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Sample records for hiv-1 drug resistant

  1. Current Perspectives on HIV-1 Antiretroviral Drug Resistance

    PubMed Central

    Iyidogan, Pinar; Anderson, Karen S.

    2014-01-01

    Current advancements in antiretroviral therapy (ART) have turned HIV-1 infection into a chronic and manageable disease. However, treatment is only effective until HIV-1 develops resistance against the administered drugs. The most recent antiretroviral drugs have become superior at delaying the evolution of acquired drug resistance. In this review, the viral fitness and its correlation to HIV-1 mutation rates and drug resistance are discussed while emphasizing the concept of lethal mutagenesis as an alternative therapy. The development of resistance to the different classes of approved drugs and the importance of monitoring antiretroviral drug resistance are also summarized briefly. PMID:25341668

  2. Sparse Representation for Prediction of HIV-1 Protease Drug Resistance.

    PubMed

    Yu, Xiaxia; Weber, Irene T; Harrison, Robert W

    2013-01-01

    HIV rapidly evolves drug resistance in response to antiviral drugs used in AIDS therapy. Estimating the specific resistance of a given strain of HIV to individual drugs from sequence data has important benefits for both the therapy of individual patients and the development of novel drugs. We have developed an accurate classification method based on the sparse representation theory, and demonstrate that this method is highly effective with HIV-1 protease. The protease structure is represented using our newly proposed encoding method based on Delaunay triangulation, and combined with the mutated amino acid sequences of known drug-resistant strains to train a machine-learning algorithm both for classification and regression of drug-resistant mutations. An overall cross-validated classification accuracy of 97% is obtained when trained on a publically available data base of approximately 1.5×10(4) known sequences (Stanford HIV database http://hivdb.stanford.edu/cgi-bin/GenoPhenoDS.cgi). Resistance to four FDA approved drugs is computed and comparisons with other algorithms demonstrate that our method shows significant improvements in classification accuracy. PMID:24910813

  3. Sparse Representation for Prediction of HIV-1 Protease Drug Resistance

    PubMed Central

    Yu, Xiaxia; Weber, Irene T.; Harrison, Robert W.

    2013-01-01

    HIV rapidly evolves drug resistance in response to antiviral drugs used in AIDS therapy. Estimating the specific resistance of a given strain of HIV to individual drugs from sequence data has important benefits for both the therapy of individual patients and the development of novel drugs. We have developed an accurate classification method based on the sparse representation theory, and demonstrate that this method is highly effective with HIV-1 protease. The protease structure is represented using our newly proposed encoding method based on Delaunay triangulation, and combined with the mutated amino acid sequences of known drug-resistant strains to train a machine-learning algorithm both for classification and regression of drug-resistant mutations. An overall cross-validated classification accuracy of 97% is obtained when trained on a publically available data base of approximately 1.5×104 known sequences (Stanford HIV database http://hivdb.stanford.edu/cgi-bin/GenoPhenoDS.cgi). Resistance to four FDA approved drugs is computed and comparisons with other algorithms demonstrate that our method shows significant improvements in classification accuracy. PMID:24910813

  4. Modeling HIV-1 Drug Resistance as Episodic Directional Selection

    PubMed Central

    Murrell, Ben; de Oliveira, Tulio; Seebregts, Chris; Kosakovsky Pond, Sergei L.; Scheffler, Konrad

    2012-01-01

    The evolution of substitutions conferring drug resistance to HIV-1 is both episodic, occurring when patients are on antiretroviral therapy, and strongly directional, with site-specific resistant residues increasing in frequency over time. While methods exist to detect episodic diversifying selection and continuous directional selection, no evolutionary model combining these two properties has been proposed. We present two models of episodic directional selection (MEDS and EDEPS) which allow the a priori specification of lineages expected to have undergone directional selection. The models infer the sites and target residues that were likely subject to directional selection, using either codon or protein sequences. Compared to its null model of episodic diversifying selection, MEDS provides a superior fit to most sites known to be involved in drug resistance, and neither one test for episodic diversifying selection nor another for constant directional selection are able to detect as many true positives as MEDS and EDEPS while maintaining acceptable levels of false positives. This suggests that episodic directional selection is a better description of the process driving the evolution of drug resistance. PMID:22589711

  5. Evolution of transmitted HIV-1 drug resistance in HIV-1-infected patients in Italy from 2000 to 2010.

    PubMed

    Colafigli, M; Torti, C; Trecarichi, E M; Albini, L; Rosi, A; Micheli, V; Manca, N; Penco, G; Bruzzone, B; Punzi, G; Corsi, P; Parruti, G; Bagnarelli, P; Monno, L; Gonnelli, A; Cauda, R; Di Giambenedetto, S

