Search

Drugs targeting DNA and RNA in mammalian cells or viruses can also affect bacteria present in the host and thereby induce the bacterial SOS system. This has the potential to increase mutagenesis and the development of antimicrobial resistance (AMR). Here, we have examined nucleoside analogues (NAs) commonly used in anti-viral and anti-cancer therapies for potential effects on mutagenesis in Escherichia coli , using the rifampicin mutagenicity assay. To further explore the mode of action of the NAs, we applied E. coli deletion mutants, a peptide inhibiting Pol V (APIM-peptide) and metabolome and proteome analyses. Five out of the thirteen NAs examined, including three nucleoside reverse transcriptase inhibitors (NRTIs) and two anti-cancer drugs, increased the mutation frequency in E. coli by more than 25-fold at doses that were within reported plasma concentration range (Pl.CR), but that did not affect bacterial growth. We show that the SOS response is induced and that the increase in mutation frequency is mediated by the TLS polymerase Pol V. Quantitative mass spectrometry-based metabolite profiling did not reveal large changes in nucleoside phosphate or other central carbon metabolite pools, which suggests that the SOS induction is an effect of increased replicative stress. Our results suggest that NAs/NRTIs can contribute to the development of AMR and that drugs inhibiting Pol V can reverse this mutagenesis.

Human immunodeficiency virus 1 (HIV-1) infection is a global health issue since neither a cure nor a vaccine is available. However, the highly active antiretroviral therapy (HAART) has improved the life expectancy for patients with acquired immunodeficiency syndrome (AIDS). Nucleoside reverse transcriptase inhibitors (NRTIs) are in almost all HAART and target reverse transcriptase (RT), an essential enzyme for the virus. Even though NRTIs are highly effective, they have limitations caused by RT resistance. The main mechanisms of RT resistance to NRTIs are discrimination and excision. Understanding the molecular mechanisms for discrimination and excision are essential to develop more potent and selective NRTIs. Using protein X-ray crystallography, we determined the first crystal structure of RT in its post-catalytic state in complex with emtricitabine, (-)FTC or stavudine (d4T). Our structural studies provide the framework for understanding how RT discriminates between NRTIs and natural nucleotides, and for understanding the requirement of (-)FTC to undergo a conformation change for successful incorporation by RT. The crystal structure of RT in post-catalytic complex with d4T provides a "snapshot" for considering the possible mechanism of how RT develops resistance for d4T via excision. The findings reported herein will contribute to the development of next generation NRTIs.

Background: Virologic failure (VF) is highly prevalent in sub-Saharan African children on antiretroviral therapy (ART) and is often associated with human immunodeficiency virus drug resistance (DR). Most children still lack access to routine viral load (VL) monitoring for early identification of treatment failure, with implications for the efficacy of second-line ART., Methods: Children aged 1 to 14 years on ART for ≥12 months at 6 public facilities in Maputo, Mozambique were consecutively enrolled after informed consent. Chart review and caregiver interviews were conducted. VL testing was performed, and specimens with ≥1000 copies/mL were genotyped., Results: Of the 715 children included, the mean age was 103 months, 85.8% had no immunosuppression, 73.1% were taking stavudine/lamivudine/nevirapine, and 20.1% had a history prevention of mother-to-child transmission exposure. The mean time on ART was 60.0 months. VF was present in 259 patients (36.3%); 248 (95.8%) specimens were genotyped, and DR mutations were found in 238 (96.0%). Severe immunosuppression and nutritional decline were associated with DR. M184V and Y181C were the most common mutations. In the 238 patients with DR, standard second-line ART would have 0, 1, 2, and 3 effective antiretrovirals in 1 (0.4%), 74 (31.1%), 150 (63.0%), and 13 (5.5%) patients, respectively., Conclusion: This cohort had high rates of VF and DR with frequent compromise of second-line ART. There is urgent need to scale-up VL monitoring and heat-stable protease inhibitor formulations or integrase inhibitorsfor a more a durable first-line regimen that can feasibly be implemented in developing settings., (© The Author(s) 2018. Published by Oxford University Press on behalf of The Journal of the Pediatric Infectious Diseases Society.)

Mice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS. Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Objective: This study aims to investigate the prevalence and types of drug resistance mutations among patients failing first-line antiretroviral therapy (ART)., Methods: Plasma samples from 112 patients with human immunodeficiency virus-1 (HIV-1) were collected for virus RNA extract and gene amplification. The mutations related to drug resistance were detected and the incidence was statistically analyzed, and the drug resistance rate against common drugs was also evaluated., Results: 103 cases were successfully amplified, and the main drug resistance mutations in the reverse transcriptase (RT) region were M184V (50.49%), K103N (28.16%), Y181C (25.24%), and K65R (27.18%), while no drug main resistance mutation was found in the protease (PR) region. The incidence of drug resistance mutations was significantly different among patients with different ages, routes of infection, duration of treatment, initial ART regimens and viral load. The drug resistance rate to the common drugs was assessed, including Efavirenz (EFV, 71.84%), Nevirapine (NVP, 74.76%), Lamivudine (3TC, 66.02%), Zidovudine (AZT, 4.85%), Stavudine (D4T, 16.51%), and Tenofovir (TDF, 21.36%)., Conclusion: The drug resistance mutations to NRTIs and NNRTIs are complex and highly prevalent, which was the leading cause of first-line ART failure. This study provides significant theoretical support for developing the second-line and third-line therapeutic schemes., (Copyright © 2019 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Stavudine (d4T, 2',3'-didehydro-2',3'-dideoxythymidine) was one of the first chain-terminating nucleoside analogs used to treat HIV infection. We present the first structure of the active, triphosphate form of d4T (d4TTP) bound to a catalytic complex of HIV-1 RT/dsDNA template-primer. We also present a new strategy for disulfide (S-S) chemical cross-linking between N 6 of a modified adenine at the second overhang base to I63C in the fingers subdomain of RT. The cross-link site is upstream of the duplex-binding region of RT, however, the structure is very similar to published RT structures with cross-linking to Q258C in the thumb, which suggests that cross-linking at either site does not appreciably perturb the RT/DNA structures. RT has a catalytic maximum at pH 7.5. We determined the X-ray structures of the I63C-RT/dsDNA/d4TTP cross-linked complexes at pH 7, 7.5, 8, 8.5, 9, and 9.5. We found small (~0.5 Å), pH-dependent motions of the fingers subdomain that folds in to form the dNTP-binding pocket. We propose that the pH-activity profile of RT relates to this motion of the fingers. Due to side effects of neuropathy and lipodystrophy, use of d4T has been stopped in most countries, however, chemical modification of d4T might lead to the development of a new class of nucleoside analogs targeting RNA and DNA polymerases., (© 2018 The Protein Society.)

Human endogenous retroviruses (HERVs) make up 8% of the human genome. The HERV type K (HERV-K) HML-2 (HK2) family contains proviruses that are the most recent entrants into the human germ line and are transcriptionally active. In HIV-1 infection and cancer, HK2 genes produce retroviral particles that appear to be infectious, yet the replication capacity of these viruses and potential pathogenicity has been difficult to ascertain. In this report, we screened the efficacy of commercially available reverse transcriptase inhibitors (RTIs) at inhibiting the enzymatic activity of HK2 RT and HK2 genomic replication. Interestingly, only one provirus, K103, was found to encode a functional RT among those examined. Several nucleoside analogue RTIs (NRTIs) blocked K103 RT activity and consistently inhibited the replication of HK2 genomes. The NRTIs zidovudine (AZT), stavudine (d4T), didanosine (ddI), and lamivudine (3TC), and the nucleotide RTI inhibitor tenofovir (TDF), show efficacy in blocking K103 RT. HIV-1-specific nonnucleoside RTIs (NNRTIs), protease inhibitors (PIs), and integrase inhibitors (IIs) did not affect HK2, except for the NNRTI etravirine (ETV). The inhibition of HK2 infectivity by NRTIs appears to take place at either the reverse transcription step of the viral genome prior to HK2 viral particle formation and/or in the infected cells. Inhibition of HK2 by these drugs will be useful in suppressing HK2 infectivity if these viruses prove to be pathogenic in cancer, neurological disorders, or other diseases associated with HK2. The present studies also elucidate a key aspect of the life cycle of HK2, specifically addressing how they do, and/or did, replicate. IMPORTANCE Endogenous retroviruses are relics of ancestral virus infections in the human genome. The most recent of these infections was caused by HK2. While HK2 often remains silent in the genome, this group of viruses is activated in HIV-1-infected and cancer cells. Recent evidence suggests that these viruses are infectious, and the potential exists for HK2 to contribute to disease. We show that HK2, and specifically the enzyme that mediates virus replication, can be inhibited by a panel of drugs that are commercially available. We show that several drugs block HK2 with different efficacies. The inhibition of HK2 replication by antiretroviral drugs appears to occur in the virus itself as well as after infection of cells. Therefore, these drugs might prove to be an effective treatment by suppressing HK2 infectivity in diseases where these viruses have been implicated, such as cancer and neurological syndromes., (Copyright © 2017 American Society for Microbiology.)

