed genes, among many other applications. The fidelity of retroviral RTs has been traditionally analyzed using enzymatic (gel-based) or reporter-based assays. However, these methods are laborious and have important limitations. The development of next-generation sequencing (NGS) technologies opened the possibility of obtaining reverse transcription error rates from a large number of sequences, although appropriate protocols had to be developed. In this review, we summarize the developments in this field that allowed the determination of RNA-dependent DNA synthesis error rates for different RTs (viral and non-viral), including methods such as PRIMER IDs, REP-SEQ, ARC-SEQ, CIR-SEQ, SMRT-SEQ and ROLL-SEQ. Their advantages and limitations are discussed. Complementary DNA (cDNA) synthesis error rates obtained in different studies, using RTs and RNAs of diverse origins, are presented and compared. Future improvements in methodological pipelines will be needed for the precise identification of mutations in the RNA template, including modified bases.

Novel RNase H Inhibitors Blocking RNA-directed Strand Displacement DNA Synthesis by HIV-1 Reverse Transcriptase

In retroviruses, strand displacement DNA-dependent DNA polymerization catalyzed by the viral reverse transcriptase (RT) is required to synthesize double-stranded proviral DNA. In addition, strand displacement during RNA-dependent DNA synthesis is critical to generate high-quality cDNA for use in molecular biology and biotechnology. In this work, we show that the loss of RNase H activity due to inactivating mutations in HIV-1 RT (e.g. D443N or E478Q) has no significant effect on strand displacement while copying DNA templates, but has a large impact on DNA polymerization in reactions carried out with RNA templates. Similar effects were observed with β-thujaplicinol and other RNase H active site inhibitors, including compounds with dual activity (i.e., characterized also as inhibitors of HIV-1 integrase and/or the RT DNA polymerase). Among them, dual inhibitors of HIV-1 RT DNA polymerase/RNase H activities, containing a 7-hydroxy-6-nitro-2H-chromen-2-one pharmacophore were found to be very potent and effective strand displacement inhibitors in RNA-dependent DNA polymerization reactions. These findings might be helpful in the development of transcriptomics technologies to obtain more uniform read coverages when copying long RNAs and for the construction of more representative libraries avoiding biases towards 5′ and 3′ ends, while providing valuable information for the development of novel antiretroviral agents.

Novel Ansa-Chain Conformation of a Semi-Synthetic Rifamycin Prepared Employing the Alder-Ene Reaction: Crystal Structure and Absolute Stereochemistry

Rifamycins are an extremely important class of antibacterial agents whose action results from the inhibition of DNA-dependent RNA synthesis. A special arrangement of unsubstituted hydroxy groups at C21 and C23, with oxygen atoms at C1 and C8 is essential for activity. Moreover, it is known that the antibacterial action of rifamycin is lost if either of the two former hydroxy groups undergo substitution and are no longer free to act in enzyme inhibition. In the present work, we describe the successful use of an Alder-Ene reaction between Rifamycin O, 1 and diethyl azodicarboxylate, yielding 2, which was a targeted introduction of a relatively bulky group close to C21 to protect its hydroxy group. Many related azo diesters were found to react analogously, giving one predominant product in each case. To determine unambiguously the stereochemistry of the Alder-Ene addition process, a crystalline zwitterionic derivative 3 of the diethyl azodicarboxylate adduct 2 was prepared by reductive amination at its spirocyclic centre C4. The adduct, as a mono chloroform solvate, crystallized in the non-centrosymmetric Sohnke orthorhombic space group, P212121. The unique conformation and absolute stereochemistry of 3 revealed through X-ray crystal structure analysis is described.

Structural basis for stabilization of human telomeric G-quadruplex [d-(TTAGGGT)]4 by anticancer drug adriamycin