    2012-08-01

    Prevalence and predictors of transmitted drug resistance (TDR), defined as the presence of at least one WHO surveillance drug resistance mutation (SDRM), were investigated in antiretroviral-naïve HIV-1-infected patients, with a genotypic resistance test (GRT) performed ≤6 months before starting cART between 2000 and 2010. 3163 HIV-1 sequences were selected (69% subtype B). Overall, the prevalence of TDR was 12% (13.2% subtype B, 9% non-B). TDR significantly declined overall and for the single drug classes. Older age independently predicted increased odds of TDR, whereas a more recent GRT, a higher HIV-RNA and C vs. B subtype predicted lower odds of TDR. PMID:22536753

  6. Insights into the mechanism of drug resistance. X-ray structure analysis of multi-drug resistant HIV-1 protease ritonavir complex

    SciTech Connect

    Liu, Zhigang; Yedidi, Ravikiran S.; Wang, Yong; Dewdney, Tamaria G.; Reiter, Samuel J.; Brunzelle, Joseph S.; Kovari, Iulia A.; Kovari, Ladislau C.

    2013-01-08

    Ritonavir (RTV) is a first generation HIV-1 protease inhibitor with rapidly emerging drug resistance. Mutations at residues 46, 54, 82 and 84 render the HIV-1 protease drug resistant against RTV. We report the crystal structure of multi-drug resistant (MDR) 769 HIV-1 protease (carrying resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84 and 90) complexed with RTV and the in vitro enzymatic IC50 of RTV against MDR HIV-1 protease. The structural and functional studies demonstrate significant drug resistance of MDR HIV-1 protease against RTV, arising from reduced hydrogen bonds and Van der Waals interactions between RTV and MDR HIV-1 protease.

  7. Assessing transmissibility of HIV-1 drug resistance mutations from treated and from drug-naive individuals

    PubMed Central

    Winand, Raf; Theys, Kristof; Eusébio, Mónica; Aerts, Jan; Camacho, Ricardo J.; Gomes, Perpetua; Suchard, Marc A.; Vandamme, Anne-Mieke; Abecasis, Ana B.

    2015-01-01

    Objectives: Surveillance drug resistance mutations (SDRMs) in drug-naive patients are typically used to survey HIV-1-transmitted drug resistance (TDR). We test here how SDRMs in patients failing treatment, the original source of TDR, contribute to assessing TDR, transmissibility and transmission source of SDRMs. Design: This is a retrospective observational study analyzing a Portuguese cohort of HIV-1-infected patients. Methods: The prevalence of SDRMs to protease inhibitors, nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs) in drug-naive and treatment-failing patients was measured for 3554 HIV-1 subtype B patients. Transmission ratio (prevalence in drug-naive/prevalence in treatment-failing patients), average viral load and robust linear regression with outlier detection (prevalence in drug-naive versus in treatment-failing patients) were analyzed and used to interpret transmissibility. Results: Prevalence of SDRMs in drug-naive and treatment-failing patients were linearly correlated, but some SDRMs were classified as outliers – above (PRO: D30N, N88D/S, L90 M, RT: G190A/S/E) or below (RT: M184I/V) expectations. The normalized regression slope was 0.073 for protease inhibitors, 0.084 for NRTIs and 0.116 for NNRTIs. Differences between SDRMs transmission ratios were not associated with differences in viral loads. Conclusion: The significant linear correlation between prevalence of SDRMs in drug-naive and in treatment-failing patients indicates that the prevalence in treatment-failing patients can be useful to predict levels of TDR. The slope is a cohort-dependent estimate of rate of TDR per drug class and outlier detection reveals comparative persistence of SDRMs. Outlier SDRMs with higher transmissibility are more persistent and more likely to have been acquired from drug-naive patients. Those with lower transmissibility have faster reversion dynamics after transmission and are associated with

  8. An international multicenter study on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing.

    PubMed

    Simen, Birgitte B; Braverman, Michael S; Abbate, Isabella; Aerssens, Jeroen; Bidet, Yannick; Bouchez, Olivier; Gabriel, Christian; Izopet, Jacques; Kessler, Harald H; Stelzl, Evelyn; Di Giallonardo, Francesca; Schlapbach, Ralph; Radonic, Aleksander; Paredes, Roger; Recordon-Pinson, Patricia; Sakwa, James; St John, Elizabeth P; Schmitz-Agheguian, Gudrun G; Metzner, Karin J; Däumer, Martin P