Treatment options for individuals infected with human immunodeficiency virus type 2 (HIV-2) are restricted by the intrinsic resistance of the virus to nonnucleoside reverse transcriptase inhibitors (NNRTIs) and the reduced susceptibility of HIV-2 to several protease inhibitors (PIs) used in antiretroviral therapy (ART). In an effort to identify new antiretrovirals for HIV-2 treatment, we evaluated the in vitro activity of the investigational nucleoside analog BMS-986001 (2',3'-didehydro-3'-deoxy-4'-ethynylthymidine; also known as censavudine, festinavir, OBP-601, 4'-ethynyl stavudine, or 4'-ethynyl-d4T). In single-cycle assays, BMS-986001 inhibited HIV-2 isolates from treatment-naive individuals, with 50% effective concentrations (EC50s) ranging from 30 to 81 nM. In contrast, EC50s for group M and O isolates of HIV-1 ranged from 450 to 890 nM. Across all isolates tested, the average EC50 for HIV-2 was 9.5-fold lower than that for HIV-1 (64 ± 18 nM versus 610 ± 200 nM, respectively; mean ± standard deviation). BMS-986001 also exhibited full activity against HIV-2 variants whose genomes encoded the single amino acid changes K65R and Q151M in reverse transcriptase, whereas the M184V mutant was 15-fold more resistant to the drug than the parental HIV-2ROD9 strain. Taken together, our findings show that BMS-986001 is an effective inhibitor of HIV-2 replication. To our knowledge, BMS-986001 is the first nucleoside analog that, when tested against a diverse collection of HIV-1 and HIV-2 isolates, exhibits more potent activity against HIV-2 than against HIV-1 in culture., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Sterile alpha motif- and histidine/aspartic acid domain-containing protein 1 (SAMHD1) limits HIV-1 replication by hydrolyzing deoxynucleoside triphosphates (dNTPs) necessary for reverse transcription. Nucleoside reverse transcriptase inhibitors (NRTIs) are components of anti-HIV therapies. We report here that SAMHD1 cleaves NRTI triphosphates (TPs) at significantly lower rates than dNTPs and that SAMHD1 depletion from monocytic cells affects the susceptibility of HIV-1 infections to NRTIs in complex ways that depend not only on the relative changes in dNTP and NRTI-TP concentrations but also on the NRTI activation pathways., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Sterile alpha motif and histidine-aspartic domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase recently recognized as an antiviral factor that acts by depleting dNTP availability for viral reverse transcriptase (RT). SAMHD1 restriction is counteracted by the human immunodeficiency virus type 2 (HIV-2) accessory protein Vpx, which targets SAMHD1 for proteosomal degradation, resulting in an increased availability of dNTPs and consequently enhanced viral replication. Nucleoside reverse transcriptase inhibitors (NRTI), one of the most common agents used in antiretroviral therapy, compete with intracellular dNTPs as the substrate for viral RT. Consequently, SAMHD1 activity may be influencing NRTI efficacy in inhibiting viral replication. Here, a panel of different RT inhibitors was analyzed for their different antiviral efficacy depending on SAMHD1. Antiviral potency was measured for all the inhibitors in transformed cell lines and primary monocyte-derived macrophages and CD4(+) T cells infected with HIV-1 with or without Vpx. No changes in sensitivity to non-NRTI or the integrase inhibitor raltegravir were observed, but for NRTI, sensitivity significantly changed only in the case of the thymidine analogs (AZT and d4T). The addition of exogenous thymidine mimicked the change in viral sensitivity observed after Vpx-mediated SAMHD1 degradation, pointing toward a differential effect of SAMHD1 activity on thymidine. Accordingly, sensitivity to AZT was also reduced in CD4(+) T cells infected with HIV-2 compared to infection with the HIV-2ΔVpx strain. In conclusion, reduction of SAMHD1 levels significantly decreases HIV sensitivity to thymidine but not other nucleotide RT analog inhibitors in both macrophages and lymphocytes., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Nucleoside analogue reverse transcriptase inhibitor (NRTI), an integral component of highly active antiretroviral therapy (HAART), was widely used to inhibit HIV replication. Long-term exposure to NRTIs can result in mitochondrial toxicity which manifests as lipoatrophy, lactic acidosis, cardiomyopathy and myopathy, as well as polyneuropathy. But the cerebral neurotoxicity of NRTIs is still not well known partly due to the restriction of blood-brain barrier (BBB) and the complex microenvironment of the central nervous system (CNS). In this study, the Balb/c mice were administered 50 mg/kg stavudine (D4T), 100 mg/kg zidovudine (AZT), 50 mg/kg lamivudine (3TC) or 50 mg/kg didanosine (DDI) per day by intraperitoneal injection, five days per week for one or four months, and primary cortical neurons were cultured and exposed to 25 µM D4T, 50 µM AZT, 25 µM 3TC or 25 µM DDI for seven days. Then, single neuron was captured from mouse cerebral cortical tissues by laser capture microdissection. Mitochondrial DNA (mtDNA) levels of the primary cultured cortical neurons, and captured neurons or glial cells, and the tissues of brains and livers and muscles were analyzed by relative quantitative real-time PCR. The data showed that mtDNA did not lose in both NRTIs exposed cultured neurons and one month NRTIs treated mouse brains. In four months NRTIs treated mice, brain mtDNA levels remained unchanged even if the mtDNA levels of liver (except for 3TC) and muscle significantly decreased. However, mtDNA deletion was significantly higher in the captured neurons from mtDNA unchanged brains. These results suggest that long-term exposure to NRTIs can result in mtDNA deletion in mouse cortical neurons.

Objective: To determine the frequency and patterns of acquired antiretroviral drug resistance in a rural primary health care programme in South Africa., Design: Cross-sectional study nested within HIV treatment programme., Methods: Adult (≥ 18 years) HIV-infected individuals initially treated with a first-line stavudine- or zidovudine-based antiretroviral therapy (ART) regimen and with evidence of virological failure (one viral load >1000 copies/ml) were enrolled from 17 rural primary health care clinics. Genotypic resistance testing was performed using the in-house SATuRN/Life Technologies system. Sequences were analysed and genotypic susceptibility scores (GSS) for standard second-line regimens were calculated using the Stanford HIVDB 6.0.5 algorithms., Results: A total of 222 adults were successfully genotyped for HIV drug resistance between December 2010 and March 2012. The most common regimens at time of genotype were stavudine, lamivudine and efavirenz (51%); and stavudine, lamivudine and nevirapine (24%). Median duration of ART was 42 months (interquartile range (IQR) 32-53) and median duration of antiretroviral failure was 27 months (IQR 17-40). One hundred and ninety one (86%) had at least one drug resistance mutation. For 34 individuals (15%), the GSS for the standard second-line regimen was <2, suggesting a significantly compromised regimen. In univariate analysis, individuals with a prior nucleoside reverse-transcriptase inhibitor (NRTI) substitution were more likely to have a GSS <2 than those on the same NRTIs throughout (odds ratio (OR) 5.70, 95% confidence interval (CI) 2.60-12.49)., Conclusions: There are high levels of drug resistance in adults with failure of first-line antiretroviral therapy in this rural primary health care programme. Standard second-line regimens could potentially have had reduced efficacy in about one in seven adults involved.