Besides inhibiting DNA duplication, DNA dependent RNA synthesis and topoisomerase-II enzyme action, anticancer drug adriamycin is found to cause telomere dysfunction and shows multiple strategies of action on gene functioning. We present evidence of binding of adriamycin to parallel stranded intermolecular [d-(TTAGGGT)]4 G-quadruplex DNA comprising human telomeric DNA by proton and phosphorus-31 nuclear magnetic resonance spectroscopy. Diffusion ordered spectroscopy shows formation of complex between the two molecules. Changes in chemical shift and line broadening of DNA and adriamycin protons suggest participation of specific chemical groups/moieties in interaction. Presence of sequential nuclear Overhauser enhancements at all base quartet steps and absence of large downfield shifts in 31P resonances give clear proof of absence of intercalation of adriamycin chromophore between base quartets. Restrained molecular dynamics simulations using observed 15 short intermolecular inter proton distance contacts depict stacking of ring D of adriamycin with terminal G6 quartet by displacing T7 base and external groove binding close to T1–T2–A3 bases. The disappearance of imino protons monitored as a function of temperature and differential scanning calorimetry experiments yield thermal stabilization of 24 °C, which is likely to come in the way of telomerase association with telomeres. The findings pave the way for design of alternate anthracycline based drugs with specific modifications at ring D to enhance induced thermal stabilization and use alternate mechanism of binding to G-quadruplex DNA for interference in functional pathway of telomere maintenance by telomerase enzyme besides their well known action on duplex DNA. Communicated by Ramaswamy H. Sarma

Distinct Mechanisms of Transcription Initiation by RNA Polymerases I and II.

RNA polymerases I and II (Pol I and Pol II) are the eukaryotic enzymes that catalyze DNA-dependent synthesis of ribosomal RNA and messenger RNA, respectively. Recent work shows that the transcribing forms of both enzymes are similar and the fundamental mechanisms of RNA chain elongation are conserved. However, the mechanisms of transcription initiation and its regulation differ between Pol I and Pol II. Recent structural studies of Pol I complexes with transcription initiation factors provided insights into how the polymerase recognizes its specific promoter DNA, how it may open DNA, and how initiation may be regulated. Comparison with the well-studied Pol II initiation system reveals a distinct architecture of the initiation complex and visualizes promoter- and gene-class-specific aspects of transcription initiation. On the basis of new structural studies, we derive a model of the Pol I transcription cycle and provide a molecular movie of Pol I transcription that can be used for teaching.

Deciphering the RNA-dependent DNA synthesis fidelity of retroviral reverse transcriptases: impact of transcriptional inaccuracy threshold.

Deciphering the RNA-dependent DNA synthesis fidelity of retroviral reverse transcriptases: impact of transcriptional inaccuracy threshold.

Transcriptional inaccuracy threshold attenuates differences in RNA-dependent DNA synthesis fidelity between retroviral reverse transcriptases

In M13mp2 lacZα forward mutation assays measuring intrinsic fidelity of DNA-dependent DNA synthesis, wild-type human immunodeficiency virus type 1 (HIV-1) RTs of group M/subtype B previously showed >10-fold higher error rates than murine leukaemia virus (MLV) and avian myeloblastosis virus (AMV) RTs. An adapted version of the assay was used to obtain error rates of RNA-dependent DNA synthesis for several RTs, including wild-type HIV-1BH10, HIV-1ESP49, AMV and MLV RTs, and the high-fidelity mutants of HIV-1ESP49 RT K65R and K65R/V75I. Our results showed that there were less than two-fold differences in fidelity between the studied RTs with error rates ranging within 2.5 × 10−5 and 3.5 × 10−5. These results were consistent with the existence of a transcriptional inaccuracy threshold, generated by the RNA polymerase while synthesizing the RNA template used in the assay. A modest but consistent reduction of the inaccuracy threshold was achieved by lowering the pH and Mg2+ concentration of the transcription reaction. Despite assay limitations, we conclude that HIV-1BH10 and HIV-1ESP49 RTs are less accurate when copying DNA templates than RNA templates. Analysis of the RNA-dependent mutational spectra revealed a higher tendency to introduce large deletions at the initiation of reverse transcription by all HIV-1 RTs except the double-mutant K65R/V75I.

Intralesional 8.33% Rifamycin infiltration; New treatment for cutaneous leishmaniasis