    2014-08-01

    The detection of mutant spectra within the viral quasispecies is critical for therapeutic management of HIV-1 infections. Routine clinical application of ultrasensitive genotyping requires reproducibility and concordance within and between laboratories. The goal of the study was to evaluate a new protocol on HIV-1 drug resistance testing by 454 ultra-deep pyrosequencing (454-UDS) in an international multicenter study. Sixteen blinded HIV-1 subtype B samples were provided for 454-UDS as both RNA and cDNA with viral titers of 88,600-573,000 HIV-1 RNA copies/ml. Eight overlapping amplicons spanning protease (PR) codons 10-99 and reverse transcriptase (RT) codons 1-251 were generated using molecular barcoded primers. 454-UDS was performed using the 454 Life Sciences/Roche GS FLX platform. PR and RT sequences were analyzed using 454 Life Sciences Amplicon Variant Analyzer (AVA) software. Quantified variation data were analyzed for intra-laboratory reproducibility and inter-laboratory concordance. Routine population sequencing was performed using the ViroSeq HIV-1 genotyping system. Eleven laboratories and the reference laboratory 454 Life Sciences sequenced the HIV-1 sample set. Data presented are derived from seven laboratories and the reference laboratory since severe study protocol execution errors occurred in four laboratories leading to exclusion. The median sequencing depth across all sites was 1364 reads per position (IQR=809-2065). 100% of the ViroSeq-reported mutations were also detected by 454-UDS. Minority HIV-1 drug resistance mutations, defined as HIV-1 drug resistance mutations identified at frequencies of 1-25%, were only detected by 454-UDS. Analysis of 10 preselected majority and minority mutations were consistently found across sites. The analysis of drug-resistance mutations detected between 1 and 10% demonstrated high intra- and inter-laboratory consistency in frequency estimates for both RNA and prepared cDNA samples, indicating robustness of the

  9. HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing

    PubMed Central

    Rhee, Soo-Yon; Jordan, Michael R.; Raizes, Elliot; Chua, Arlene; Parkin, Neil; Kantor, Rami; Van Zyl, Gert U.; Mukui, Irene; Hosseinipour, Mina C.; Frenkel, Lisa M.; Ndembi, Nicaise; Hamers, Raph L.; Rinke de Wit, Tobias F.; Wallis, Carole L.; Gupta, Ravindra K.; Fokam, Joseph; Zeh, Clement; Schapiro, Jonathan M.; Carmona, Sergio; Katzenstein, David; Tang, Michele; Aghokeng, Avelin F.; De Oliveira, Tulio; Wensing, Annemarie M. J.; Gallant, Joel E.; Wainberg, Mark A.; Richman, Douglas D.; Fitzgibbon, Joseph E.; Schito, Marco; Bertagnolio, Silvia; Yang, Chunfu; Shafer, Robert W.

    2015-01-01

    The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy. PMID:26717411

  10. HIV-1 Drug Resistance Mutations: Potential Applications for Point-of-Care Genotypic Resistance Testing.

    PubMed

    Rhee, Soo-Yon; Jordan, Michael R; Raizes, Elliot; Chua, Arlene; Parkin, Neil; Kantor, Rami; Van Zyl, Gert U; Mukui, Irene; Hosseinipour, Mina C; Frenkel, Lisa M; Ndembi, Nicaise; Hamers, Raph L; Rinke de Wit, Tobias F; Wallis, Carole L; Gupta, Ravindra K; Fokam, Joseph; Zeh, Clement; Schapiro, Jonathan M; Carmona, Sergio; Katzenstein, David; Tang, Michele; Aghokeng, Avelin F; De Oliveira, Tulio; Wensing, Annemarie M J; Gallant, Joel E; Wainberg, Mark A; Richman, Douglas D; Fitzgibbon, Joseph E; Schito, Marco; Bertagnolio, Silvia; Yang, Chunfu; Shafer, Robert W

    2015-01-01

    The increasing prevalence of acquired and transmitted HIV-1 drug resistance is an obstacle to successful antiretroviral therapy (ART) in the low- and middle-income countries (LMICs) hardest hit by the HIV-1 pandemic. Genotypic drug resistance testing could facilitate the choice of initial ART in areas with rising transmitted drug resistance (TDR) and enable care-providers to determine which individuals with virological failure (VF) on a first- or second-line ART regimen require a change in treatment. An inexpensive near point-of-care (POC) genotypic resistance test would be useful in settings where the resources, capacity, and infrastructure to perform standard genotypic drug resistance testing are limited. Such a test would be particularly useful in conjunction with the POC HIV-1 viral load tests that are currently being introduced in LMICs. A POC genotypic resistance test is likely to involve the use of allele-specific point mutation assays for detecting drug-resistance mutations (DRMs). This study proposes that two major nucleoside reverse transcriptase inhibitor (NRTI)-associated DRMs (M184V and K65R) and four major NNRTI-associated DRMs (K103N, Y181C, G190A, and V106M) would be the most useful for POC genotypic resistance testing in LMIC settings. One or more of these six DRMs was present in 61.2% of analyzed virus sequences from ART-naïve individuals with intermediate or high-level TDR and 98.8% of analyzed virus sequences from individuals on a first-line NRTI/NNRTI-containing regimen with intermediate or high-level acquired drug resistance. The detection of one or more of these DRMs in an ART-naïve individual or in a individual with VF on a first-line NRTI/NNRTI-containing regimen may be considered an indication for a protease inhibitor (PI)-containing regimen or closer virological monitoring based on cost-effectiveness or country policy. PMID:26717411