Background: Limited HIV-1 drug-resistance surveillance has been carried out in Ghana since the implementation of antiretroviral therapy (ART). This study sought to provide data on the profile of HIV-1 drug resistance in ART-experienced and newly diagnosed individuals in Ghana., Methods: Samples were collected from 101 HIV-1-infected patients (32 ART-experienced cases with virological failure and 69 newly diagnosed ART-naïve cases, including 11 children), in Koforidua, Eastern region of Ghana, from February 2009 to January 2010. The pol gene sequences were analyzed by in-house HIV-1 drug-resistance testing., Results: The most prevalent HIV-1 subtype was CRF02&#95;AG (66.3%, 67/101) followed by unique recombinant forms (25.7%, 26/101). Among 31 ART-experienced adults, 22 (71.0%) possessed at least one drug-resistance mutation, and 14 (45.2%) had two-class-resistance to nucleoside and non-nucleoside reverse-transcriptase inhibitors used in their first ART regimen. Importantly, the number of accumulated mutations clearly correlated with the duration of ART. The most prevalent mutation was lamivudine-resistance M184V (n = 12, 38.7%) followed by efavirenz/nevirapine-resistance K103N (n = 9, 29.0%), and zidovudine/stavudine-resistance T215Y/F (n = 6, 19.4%). Within the viral protease, the major nelfinavir-resistance mutation L90M was found in one case. No transmitted HIV-1 drug-resistance mutation was found in 59 ART-naïve adults, but K103N and G190S mutations were observed in one ART-naïve child., Conclusions: Despite expanding accessibility to ART in Eastern Ghana, the prevalence of transmitted HIV-1 drug resistance presently appears to be low. As ART provision with limited options is scaled up nationwide in Ghana, careful monitoring of transmitted HIV-1 drug resistance is necessary.

Objective: To evaluate the impact of the active metabolite of vitamin D, 1α,25-dihydroxycholecalciferol (1,25D3), on nucleoside reverse transcriptase inhibitor (NRTI) induced mitochondrial DNA (mtDNA) depletion in human skeletal muscle myoblasts and myotubes., Design: mtDNA was quantified in human skeletal muscle myoblasts and myotubes following 1,25D3 and NRTI treatment using real-time PCR., Methods: Human skeletal muscle myoblasts and myotubes were treated with didanosine (ddI), stavudine (d4T), zidovudine (ZDV), lamivudine (3TC) and abacavir (ABC) alone or in combination either in the presence or absence of 1,25D3 for 5 days. Cells were harvested, DNA extracted and mtDNA quantified., Results: ddI and ddI-d4T significantly decreased both myoblast and myotube mtDNA in the absence of 1,25D3 compared with untreated controls (P≤0.029). In addition, the ZDV-3TC combination resulted in a 47% decrease in myotube mtDNA (P=0.005). 1,25D3 increased myotube mtDNA levels in ddI, ZDV, 3TC, ABC, ddI-d4T, d4T-3TC, ZDV-3TC, ZDV-ABC and ZDV-3TC-ABC-containing regimens and myoblast mtDNA levels in ddI, d4T, ZDV, 3TC, ddI-d4T, ZDV-3TC and ZDV-ABC-containing regimens. Of note, 1,25D3 protected against myotube mtDNA depletion following ZDV-3TC treatment, rendering them similar to 1,25D3 untreated controls (P=0.62), and increased both myotube and myoblast mtDNA two to three-fold in ddI-containing regimens (P<0.05)., Conclusion: 1,25D3 confers a protective effect against NRTI-induced mitochondrial toxicity in skeletal muscle myoblasts and myotubes. These findings support a protective role for vitamin D in preventing mitochondrial toxicity and suggest that supplemental vitamin D may protect against NRTI-associated mitochondrial toxicity.

Background: Tenofovir (TDF) is part of the WHO recommended first-line antiretroviral therapy (ART); however, there are limited data comparing TDF to other nucleoside reverse transcriptase inhibitors in resource-limited-settings. Using a routine workplace and community-based ART cohort in South Africa, we assessed single drug substitution, HIV RNA suppression, CD4 count increase, loss-from-care, and mortality between TDF, stavudine (d4T) 30 mg dose, and zidovudine (AZT)., Methods: In a prospective cohort study we included ART naïve patients aged ≥17 years-old who initiated ART containing TDF, d4T, or AZT between 2007 and 2009. For analysis of single drug substitutions we used a competing-risks time-to-event analysis; for loss-from-care, mixed-effect Poisson modeling; for HIV RNA suppression, competing-risks logistic regression; for CD4 count slope, mixed-effects linear regression; and for mortality, proportional hazards modeling., Results: Of 6,196 patients, the initial drug was TDF for 665 (11%), d4T for 4,179 (68%), and AZT for 1,352 (22%). During the first 6 months of ART, the adjusted hazard ratio for a single drug substitution was 2.3 for d4T (95% confidence interval [CI]: 0.27, 19) and 5.2 for AZT (95% CI: 1.1, 23), compared to TDF; whereas, after 6 months, it was 10 (95% CI: 5.8, 18) and 4.4 (95% CI: 2.5, 7.8) for d4T and AZT, respectively. Virologic suppression was similar by agent; however, CD4 count rise was lowest for AZT. The adjusted hazard ratio for loss-from-care, when compared to TDF, was 1.5 (95% CI: 1.1, 1.9) for d4T and 1.2 (95% CI: 1.1, 1.4) for AZT. The adjusted hazard ratio for mortality, when compared to TDF, was 2.7 (95% CI: 2.0, 3.5) and 1.4 (95% CI: 1.3, 1.5) and for d4T and AZT, respectively., Discussion: In routine care, TDF appeared to perform better than either d4T or AZT, most notably with less drug substitution and mortality than for either other agent.

HIV-1 establishes infection in astrocytes and macroage-lineage cells of the central nervous system (CNS). Certain antiretroviral drugs (ARVs) can penetrate the CNS, and are therefore often used in neurologically active combined antiretroviral therapy (Neuro-cART) regimens, but their relative activity in the different susceptible CNS cell populations is unknown. Here, we determined the HIV-1 inhibitory activity of CNS-penetrating ARVs in astrocytes and macrophage-lineage cells. Primary human fetal astrocytes (PFA) and the SVG human astrocyte cell line were used as in vitro models for astrocyte infection, and monocyte-derived macrophages (MDM) were used as an in vitro model for infection of macrophage-lineage cells. The CNS-penetrating ARVs tested were the nucleoside reverse transcriptase inhibitors (NRTIs) abacavir (ABC), lamivudine (3TC), stavudine (d4T) and zidovudine (ZDV), the non-NRTIs efavirenz (EFV), etravirine (ETR) and nevirapine (NVP), and the integrase inhibitor raltegravir (RAL). Drug inhibition assays were performed using single-round HIV-1 entry assays with luciferase viruses pseudotyped with HIV-1 YU-2 envelope or vesicular stomatitis virus G protein (VSV-G). All the ARVs tested could effectively inhibit HIV-1 infection in macrophages, with EC90s below concentrations known to be achievable in the cerebral spinal fluid (CSF). Most of the ARVs had similar potency in astrocytes, however the NRTIs 3TC, d4T and ZDV had insufficient HIV-1 inhibitory activity in astrocytes, with EC90s 12-, 187- and 110-fold greater than achievable CSF concentrations, respectively. Our data suggest that 3TC, d4T and ZDV may not adequately target astrocyte infection in vivo, which has potential implications for their inclusion in Neuro-cART regimens.

Background: HIV and combination antiretroviral therapy (cART) may increase cardiovascular disease (CVD) risk. We assessed the early effects of cART on CVD risk markers in a population with presumed low CVD risk., Methods: Adult patients (n=118) in Lusaka, Zambia were recruited at the time of initiation of cART for HIV/AIDS. Cardiometabolic risk factors were measured before and 90 days after starting cART. Participants were grouped according to cART regimens: Zidovudine + Lamivudine + Nevirapine (n=58); Stavudine + Lamivudine + Nevirapine (n=43); and 'other' (Zidovudine + Lamivudine + Efavirenz, Stavudine + Lamivudine + Efavirenz, Tenofovir + Emtricitabine + Efavirenz or Tenofovir + Emtricitabine + Nevirapine, n=17). ANOVA was used to test whether changes in cardiometabolic risk markers varied by cART regimen., Results: From baseline to 90 days after initiation of cART, the prevalence of low levels of high-density lipoprotein cholesterol (<1.04 mmol/L for men and <1.30 mmol/L for women) significantly decreased (78.8% vs. 34.8%, P<0.001) while elevated total cholesterol (TC ≥5.18 mmol/L, 5.1% vs. 11.9%, P=0.03) and the homeostasis model assessment of insulin resistance ≥3.0 (1.7% vs. 17.0%, P<0.001) significantly increased. The prevalence of TC:HDL-c ratio ≥5.0 significantly decreased (44.9% vs. 6.8%, P<0.001). These changes in cardiometabolic risk markers were independent of the cART regimen., Conclusion: Our results suggest that short-term cART is associated with a cardioprotective lipid profile in Zambia and a tendency towards insulin resistance regardless of the cART regimen.