BackgroundCutaneous leishmaniasis (CL) is an endemic disease in Iraq, for which many therapies had been tried, aiming to induce cure with no or minimal scaring. Rifamycin is an antibacterial agent, with the ability to inhibit bacterial DNA-dependent RNA synthesis and it is effective against extra- and intracellular microorganisms. ObjectiveTo evaluate the effectiveness and safety of intralesional 8.33% Rifamycin infiltration in treatment of cutaneous leishmaniasis. Patients and methodsThis is an open controlled therapeutic trial, carried out in the outpatient clinic of Dermatology & Venereology, Al-Yarmouk Teaching Hospital during the period between the first of December 2013 to the end of June 2014. A total of 29 patients have been enrolled in this therapeutic trial, however 4 patients were defaulted and 25 patients have completed the therapeutic trial. Fourteen (56%) were males and 11 (44%) were females, with a male to female ratio 1.27. Their ages ranged from 6 to 60years, with a mean±SD of 28.6±14.9years. The total number of lesions was 56 and the duration of lesions ranged between 4 and 18weeks with a mean±SD of 9.9±4.4weeks. The size of lesions ranged from 0.5 to 6cm in diameter with a mean±SD of 2.46±1.17cm. All patients have been diagnosed through history and clinical examination and the diagnosis was confirmed by skin smear and biopsy. The lesions were divided into two groups; group A treated with intralesional 8.33% Rifamycin infiltration every 2weeks for a maximum of 5 sessions, while group B was left untreated as a control group. Follow up was every 2weeks during the treatment period, and for 6weeks after completing the therapeutic sessions. ResultsTwenty five patients with a total of 56 lesions were completed the study period. Twenty one (37%) lesions were ulcerative and 35 (63%) were non ulcerative lesions. Ten (40%) patients had single lesion whereas 15 (60%) patients had multiple lesions. In group A, 38 (92.7%) out of 41 lesions showed a cure with 2–5 sessions with a mean±SD of 4.9±0.79 sessions, while in the control group no lesion had significantly improved or cured. At the end of the study period, only 8 out of 41 treated lesions have left trivial scars. Most of the lesions showed transient hyperpigmentation at the end of the study, but fortunately, it had disappeared in all cases few weeks later. ConclusionRifamycin 8.33% solution given intralesionally in cutaneous leishmaniasis is a highly effective agent with a success rate of 92.7%. For cure, it needs 2–5 local infiltrations given every two weeks. In addition to being effective, it is a safe, and less costly therapeutic option for cutaneous leishmaniasis.

LECTINS OF SAMBUCUS NIGRA AS BIOLOGICALLY ACTIVE AND DNA-PROTECTIVE SUBSTANCES

The human genome is affected by diverse environmental pollution factors and this is particularly acute for Ukrainians who have suffered from the Chernobyl radiation disaster. At the same time many natural compounds, especially from medicinal plants, are known as agents that prevent DNA lesions which often result in increasing mutagenesis and carcinogenesis. The aim of our work was to study the DNA-protective potential of biologically active compounds found in Sambucus nigra called lectins, a very large group of universally occurring proteins that recognize and specifically bind to carbohydrates/glycoconjugates. Various physiological activities of lectins based on protein-carbohydrate interactions have been demonstrated in all living forms. For example, lectins participate in host defense in plants against stress-related conditions, the attack of phytopathogens and phytophagous insects, as well as modulation of immune response, mitogenic stimulation or induction of apoptosis in animals. Antiviral, immunomodulating, antioxidant and insulin-stimulating properties of S. nigra fruit and flower extracts have been described in scientific literature. Also the elderberry lectins were found in roots, leaves, bark, seeds and fruits, the latter (SNA-IV) being the predominant protein in the juice. In Ukraine, various parts of S. nigra plants are used in folk medicine. Using methods of isoelectric focusing and chromatography, we determined that elderberry flowers and pollen contain rather high levels of lectins agglutinating animal and human erythrocytes, which differ from a commercial preparation of S. nigra bark lectin (SNA-I). The major lectin found in inflorescences, named SNAflu-I, is GalNAc/Gal specific and is supposed to be a heterotetramer with subunits of about 30 and 33 kDa. Sambucus nigra lectins demonstrated protective effects against heavy metals (nickel ions) in soil bacteria Bacillus subtilis. Also it was shown that lectins under study can modulate, in a concentration-dependent manner, the frequency of mutations and genotoxic activity of alkylating agents in eukaryotic cell cultures. Studies focused on the elucidation of cell targets sensitive to S. nigra lectins and demonstrated that at least one of the targets may be the DNA-dependent synthesis of RNA – the way to modulate gene expression. The results obtained lead to the conlusion that the protective functions of lectins both in pro- and eukaryotes involve complex mechanisms including components of the DNA repair system.