Lipoatrophy in HIV patients can result from prolonged exposure to thymidine analogues. Mitochondrial toxicity leading to dysregulated adipogenesis and increased cell death has been proposed as a leading factor in the etiology of peripheral fat loss. We hypothesized that thymidine analogues interfere with autophagy, a lysosomal degradation pathway, which is important for mitochondrial quality control, cellular survival, and adipogenesis. We assessed the effects of zidovudine (AZT), stavudine (d4T), and lamivudine (3TC) on autophagy in eukaryotic cells and adipocytes (3T3-F442A) by fluorescence microscopy and flow cytometry. The effects were compared to interventions with established genetic and pharmacological inhibitors of autophagy and correlated to assessments of cell viability, proliferation, and differentiation. AZT and d4T, but not 3TC, inhibited both constitutive and induced autophagic activity in adipocytes. This inhibition was associated with accumulation of dysfunctional mitochondria, increased reactive oxygen species (ROS) production, increased apoptosis, decreased proliferation, and impaired adipogenic conversion. Autophagy inhibition was dose and time dependent and detectable at therapeutic drug concentrations. Similar phenotypic changes were obtained when genetic or pharmacological inhibition of autophagy was employed. Our data suggest that thymidine analogues disturb adipocyte function through inhibition of autophagy. This novel mechanism potentially contributes to peripheral fat loss in HIV-infected patients.

Objectives: Prolonged nucleoside reverse transcriptase inhibitors (NRTI) exposure can lead to microvesicular steatosis. We hypothesized that thymidine analogues might interfere with autophagy in hepatocytes, a lysosomal degradation pathway implicated in cell survival and regulation of hepatocyte lipid metabolism., Design: Using HepG2 and HUH7 cell lines and primary human hepatocytes, we performed a comprehensive analysis of NRTI-mediated effects on autophagy., Methods: The impact of zidovudine (ZDV), stavudine (d4T) and lamivudine (3TC) on constitutive and induced autophagy was analyzed by fluorescent and electron microscopy, western blotting and flow cytometry. Effects on hepatocyte autophagy were correlated to cellular viability, mitochondrial dysfunction and intracellular lipid accumulation., Results: ZDV and d4T, but not 3TC, significantly inhibited both constitutive as well as stimulated autophagic activity in a dose-dependent and time-dependent manner. Inhibition of autophagy at therapeutic drug concentrations led to accumulation of dysfunctional mitochondria, increased ROS production, increased apoptosis, decreased proliferation and increased intracellular lipid accumulation. These NRTI effects could be readily resembled by pharmacological and genetic inhibition of hepatocyte autophagy., Conclusion: Our data suggest that thymidine analogues inhibit autophagy in hepatocytes, which in turn leads to increased ROS production, lipid accumulation and hepatic dysfunction. This novel mechanism could contribute to nonalcoholic fatty liver disease in HIV-infected patients.

The accumulation of mutations causes cell lethality and can lead to carcinogenesis. An important class of mutations, which are associated with mutational hotspots in many organisms, are those that arise by nascent strand misalignment and template-switching at the site of short repetitive sequences in DNA. Mutagens that strongly and specifically affect this class, which is mechanistically distinct from other mutations that arise from polymerase errors or by DNA template damage, are unknown. Using Escherichia coli and assays for specific mutational events, this study defines such a mutagen, 3'-azidothymidine [zidovudine (AZT)], used widely in the treatment and prevention of HIV/AIDS. At sublethal doses, AZT has no significant effect on frame shifts and most base-substitution mutations. AT-to-CG transversions and deletions at microhomologies were enhanced modestly by AZT. AZT strongly stimulated the "template-switch" class of mutations that arise in imperfect inverted repeat sequences by DNA-strand misalignments during replication, presumably through its action as a chain terminator during DNA replication. Chain-terminating 2'-3'-didehydro 3'-deoxythymidine [stavudine (D4T)] and 2'-3'-dideoxyinosine [didanosine (ddI)] likewise stimulated template-switch mutagenesis. These agents define a specific class of mutagen that promotes template-switching and acts by stalling replication rather than by direct nucleotide base damage.

In the present study, we investigated the ability of anti-HIV drugs to interfere with normal cell cycle progression and to induce oxidative stress by perturbing the redox environment. Our results provide evidence that anti-HIV drugs have a differential effect on adipocyte cell cycle and differentiation, being able to modify the response to oxidative stress through an increase of reactive oxygen species (ROS) that compromises the induction of phase-2 and antioxidant enzymes. In detail, saquinavir, efavirenz, and stavudine exert antiadipogenic influences on the model 3T3-L1 cell line, perturbing the oxidative response and inducing of apoptosis. When considered together, the effects of anti-HIV drugs on 3T3-L1 pre adipocytes are distinct but commonly antiadipogenic, thus suggesting another additional possible mechanism by which antiretroviral therapies could contribute to lipoatrophy.

This study investigates the capability of methylmethacrylate-sulfopropylmethacrylate (MMA-SPM) nanoparticles (NPs) with grafted RMP-7 (RMP-7/MMA-SPM NPs) to deliver stavudine (D4T), delavirdine (DLV), and saquinavir (SQV) across the blood-brain barrier (BBB). The permeability coefficients of the three drugs across the BBB were evaluated by a co-culture model containing human brain-microvascular endothelial cells and human astrocytes. An increase in the concentration of ammonium persulfate (APS), the polymerization initiator, enhanced the particle size of drug-loaded RMP-7/MMA-SPM NPs. When the concentration of APS was 0.6%, the average particle diameter was smaller than 50 nm. These spherical drug carriers were uniform in size and displayed a dominant topography of discrete hillocks and deep pits in deposited film. Smaller RMP-7/MMA-SPM NPs yielded a larger drug loading efficiency. The order of drug in the loading efficiency and in the particle uptake was, respectively, D4T>DLV>SQV and D4T>SQV>DLV. Endocytosis of RMP-7/MMA-SPM NPs and tight junction mediation can improve the permeability of D4T, DLV, and SQV across the BBB., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Objective: To examine HIV and hepatitis B virus (HBV)-related outcomes in HIV/HBV-coinfected participants in the PHIDISA II study by use of HBV-active vs. non-HBV-active antiretroviral therapy (ART)., Design and Methods: PHIDISA II was a randomized study of ART therapy in HIV-infected adults employing zidovudine along with didanosine, or lamivudine along with stavudine in a factorial 2x2 design. HIV/HBV-coinfected participants by randomization received HBV-active or non-HBV-active ART. The following outcomes of interest were examined: immunological recovery and HIV RNA suppression; hepatic flare; HBV DNA suppression; and mortality., Results: HIV/HBV coinfection was present in 106 of 1771 (6%) of participants. Participants with HIV/HBV coinfection were more likely to be men, and have higher baseline alanine aminotransferase, lower albumin, and lower platelets than those with HIV monoinfection. Median CD4 cell gain and HIV RNA suppression was similar across all groups. Hepatic flare was observed in 9.4% of coinfected and 0.02% monoinfected participants. HBV DNA suppression (<55 IU/ml) at week 48 was observed in only 33% of those on lamivudine vs. 13% in those on no HBV-active drugs (P = 0.13). Mortality over follow-up was significantly greater in coinfected (17%) than monoinfected (11%) participants (P = 0.04)., Conclusion: In summary, the use of lamivudine-containing ART in HIV/HBV participants in PHIDISA II resulted in little additional benefit over that of ART itself and failed to impact on the greater mortality in this group. These data provide strong support for recent guidelines advocating the use of tenofovir in all HIV-HBV-coinfected individuals initiating ART.

Background: The identification and management of first-line antiretroviral therapy (ART) failure is a key challenge for HIV programs in resource-limited settings. In 2008, the National AIDS Control Organisation, India piloted a national strategy to provide second-line ART. We assessed the National AIDS Control Organisation second-line ART evaluation algorithm., Methods: Adult patients who had received 6 months or more of standard first-line ART were referred for second-line ART evaluation if they demonstrated CD4 decline to pre-ART values, CD4 drop to less than 50% of peak on-treatment value, failure to achieve CD4 greater than 100 c/mm(3), or development of a new World Health Organization Stage 3 or 4 AIDS-defining illness. Patients received HIV RNA testing, and those with HIV RNA 10,000 c/mL or greater qualified to switch to second-line ART. World Health Organization-defined clinical and CD4 criteria for ART failure were compared against virologic failure criteria., Results: Between January and June 2008, 154 patients met criteria for evaluation. Of 122 (79%) patients who had HIV RNA testing, 87 (71%) had viral load 10,000 c/mL or greater and were recommended to start second-line ART, 29 (24%) had viral load less than 400 c/mL, and six (5%) had viral load between 400 and 10,000 c/mL. The positive predictive value of World Health Organization clinical/immunologic criteria to detect virologic failure was 71% (95% confidence interval, 63% to 79%)., Conclusions: Second-line ART was initiated in the public sector in India using an approach combining clinical and immunologic evaluation with confirmation of virologic failure. Almost 25% of patients who met clinical/immunologic failure criteria demonstrated virologic suppression. Inclusion of targeted HIV RNA testing in the evaluation of treatment failure can prevent unnecessary switches to second-line ART.

Persistent genital human immunodeficiency virus type 1 (HIV-1) shedding among women receiving antiretroviral therapy (ART) may present a transmission risk. We investigated the associations between genital HIV-1 suppression after ART initiation and adherence, resistance, pretreatment CD4 cell count, and hormonal contraceptive use. First-line ART was initiated in 102 women. Plasma and genital HIV-1 RNA levels were measured at months 0, 3, and 6. Adherence was a strong and consistent predictor of genital HIV-1 suppression (P < .001), whereas genotypic resistance was associated with higher vaginal HIV-1 RNA level at month 6 (P = .04). These results emphasize the importance of adherence to optimize the potential benefits of ART for reducing HIV-1 transmission risk.

Background: Fixed-dose combination scored dispersible stavudine, lamivudine, and nevirapine minitablets (Triomune Baby and Junior; Cipla Ltd) are simpler and cheaper than liquid formulations and have correct dose ratios for human immunodeficiency virus-infected children. However, they cannot be used for dose escalation (DE) of nevirapine., Methods: Children were randomized to initiate antiretroviral therapy with full-dose (FD) nevirapine (Triomune Baby or Junior in the morning and evening) versus DE (half-dose nevirapine for 14 days [Triomune in the morning and stavudine-lamivudine {Lamivir-S} in the evening], then FD), in accordance with World Health Organization weight-band dosing tables. The primary end point was nevirapine-related clinical or laboratory grade 3 or 4 adverse events (AEs)., Results: In total, 211 children (median [interquartile range {IQR}] age, 5 [ 2-9 ] years; median [IQR] CD4 cell percentage, 13% [8%-18%]) were enrolled and followed up for a median (IQR) of 92 (68-116) weeks. There were 31 grade 3 or 4 AEs that were definitely/probably or uncertainly related to nevirapine in the FD group (18.0 per 100 child-years), compared with 29 in the DE group (16.5 per 100 child-years) (incidence rate ratio, 1.09; 95% confidence interval, 0.63&#x2013;1.87; P = .74). All were asymptomatic; 11 versus 3 were single grade 3 or 4 elevations in alanine aminotransferase (ALT) or aspartate aminotransferase (AST) levels, all of which resolved without a change in nevirapine dose or interruption. Thirteen (12%) FD versus 2 (2%) DE children had grade 1 (2 in FD) or grade 2 (11 in FD and 2 in DE) rashes. Three (2 in FD and 1 in DE) substituted efavirenz, 3 (FD) continued FD nevirapine, and 9 (8 in FD and 1 in DE) temporarily interrupted nevirapine, followed by successful DE. Predictors of nevirapine rash were older age (P = .003) and higher CD4 cell count for age (P = .03). Twenty-two children died (12 in FD and 10 in DE), 1 FD and 5 DE children at <4 weeks; none were considered to be drug related by independent review., Conclusions: Rash was more frequent with FD nevirapine, but 88% had no clinical toxicity; elevated AST or ALT levels were transient and resolved spontaneously, suggesting that routine laboratory monitoring has limited value. Dual pediatric stavudine-lamivudine minitablets are preferred for safe and simple DE; if unavailable, initiating FD Triomune requires timely review for rash, which could be managed by temporary reduction to half-dose Triomune or efavirenz substitution., Trial Registration: Current Controlled Trials identifier: ISRCTN31084535 .

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a derivative of stavudine (d4T), has potent activity against human immunodeficiency virus and is much less inhibitory to mitochondrial DNA synthesis and cell growth than its progenitor, d4T. 4'-Ed4T triphosphate was a better reverse transcriptase (RT) inhibitor than d4T triphosphate, due to the additional binding of the 4'-ethynyl group at a presumed hydrophobic pocket in the RT active site. Previous in vitro selection for 4'-Ed4T-resistant viral strains revealed M184V and P119S/T165A/M184V mutations on days 26 and 81, respectively; M184V and P119S/T165A/M184V conferred 3- and 130-fold resistance to 4'-Ed4T, respectively. We investigated the relative contributions of these mutations, engineered into the strain NL4-3 background, to drug resistance, RT activity, and viral growth. Viral variants with single RT mutations (P119S or T165A) did not show resistance to 4'-Ed4T; however, M184V and P119S/T165A/M184V conferred three- and fivefold resistance, respectively, compared with that of the wild-type virus. The P119S/M184V and T165A/M184V variants showed about fourfold resistance to 4'-Ed4T. The differences in the growth kinetics of the variants were not more than threefold. The purified RT of mutants with the P119S/M184V and T165A/M184V mutations were inhibited by 4'-Ed4TTP with 8- to 13-fold less efficiency than wild-type RT. M184V may be the primary resistance-associated mutation of 4'-Ed4T, and P119S and T165A are secondary mutations. On the basis of our findings and the results of structural modeling, a virus with a high degree of resistance to 4'-Ed4T (e.g., more than 50-fold resistance) will be difficult to develop. The previously observed 130-fold resistance of the virus with P119S/T165A/M184V to 4'-Ed4T may be partly due to mutations both in the RT sequence and outside the RT sequence.

Brain-Derived Neurotrophic Factor (BDNF) is a central nervous system modulator of nociception. In animal models of chronic pain, BDNF exerts its effects on nociceptive processing by binding to the full-length receptor tropomyosin-related kinase B (trkB.FL) and transducing intracellular signaling to produce nocifensive behaviors. In addition to trkB.FL, the trkB locus also produces a widely-expressed alternatively-spliced truncated isoform, trkB.T1. TrkB.T1 binds BDNF with high affinity; however the unique 11 amino acid intracellular cytoplasmic tail lacks the kinase domain of trkB.FL. Recently, trkB.T1 was shown to be specifically up-regulated in a model of HIV-associated neuropathic pain, potentially implicating trkB.T1 as a modulator of nociception. Here, we report that trkB.T1 mRNA and protein is up-regulated in the spinal dorsal horn at times following antiretroviral drug treatment and hind paw inflammation in which nocifensive behaviors develop. While genetic depletion of trkB.T1 did not affect baseline mechanical and thermal thresholds, the absence of trkB.T1 resulted in significant attenuation of inflammation- and antiretroviral-induced nocifensive behaviors. Our results suggest that trkB.T1 up-regulation following antiretroviral treatment and tissue inflammation participates in the development and maintenance of nocifensive behavior and may represent a novel therapeutic target for pain treatment.

Human immunodeficiency virus type 1 containing the reverse transcriptase mutation M184V or K65R or mutations M41L, M184V, and T215Y did not accumulate additional resistance mutations in the reverse transcriptase when increasing amounts of apricitabine drug pressure were applied. The original mutations were maintained by the presence of apricitabine but were lost when cultured without drug pressure.

To test whether zidovudine (3'-azido-3'-deoxythymidine) (AZT) inhibition of thymidine phosphorylation causes depletion of the TTP pool resulting in mitochondrial DNA depletion, 3T3-F442a cells were differentiated in the presence of AZT and analyzed to determine mitochondrial DNA content and deoxynucleotide levels. These results suggest that AZT toxicity may not be related to deoxynucleotide pool alterations.