17β-Estradiol positively modulates growth hormone signaling through the reduction of SOCS2 negative feedback in human osteoblasts

Recent evidence demonstrated an interplay between estrogens and growth hormone (GH) at cellular level. To investigate the possible mechanism/s involved, we studied the effect of 17β-estradiol (E2) on GH signaling pathways in primary culture of human osteoblasts (hOBs). Exposure of hOBs to E2 (10−8M) 60min before GH (5ng/ml) significantly increased phosphorylated STAT5 (P-STAT5) levels compared with GH alone. E2 per se had no effect on P-STAT5. E2-enhanced GH signaling was effective in increasing osteopontin, bone-sialoprotein, and IGF II mRNA expression to a greater extent than GH alone. We then studied the effect of E2 on the protein levels of the negative regulator of GH signaling, suppressor of cytokine signaling-2 (SOCS2). E2 (10−11M–10−7M) reduced dose-dependently SOCS2 protein levels without modifying its mRNA expression. The silencing of SOCS2 gene prevented E2 positive effect on GH induced P-STAT5 and on GH induced bone-sialoprotein and osteopontin mRNA expression. Treatment with the inhibitor of DNA-dependent RNA synthesis, actinomycin-D, did not prevent E2 induced decrease of SOCS2, thus suggesting a non-genomic effect. E2 promoted an increase in SOCS2 ubiquitination. To determine if increased ubiquitination of SOCS2 by E2 led to degradation by proteasome, hOBs were pretreated with the proteasome inhibitor MG132 (5μM) which blocked E2 reduction of SOCS2. These findings demonstrate for the first time that E2 can amplify GH intracellular signaling in hOBs with an essential role played by the reduction of the SOCS2 mediated feedback loop.

Total Proteins and Enzymes Alterations Associated with Induction of Antiviral Resistance in Treated Plants

Systemic resistance inducer, extracted from the leaves Pseuderanthemum bicolor Radlk (PB-SRI) had the ability to prevent sunnhemp rosette virus infection in Cyamopsis tetragonoloba test plants. The systemic resistance induced was maximum at 24 h after the PB-SRI treatment. The alterations of enzymes catalase, peroxidase and total proteins activities exhibited a definite pattern with their concentration peak values at 24 h of treatment, corresponded with highest antiviral activity which thereafter decreased gradually. A direct relationship between alterations of total proteins and enzymes activities was observed in treated hosts. The product of these enzymes catalyze reactions may have a role in observed antiviral effects. Apart from that induction of resistance is an active host mediated phenomenon with the involvement of DNA-dependent RNA synthesis and consequently the formation of newer antiviral proteins within treated test host involved in induction of antiviral resistance in C. tetragonoloba test plants.

Transcription Factor-dependent DNA Bending Governs Promoter Recognition by the Mitochondrial RNA Polymerase

Promoter recognition is the first and the most important step during gene expression. Our studies of the yeast (Saccharomyces cerevisiae) mitochondrial (mt) transcription machinery provide mechanistic understandings on the basic problem of how the mt RNA polymerase (RNAP) with the help of the initiation factor discriminates between promoter and non-promoter sequences. We have used fluorescence-based approaches to quantify DNA binding, bending, and opening steps by the core mtRNAP subunit (Rpo41) and the transcription factor (Mtf1). Our results show that promoter recognition is not achieved by tight and selective binding to the promoter sequence. Instead, promoter recognition is achieved by an induced-fit mechanism of transcription factor-dependent differential conformational changes in the promoter and non-promoter DNAs. While Rpo41 induces a slight bend upon binding both the DNAs, addition of the Mtf1 results in severe bending of the promoter and unbending of the non-promoter DNA. Only the sharply bent DNA results in the catalytically active open complex. Such an induced-fit mechanism serves three purposes: 1) assures catalysis at promoter sites, 2) prevents RNA synthesis at non-promoter sites, and 3) provides a conformational state at the non-promoter sites that would aid in facile translocation to scan for specific sites.

Anastomotic Strictures: Conservative Treatment

Anastomotic Strictures: Conservative Treatment

Inhibition of translation by cytotrienin A—a member of the ansamycin family

The ansamycins are a diverse and often physiologically active group of compounds that include geldanamycin and rifamycin, inhibitors of heat shock protein 90 and prokaryotic DNA-dependent RNA synthesis, respectively. Cytotrienin A is an ansamycin-type small molecule with potent antiproliferative and proapoptotic properties. Here, we report that this compound inhibits eukaryotic protein synthesis by targeting translation elongation and interfering with eukaryotic elongation factor 1A function. We also find that cytotrienin A prevents HUVEC tube formation and diminishes microvessel formation in the chorioallantoic membrane assay. These results provide a molecular understanding into cytotrienin A's previously reported properties as an anticancer apoptosis-inducing drug.

The effect of mitomycin C in reducing intraarticular adhesion after knee surgery in rabbits

Mitomycin C (MMC) is known to inhibit fibroblast proliferation through suppressing DNA dependent RNA synthesis. Based on this knowledge, we illustrated the effect of MMC on inhibiting fibroblast collagen synthesis and reducing intraarticular fibrous adhesion in a rabbit model. Forty-eight New Zealand white rabbits were randomly divided into four groups. Approximately 10 × 10 mm squares of the cortical bone was removed from both sides of the femoral condyle, and the underneath cancellous bone was exposed. MMC in various concentrations or saline were then applied to the decorticated areas. The intraarticular adhesions were evaluated after four weeks by macroscopic evaluation, histological evaluation and biochemical analysis of hydroxyproline content. The results demonstrated that MMC could suppress the formation of intraarticular fibrous adhesion in a dose-dependent manner. The intraarticular adhesion score, the hydroxyproline content and the fibroblast number in 0.1 mg/ml MMC group were significantly less than those in 0.05 mg/ml MMC group, 0.01 mg/ml MMC group and control group. However, dense adhesions were found in 0.01 mg/ml MMC group and control group. These results indicated that topical application of 0.1 mg/ml MMC could reduce intraarticular adhesion through inhibiting fibroblast proliferation in rabbits.

Molecular basis of RNA-dependent RNA polymerase II activity

RNA polymerase (Pol) II catalyses DNA-dependent RNA synthesis during gene transcription. There is, however, evidence that Pol II also possesses RNA-dependent RNA polymerase (RdRP) activity. Pol II can use a homopolymeric RNA template, can extend RNA by several nucleotides in the absence of DNA, and has been implicated in the replication of the RNA genomes of hepatitis delta virus (HDV) and plant viroids. Here we show the intrinsic RdRP activity of Pol II with only pure polymerase, an RNA template-product scaffold and nucleoside triphosphates (NTPs). Crystallography reveals the template-product duplex in the site occupied by the DNA-RNA hybrid during transcription. RdRP activity resides at the active site used during transcription, but it is slower and less processive than DNA-dependent activity. RdRP activity is also obtained with part of the HDV antigenome. The complex of transcription factor IIS (TFIIS) with Pol II can cleave one HDV strand, create a reactive stem-loop in the hybrid site, and extend the new RNA 3' end. Short RNA stem-loops with a 5' extension suffice for activity, but their growth to a critical length apparently impairs processivity. The RdRP activity of Pol II provides a missing link in molecular evolution, because it suggests that Pol II evolved from an ancient replicase that duplicated RNA genomes.

Biochemical and biological properties of 5-bromotubercidin: Differential effects on cellular DNA-directed and viral RNA-directed RNA synthesis

We have studied the biochemical and biological properties of 5-bromotubercidin (4-amino-5-bromo-7-β- d-ribofuranosyl-pyrrolo [2,3- d]pyrimidine) (BrTu), a synthetic analogue of the highly cytotoxic pyrrolo[2,3- d]pyrimidine ribonucleoside antibiotic tubercidin (Tu) that interferes with numerous cellular processes, and has been shown to possess biological specificity and selectivity. Thus, BrTu entered the mammalian cell nucleotide pool by phosphorylation, was incorporated into RNA in an unmodified form and, as a consequence, reversibly inhibited (15 μM) mammalian cell growth and the synthesis of high-molecular-weight cellular RNA species (i.e., mRNA and rRNA). However, BrTu (300 μM) did not inhibit picornavirus RNA synthesis or multiplication, and thus discriminated between virus RNA-dependent and all forms of DNA-dependent RNA synthesis whether of cellular or viral origin; because of this BrTu should prove valuable as a metabolic probe for studying the cell–virus relationship. Furthermore, BrTu is a substrate for adenosine kinase ( K m = 24 μM), and is also its potent inhibitor ( K i = 0.93 μM); thus, low concentrations of BrTu (1.5 μM), which did not inhibit cell growth, blocked phosphorylation and the cellular uptake of other, highly cytotoxic pyrrolo-pyrimidine nucleoside analogues (e.g., tubercidin). This block in cellular uptake and incorporation of toxic analogues was associated with the protective effect of BrTu against cell killing by the analogues, providing a mechanism by which BrTu and these analogues can, as we reported elsewhere [ J. Virol. 1999, 73, 6444], be used for the selective inactivation of replicating picornaviruses.