Objective: To evaluate the safety and virological response to lopinavir/ritonavir containing second-line therapy after failing a first line nonnucleoside reverse transcriptase inhibitor (NNRTI) based regimen., Design: Prospective 36 months cohort study of patients switched to zidovudine/stavudine plus didanosine plus lopinavir/ritonavir capsules as second-line regimen., Methodology: Structured interview, medical examination, and laboratory assessment performed every 6 months., Results: We enrolled 40 patients; 1 died and 3 were lost to follow-up. Median CD4+ count at baseline was 108 cell/microL, median log viral load was 4.8 copies/mL. Sixteen (40%) patients had baseline genotypic resistant test, 14 (87%) had lamivudine resistance mutations, and all had NNRTIs resistance mutations. At month 36, 82% of the patients achieved viral suppression (<400 copies/ mL) and the median increase in CD4+ count was 214 cell/microL, (interquartile range: 128-295). Twenty-five patients (62%) experienced at least one adverse event., Conclusions: Our study confirms lopinavir/ ritonavir-based second-line regimen but with a high rate of toxicities.

A cross-sectional study of patients living with HIV/ AIDS treated during 2003 to 2007 in decentralized, rural health centers in Zambia was performed to measure virological outcomes after 12 months of antiretroviral therapy and identify factors associated with virological failure. Data from 228 patients who started antiretroviral therapy >12 months prior were analyzed. In all, 93% received stavudine + lamivudine + nevirapine regimens, and median antiretroviral therapy duration was 23.5 months (interquartile range 20-28). Of the 205 patients tested for viral load, 177 (86%) had viral load <1000 copies/mL. Probability of developing virological failure (viral load >1000 copies/mL) was 8.9% at 24 months and 19.6% at 32 months. Predictors for virological failure were <100% adherence, body mass index <18.5 kg/m(2), and women <40 years old. Of those with virological failure who underwent 3 to 6 months of intensive adherence counseling, 45% obtained virological success. In a remote, resource-limited setting in decentralized health centers, virological and immunological assessments of patients on antiretroviral therapy >12 months showed that positive health outcomes are achievable.

Objective: The role of oxidative stress in disease progression has been shown to be more complicated in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) compared to those who remain treatment-naïve. This study examined the changes in the antioxidant profile of HIV-infected subjects who remained HAART-naïve due to a high CD4 cell count and HIV-negative controls, over a 12-month follow-up period at YRG CARE, a tertiary HIV referral centre in southern India., Methods: We prospectively studied 35 HIV-infected participants (18 on d4T+3TC+EFV (stavudine+lamivudine+efavirenz), eight on AZT+3TC+EFV (zidovudine+lamivudine+efavirenz), and nine who were antiretroviral therapy-naïve) and 20 HIV-negative controls. Antioxidant profile (total antioxidant status, glutathione reductase, glutathione peroxidase, uric acid, ceruloplasmin, zinc, and albumin), CD4 cell count, plasma viral load, dietary intake, and history of smoking and alcohol use were determined at baseline and at twelve months., Results: At 12 months, participants on HAART showed a significant increase in glutathione peroxidase (baseline: 1765 vs. 12 months: 2850U/l; p<0.001) and albumin (3.6 vs. 4.4g/dl; p<0.001), and a significant decrease in glutathione reductase (52.6 vs. 50.5U/l; p=0.054) and uric acid (5.4 vs. 4.8mg/dl; p=0.027) compared to baseline. Also HAART-naïve participants had a significant increase in albumin (baseline: 3.7 vs.12 months: 4.3g/dl; p=0.023) and a significant decrease in zinc levels (baseline: 79.0 vs.12 months: 74.5microg/dl; p=0.052) from baseline to 12 months. HIV-negative subjects had a significant increase in glutathione reductase at 12 months from baseline (baseline: 37 vs.12 months: 39U/l; p=0.002). No significant difference in total antioxidant status, ceruloplasmin, and zinc levels were observed in HAART-experienced subjects and negative controls over the 12-month follow-up period., Conclusion: This study documents changes in antioxidants over a period of time in HAART-experienced subjects in a southern India setting.

Background: Cases of renal dysfunction in patients receiving tenofovir disoproxil fumarate (TDF) have been reported. We analyzed the renal safety of TDF compared with thymidine analogue-containing (control) regimens through 144 weeks from two clinical trials in antiretroviral-naive HIV-infected patients., Methods: We evaluated the changes in renal parameters in 1111 patients (TDF, n = 556; control, n = 555) who were enrolled in two randomized, controlled trials (Studies 903 and 934) comparing TDF vs. either stavudine or zidovudine in combination with efavirenz and either lamivudine or emtricitabine. The studies included patients with serum creatinine less than 1.5 mg/dl, serum phosphorus at least 2.2 mg/dl and estimated glomerular filtration rate by Cockcroft-Gault at least 60 and at least 50 ml/min at screening., Results: Baseline characteristics were similar between groups. No patient discontinued due to renal abnormalities in the TDF arm. Through 144 weeks, the proportion of patients who experienced confirmed abnormalities in serum creatinine (>1.5 mg/dl) or serum phosphorus (<2.0 mg/dl) was less than 1% in both groups; a similar proportion of patients experienced urine proteinuria at least 100 mg/dl (TDF, 5%; control, 6%). The median change from baseline to week 144 in glomerular filtration rate was -2 and 3 ml/min by Cockcroft-Gault, and -2 and -1 ml/min per 1.73 m by modification of diet in renal disease in the TDF and control groups (P < 0.05), respectively., Conclusion: In two randomized, controlled trials, small differences in glomerular filtration rate over time were noted but no clinically relevant renal disease or adverse events were demonstrated in antiretroviral-naive patients treated with TDF through 144 weeks. Additional studies on renal health and renal safety in HIV are important goals for future clinical trials.

Background: Female hormones are known to play an important role in predisposition for many infectious diseases. Recent work suggests there are gender effects in HIV/AIDS progression. Here we ask whether the sex steroid hormone beta-estradiol affects the replication of HIV-1 or the efficacy of a common anti-retroviral drug, Stavudine (D4T)., Results: Human PBL were infected with HIV-1 in the presence or absence of combinations of sex steroid hormones and the anti-retroviral drug, D4T. After seven days in culture, viral supernatants were assayed for HIV-1 p24 protein. beta-estradiol resulted in a modest inhibition of HIV-1 replication of approximately 26%. However, 2 nM beta-estradiol increased the amount of HIV-1 replication in the presence of 50 nM D4T from a baseline of 33% (+/- SE = 5.4) to 74% (+/- SE = 5.4) of control virus levels in the absence of drug. Both results were statistically highly significant (p < 0.001). beta-estradiol did not increase the replication of a D4T-resistant strain of HIV in the presence of D4T. The effects were unlikely to be due to general cell inhibition or toxicity because these concentrations of drug and hormone cause no cytotoxicity in PBL as measured by trypan blue exclusion., Conclusion: beta-estradiol inhibited both HIV-1 replication in primary human PBL and the antiretroviral efficacy of D4T in PBL cultures. To optimize antiretroviral drug therapy, it may be necessary to monitor patient hormonal status.

We previously reported that 2',3'-dideoxyinosine (didanosine, or ddI) significantly altered mitochondrial DNA (mtDNA) in peripheral blood mononuclear cells in human immunodeficiency virus type 1 (HIV-1)-infected children who had undetectable plasma HIV-1 RNA for more than 2 years while receiving highly active antiretroviral therapy. This research examines the in vitro effects of nucleoside reverse transcriptase inhibitors (NRTIs) on mitochondria of human skeletal muscle cells (HSMCs), including myoblasts and differentiated myotubes. mtDNA, mitochondrial RNA (mtRNA), and mRNA levels for nuclear mitochondrial regulatory factors were quantified in vitro using HSMCs, including myoblasts and differentiated myotubes, treated with NRTIs singly and in combination. After 5 days of treatment, mtDNA was significantly decreased in myoblasts and myotubes treated with ddI (P < 0.001 and P = 0.01, respectively) and ddI-containing regimens (P < 0.001 and P < 0.001, respectively) compared to levels in untreated cells. mtRNA (MTCYB) was also significantly decreased in the myoblasts and myotubes treated with ddI (P = 0.004) and ddI-containing regimens (P < 0.001). Regardless of the NRTI regimens examined, NRTI combinations significantly decreased mtRNA (MTCO3) in myoblasts and myotubes (P = 0.02 and P = 0.01, respectively). No significant differences were observed for nuclear mitochondrial regulatory factor mRNA in myoblasts or myotubes when treated with NRTIs (P > 0.07). ddI and ddI-containing regimens significantly decrease mtDNA and mtRNA in HSMCs, most notably in myoblasts. These findings may be of particular importance in developing countries, where ddI is widely used for first-line treatment of HIV-infected children.