Genes associated with opening and senescence of Mirabilis jalapa flowers

A modest ethylene climacteric accompanies flower senescence in Mirabilis jalapa L., and exogenous ethylene accelerates the process. However, inhibitors of ethylene action and synthesis have little effect on the life-span of these ephemeral flowers. Treatment with alpha-amanitin, an inhibitor of DNA-dependent RNA synthesis, substantially delays the onset of senescence. This effect falls linearly between 7 h and 8 h after the start of flower opening. Subtractive hybridization was used to isolate transcripts that were up- and down-regulated during this critical period. Eighty-two up-regulated and 65 down-regulated transcripts were isolated. The genes identified encode homologues of a range of transcription factors, and of proteins involved in protein turnover and degradation. Real-time quantitative RT-PCR was used to examine expression patterns of these genes during flower opening and senescence. Genes that were identified as being down-regulated during senescence showed a common pattern of very high expression during floral opening. These genes included a homologue of CCA1, a 'clock' gene identified in Arabidopsis thaliana and an aspartyl protease. Up-regulated genes commonly showed a pattern of increase during the critical period (4-9 h after opening), and some showed very strong up-regulation. For example, the abundance of transcripts encoding a RING zinc finger protein increased >40 000 fold during the critical period.

Telbivudine

Telbivudine, the unmodified L-enantiomer of the naturally occurring nucleoside D-thymidine, is a potent synthetic nucleoside analogue. It acts as a hepatitis B virus (HBV) polymerase inhibitor and preferentially inhibits HBV second strand (DNA-dependent) compared with first strand (RNA-dependent) DNA synthesis. More telbivudine than lamivudine recipients with hepatitis B e antigen (HBeAg)-positive chronic hepatitis B and similar proportions of telbivudine or lamivudine recipients with HBeAg-negative disease achieved a therapeutic response at 52 weeks in the large 2-year GLOBE trial. In a phase III trial in Chinese patients, greater reductions in serum HBV DNA occurred with telbivudine than lamivudine at 52 weeks. Reductions in serum HBV DNA at 24 weeks were greater with telbivudine than adefovir in the 1-year switching trial. A lower residual viral load at 52 weeks was seen in patients who received telbivudine or who switched from adefovir to telbivudine at 24 weeks than in patients receiving adefovir. In the 1-year lamivudine switching trial in patients with serum HBV DNA levels >3 log10 copies/mL despite having received prior treatment with lamivudine for a mean of [almost equal or equal to]7 months, those randomised to telbivudine therapy achieved greater reductions in serum HBV DNA levels at 24 weeks than patients randomised to continue lamivudine therapy. Telbivudine was generally well tolerated and most adverse events were of mild or moderate severity. The incidence of severe ALT flares with telbivudine was half that seen with lamivudine at both 52 and 104 weeks in the GLOBE trial.

Highly selective action of triphosphate metabolite of 4′-ethynyl D4T: A novel anti-HIV compound against HIV-1 RT

2′,3′-Didehydro-3′-deoxy-4′-ethynylthymidine (4′-Ed4T), is a recently discovered nucleoside reverse transcriptase inhibitor (NRTI) showing a 5- to 10-fold greater anti-human immunodeficiency virus type 1 (HIV-1) activity and less cellular and mitochondrial toxicity than its parental compound, stavudine (D4T). It is also active against a variety of NRTI-resistant HIV-1 mutants under non-cytotoxic concentrations. In this study, the effects of 4′-Ed4TTP, which is the triphosphate metabolite of 4′-Ed4T, on HIV-1 reverse transcriptase (RT) activity were investigated. We found that 4′-Ed4TTP was a substrate of HIV-1 RT serving as a DNA chain terminator, and it inhibited the DNA polymerase activity of RT more efficiently than D4TTP. The value of K i(4′-Ed4TTP)/ K m(dTTP) is 0.15 for DNA/RNA primer/template duplex (P/T), but 0.7 for DNA/DNA P/T, suggesting 4′-Ed4TTP inhibits RT more efficiently during RNA-dependent DNA synthesis than DNA-dependent DNA synthesis. 4′-Ed4TTP was also found to inhibit the 3TC (Lamivudine)-resistant RT mutant, M184V, with 3-fold less efficiency than the wild type (wt) RT. 4′-Ed4TTP showed much less inhibitory effects toward major host DNA polymerases. Overall, our results suggest that 4′-Ed4TTP is the active form for anti-HIV-1 activity via its inhibitory effect against RT.