Background: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations., Methods: The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses., Results: Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (kpol) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels., Conclusions: A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.

Lipoatrophy is a prevalent side effect of treatment with thymidine analogues. We wished to confine the time point of the antiadipogenic effect of zidovudine (AZT) during adipogenesis and to evaluate the antiproliferative effect of AZT on adipocyte homeostasis. We investigated the effects of AZT on adipogenesis in 3T3-F442A cells and studied their proliferation, differentiation, viability, and adiponectin expression. Cells were exposed to AZT (1 microM, 3 microM, 6 microM, and 180 microM), stavudine (d4T; 3 microM), or dideoxycytosine (ddC; 0.1 microM) for up to 15 days. Differentiation was assessed by real-time PCR and quantification of triglyceride accumulation. Proliferation and clonal expansion were determined by a [(3)H]thymidine incorporation assay. When they were induced to differentiate in the presence of AZT at the maximum concentration in plasma (C(max)) and lower concentrations, 3T3-F442A preadipocytes failed to accumulate cytoplasmic triacylglycerol and failed to express normal levels of the later adipogenic transcription factors, CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. AZT exerted an inhibitory effect on the completion of the mitotic clonal expansion, which resulted in incomplete 3T3-F442A differentiation and, finally, a reduction in the level of adiponectin expression. In addition, AZT impaired the constitutive proliferation in murine and primary human subcutaneous preadipocytes. In contrast, incubation with d4T and ddC at the C(max) did not affect either preadipocyte proliferation or clonal expansion and differentiation. We conclude that the antiproliferative and antiadipogenetic effects of AZT on murine and primary human preadipocytes reveal the impact of the drug on fat tissue regeneration. These effects of the drug are expected to contribute to disturbed adipose tissue homeostasis and to be influenced by differential drug concentration and penetration in individual patients.

2',3'-Didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a recently discovered nucleoside reverse transcriptase (RT) inhibitor, exhibits 5- to 10-fold-higher activity against human immunodeficiency virus type 1 (HIV-1) and less cytotoxicity than does its parental compound d4T (stavudine). Using steady-state kinetic approaches, we have previously shown that (i) 4'-ethynyl-d4T triphosphate (4'-Ed4TTP) inhibits HIV-1 RT more efficiently than d4TTP does and (ii) its inhibition efficiency toward the RT M184V mutant is threefold less than that toward wild-type (wt) RT. In this study we used pre-steady-state kinetic approaches in an attempt to understand its mechanism of inhibition. With wt and the M184V mutant RTs, 4'-Ed4TTP has three- to fivefold-lower K(d) (dissociation constant) values than d4TTP, while d4TTP has up to eightfold-higher K(d) values than dTTP. Inhibition is more effective in DNA replication with RNA template than with DNA template. In general, the M184V mutant exhibits poorer binding for all three nucleoside triphosphates than does wt RT. The structural basis for the lower binding affinity of d4TTP than of dTTP could be the lack of hydrogen bonds from the missing 3'-hydroxyl group in d4TTP to the backbone amide of Y115 and also to the side chain of Q151. The structural basis for the higher binding affinity of 4'-Ed4TTP than of d4TTP could be the additional binding of the 4'-ethynyl group in a preformed hydrophobic pocket by A114, Y115, M184, F160, and part of D185.

Lipodystrophic syndrome is a major side effect of antiviral therapy leading to profound disturbances in adipose tissue. Human preadipocyte primary culture represents a model to understand mechanisms by which antiretroviral drugs alter adipocyte biology. The aim of this study was to evaluate the effects of various protease and nucleoside reverse transcriptase inhibitors in this model. We tested the effect of drugs on triglyceride accumulation and expression of specific genes by real-time polymerase chain reaction. To determine differential mechanisms by which the efficient drugs operate, we studied mitochondrial effects by evaluating oxygen consumption rates and nuclear lamina alteration by immunocytology. Only stavudine and nelfinavir, both at 10 microM, altered human adipose cell differentiation, as shown by reduced triglyceride accumulation. Our studies revealed that stavudine increased expression of genes such as PGC1 and LPL and affected mitochondrial respiration. Cells treated with nelfinavir had a lower expression of PPARgamma, LPL, and ap2 and presented disorganization of lamin A/C. Our data suggest for the first time in a model of human adipocytes differentiated in vitro that stavudine and nelfinavir interfere with the process of differentiation by 2 distinct mechanisms. This may be particularly relevant in understanding the physiopathologic mechanisms underlying the lipodystrophic syndrome.

We describe an unusual pathway of human immunodeficiency virus type 1 reverse transcriptase resistance during therapy with tenofovir-emtricitabine, characterized initially by the mutations K70E and M184V and later by K70G and M184V, with the two mutations coexisting on the same viral genome. Phenotypic resistance to lamivudine, emtricitabine, abacavir, didanosine, and tenofovir was observed, whereas susceptibility to zidovudine and stavudine was preserved.

The therapeutic benefits of current antiretroviral therapy are limited by the evolution of drug-resistant virus and long-term toxicity. Novel antiretroviral compounds with activity against drug-resistant viruses are needed. 2',3'-didehydro-3'-deoxy-4'-ethynylthymidine (4'-Ed4T), a novel thymidine analog, has potent anti-human immunodeficiency virus (HIV) activity, maintains considerable activity against multidrug-resistant HIV strains, and is less inhibitory to mitochondrial DNA synthesis in cell culture than its progenitor stavudine (D4T). We investigated the intracellular metabolism and anti-HIV activity of 4'-Ed4T. The profile of 4'-Ed4T metabolites was qualitatively similar to that for zidovudine (AZT), with the monophosphate metabolite as the major metabolite, in contrast to that for D4T, with relatively poor formation of total metabolites. The first phosphorylation step for 4'-Ed4T in cells was more efficient than that for D4T but less than that for AZT. The amount of 4'-Ed4T triphosphate (4'-Ed4TTP) was higher than that of AZTTP at 24 h in culture. There was a dose-dependent accumulation of 4'-Ed4T diphosphate and 4'-Ed4TTP on up-regulation of thymidylate kinase and 3-phosphoglycerate kinase expression in Tet-On RKO cells, respectively. The anti-HIV activity of 4'-Ed4T in cells persisted even after 48 h of drug removal from culture in comparison with AZT, D4T, and nevirapine (NVP). The order of increasing persistence of anti-HIV activity of these compounds after drug removal was 4'-Ed4T > D4T > AZT > NVP. In conclusion, with the persistence of 4'-Ed4TTP and persistent anti-HIV activity in cells, we anticipate less frequent dosing and fewer patient compliance issues than for D4T. 4'-Ed4T is a promising antiviral candidate for HIV type 1 chemotherapy.

We conducted molecular dynamics simulations of complexes of HIV-1 reverse transcriptase (RT) with the substrate and the antiretrovirals AZT and d4T for which resistance emerges via the excision mechanism. It is currently believed that excision results from the inability of AZT to translocate to the P site because of the steric hindrances imposed by the azide group. However, such explanation is far from satisfactory as d4T does not have such steric hindrances and still suffers from excision. Such contradiction motivated us for the present study. The results point to a new explanation for excision. RT preferably excises these inhibitors over the substrate as a consequence of a different pattern of hydrogen bridges they establish with the N site after incorporation. In the complexes with normal nucleotides, the fingers residues K65 and R72 establish hydrogen bonds mainly with the leaving PPi. With the inhibitors, those same residues establish hydrogen bonds primarily with the substituted nucleotides. Consequently, pyrophosphate is eliminated before the opening of the fingers domain, which allows ATP binding, with subsequent excision and development of drug resistance.

Background: Low testosterone levels are commonly reported in patients with advanced human immunodeficiency virus disease. The effects of initiation of different antiretroviral regimens on testosterone levels and changes in fat-free mass have not been reported., Methods: Antiretroviral-naive men (n=213) were randomized to receive nelfinavir, efavirenz, or both plus either zidovudine and lamivudine or stavudine and didanosine. Patients underwent measurements of metabolic parameters, including determination of free testosterone level by equilibrium dialysis and bioelectrical impedance analysis, over a 64-week period., Results: At baseline, the median free testosterone level was 92 pg/mL; the level was subnormal (i.e., <50 pg/mL) in 6%. Lower CD4 cell count at the time of study entry, higher weight, and greater age were independently associated with lower baseline free testosterone level. At week 64, the median free testosterone level increased more in zidovudine-lamivudine recipients (48 of whom had paired values available; change, +31 pg/mL) than in stavudine-didanosine recipients (57 of whom had paired values; change, +3 pg/mL; P=.001, by Wilcoxon rank sum test), and it increased more in efavirenz recipients (37 of whom had paired values; change, +30 pg/mL) than in nelfinavir recipients (28 of whom had paired values; change, -3 pg/mL; P=.05). The median fat-free mass for the entire group increased by 1.2 kg at week 64 (change, +2.0%; P<.001); the increase was greater in the zidovudine-lamivudine group (n=70; change, +1.8 kg) than in the stavudine-didanosine group (n=79; change, +0.5 kg; P=.04), and the increase was also greater for efavirenz recipients (n=53; change, +2.1 kg) than among nelfinavir recipients (n=47; change, +0.4 kg; P=.003). White race, lower CD4 cell count at study entry, assignment to the efavirenz treatment arm, and assignment to the zidovudine-lamivudine treatment arm independently predicted greater absolute change in fat-free mass at week 64., Conclusions: Subnormal free testosterone levels occurred infrequently among these antiretroviral-naive men. Free testosterone and fat-free mass levels increased after initiation of antiretroviral therapy, with greater increases at 64 weeks among zidovudine-lamivudine recipients than among stavudine-didanosine recipients and among efavirenz recipients than among nelfinavir recipients.

Several anti-human immunodeficiency virus type 1 reverse transcriptase inhibitors were evaluated for their antiviral activities against porcine endogenous retrovirus in human cells. Among the test compounds, zidovudine was found to be the most active. The order of potency was zidovudine > phosphonylmethoxyethoxydiaminopyrimidine = phosphonylmethoxypropyldiaminopurine > tenofovir > or = adefovir > stavudine.

Mitochondrial dysfunction may contribute to the development of insulin resistance and type 2 diabetes. Nucleoside reverse transcriptase inhibitors (NRTIs), specifically stavudine, are known to alter mitochondrial function in human immunodeficiency virus (HIV)-infected individuals, but the effects of stavudine on glucose disposal and mitochondrial function in muscle have not been prospectively evaluated. In this study, we investigated short-term stavudine administration among healthy control subjects to determine effects on insulin sensitivity. A secondary aim was to determine the effects of stavudine on mitochondrial DNA (mtDNA) and function. Sixteen participants without personal or family history of diabetes were enrolled. Subjects were randomized to receive stavudine, 30-40 mg, twice a day, or placebo for 1 mo. Insulin sensitivity determined by glucose infusion rate during the hyperinsulinemic euglycemic clamp was significantly reduced after 1-mo exposure in the stavudine-treated subjects compared with placebo (-0.8 +/- 0.5 vs. +0.7 +/- 0.3 mg.kg(-1).min(-1), P = 0.04, stavudine vs. placebo). In addition, muscle biopsy specimens in the stavudine-treated group showed significant reduction in mtDNA/nuclear DNA (-52%, P = 0.005), with no change in placebo-treated subjects (+8%, P = 0.9). (31)P magnetic resonance spectroscopy (MRS) studies of mitochondrial function correlated with insulin sensitivity measures (r2 = 0.5, P = 0.008). These findings demonstrate that stavudine administration has potent effects on insulin sensitivity among healthy subjects. Further studies are necessary to determine whether changes in mtDNA resulting from stavudine contribute to effects on insulin sensitivity.

Thymidine analogs, including 3'-azido-3'-deoxythymidine (AZT) and 2',3'-dideoxy-3'-deoxythymidine (D4T), are important antiretroviral agents. To exert antiretroviral activity, these analogs undergo a stepwise phosphorylation intracellularly to the active triphosphate metabolites. We previously reported that 4'-substituted D4T with an ethynyl group (i.e., 4'-ethynyl D4T) increased the anti-human immunodeficiency virus (HIV) activity and was active against multidrug-resistant HIV strains. 4'-Ethynyl D4T is a better substrate for phosphorylation by human thymidine kinase 1 than D4T is. In this report, we first studied the enzymes involved in the phosphorylation of 4'-ethynyl D4T from monophosphate to triphosphate metabolites. The 4'-ethynyl D4TMP is phosphorylated by recombinant human TMP kinase with a K(m) of 19 +/- 4 microM and a k(cat) of 0.007 +/- 0.001 s(-1); the relative efficiency is about 9 and 15% of those of D4TMP and AZTMP, respectively. Several enzymes from crude cellular extracts, including nucleoside diphosphate kinase, pyruvate kinase, creatine kinase, and 3-phosphoglycerate kinase, could phosphorylate 4'-ethynyl D4T-diphosphate. The relative phosphorylation efficiencies of 4'-ethynyl D4TDP were about 3 to 25% of those of D4TDP and were generally similar to those of AZTDP. In T-lymphoid cell lines, there was a preponderant accumulation of 4'-ethynyl D4TMP, suggesting that TMP kinase could be the rate-limiting enzyme in the metabolism of 4'-ethynyl D4T. Although the same enzymes are involved in the stepwise phosphorylation of thymidine analogs, their behaviors in phosphorylating metabolites of 4'-ethynyl D4T are different from those of D4T and AZT. Qualitatively, the metabolism of 4'-ethynyl D4T is more similar to that of AZT than to that of its progenitor, D4T.

Background: A fixed-dose combination of stavudine, lamivudine, and nevirapine is extensively used as an antiretroviral regimen in developing countries because of its affordability. Virological failure with this regimen has become more common, and a second-line regimen needs to be prepared in the national program., Methods: Genotypic resistance testing was conducted among human immunodeficiency virus type 1 (HIV-1)-infected patients who experienced treatment failure with their first antiretroviral regimen (a fixed-dose combination of stavudine, lamivudine, and nevirapine) during 2003-2005. Patterns of resistance mutations and options for a second-line regimen were studied., Results: We studied 98 patients (mean age, 35.2 years), of whom, 63% were male. The median duration of antiretroviral therapy was 20 months. The median HIV-1 RNA load at the time of virological failure detection was 4.1 log copies/mL. The prevalences of patients with > or =1 major mutation conferring drug resistance to nucleoside reverse-transcriptase inhibitors and nonnucleoside reverse-transcriptase inhibitors were 95% and 92%, respectively. M184V was the most common nucleoside reverse-transcriptase inhibitor resistance mutation (observed in 89% of patients). Thymidine analogue mutations, K65R, and Q151M were observed in 37%, 6%, and 8% of patients, respectively. Patients with an HIV-1 RNA load of >4 log copies/mL at the time of treatment failure had higher prevalence of thymidine analogue mutations (P=.041), K65R (P=.031), and Q151M (P=.008) mutations. The second-line regimen was determined in a resource-limited setting where tenofovir and enfuvirtide are not available; the options were limited for 48% of patients., Conclusions: After experiencing treatment failure with a fixed-dose combination of stavudine, lamivudine, and nevirapine, almost all patients have lamivudine and nonnucleoside reverse-transcriptase inhibitor resistance. The options for a second-line regimen are limited for one-half of these patients. In resource-limited settings where availability of antiretroviral agents is limited, strategies for prevention of HIV-1 resistance are crucial. Early detection of virological failure may provide more options and better treatment outcomes.

Patients infected with HIV-1 with more than 1000 HIV-1 RNA copies/mL, who were genotyped within 3 months before starting stavudine and treated for at least 3 months with a stable stavudine-containing highly active antiretroviral therapy, were selected from our database to identify the determinants of response to stavudine. Nonresponse was defined as a failure to achieve HIV-1 RNA level of less than 400 copies/mL or a reduction of more than 2 log10 by week 12. Univariate logistic analysis was used to elicit the failure-associated reverse transcriptase mutations (scored 1 to develop a genotype score). Eighty-one patients were eligible for the analysis, including 75 (93%) who previously received zidovudine. Thirty-five (43%) were nonresponders. Univariate logistic analysis revealed the following failure-associated mutations: 41L (P = 0.0001), 44D (P = 0.02), 118I (P = 0.0006), 184V (P = 0.04), 210W (P = 0.0004), and 215Y (P = 0.002) for a median stavudine score of 2. Failure was observed in 7 (18.9%) of 37 patients with a score less than 2, compared with 28 (63.6%) of 44 patients with a score of 2 or greater (P < 0.0001). The multivariable analysis showed that the 118I mutation (P = 0.04) was the only independent genotypic predictor of failing on a stavudine- containing highly active antiretroviral therapy.