  11. Decomposing the Energetic Impact of Drug-Resistant Mutations: The Example of HIV-1 Protease - DRV Binding

    PubMed Central

    Cai, Yufeng; Schiffer, Celia

    2016-01-01

    Summary HIV-1 protease is a major drug target for AIDS therapy. With the appearance of drug-resistant HIV-1 protease variants, understanding the mechanism of drug resistance becomes critical. Computational methods can provide more details about inhibitor-protease binding other than crystallography and isothermal titration calorimetry. Darunavir is the latest FDA approved HIV-1 protease inhibitor. In this context, the free energy component analysis is performed on the DRV binding to WT protease and ACT, a drug resistant variant, to evaluate contribution of each atoms of DRV to the binding affinity. This information can contribute to the rationale design of new HIV-1 protease inhibitors. PMID:22183557

  12. Perinatal acquisition of drug-resistant HIV-1 infection: mechanisms and long-term outcome

    PubMed Central

    Delaugerre, Constance; Chaix, Marie-Laure; Blanche, Stephane; Warszawski, Josiane; Cornet, Dorine; Dollfus, Catherine; Schneider, Veronique; Burgard, Marianne; Faye, Albert; Mandelbrot, Laurent; Tubiana, Roland; Rouzioux, Christine

    2009-01-01

    Background Primary-HIV-1-infection in newborns that occurs under antiretroviral prophylaxis that is a high risk of drug-resistance acquisition. We examine the frequency and the mechanisms of resistance acquisition at the time of infection in newborns. Patients and Methods We studied HIV-1-infected infants born between 01 January 1997 and 31 December 2004 and enrolled in the ANRS-EPF cohort. HIV-1-RNA and HIV-1-DNA samples obtained perinatally from the newborn and mother were subjected to population-based and clonal analyses of drug resistance. If positive, serial samples were obtained from the child for resistance testing. Results Ninety-two HIV-1-infected infants were born during the study period. Samples were obtained from 32 mother-child pairs and from another 28 newborns. Drug resistance was detected in 12 newborns (20%): drug resistance to nucleoside reverse transcriptase inhibitors was seen in 10 cases, non-nucleoside reverse transcriptase inhibitors in two cases, and protease inhibitors in one case. For 9 children, the detection of the same resistance mutations in mothers' samples (6 among 10 available) and in newborn lymphocytes (6/8) suggests that the newborn was initially infected by a drug-resistant strain. Resistance variants were either transmitted from mother-to-child or selected during subsequent temporal exposure under suboptimal perinatal prophylaxis. Follow-up studies of the infants showed that the resistance pattern remained stable over time, regardless of antiretroviral therapy, suggesting the early cellular archiving of resistant viruses. The absence of resistance in the mother of the other three children (3/10) and neonatal lymphocytes (2/8) suggests that the newborns were infected by a wild-type strain without long-term persistence of resistance when suboptimal prophylaxis was stopped. Conclusion This study confirms the importance of early resistance genotyping of HIV-1-infected newborns. In most cases (75%), drug resistance was archived in

  13. Predicting drug resistance of the HIV-1 protease using molecular interaction energy components.

    PubMed

    Hou, Tingjun; Zhang, Wei; Wang, Jian; Wang, Wei

    2009-03-01

    Drug resistance significantly impairs the efficacy of AIDS therapy. Therefore, precise prediction of resistant viral mutants is particularly useful for developing effective drugs and designing therapeutic regimen. In this study, we applied a structure-based computational approach to predict mutants of the HIV-1 protease resistant to the seven FDA approved drugs. We analyzed the energetic pattern of the protease-drug interaction by calculating the molecular interaction energy components (MIECs) between the drug and the protease residues. Support vector machines (SVMs) were trained on MIECs to classify protease mutants into resistant and nonresistant categories. The high prediction accuracies for the test sets of cross-validations suggested that the MIECs successfully characterized the interaction interface between drugs and the HIV-1 protease. We conducted a proof-of-concept study on a newly approved drug, darunavir (TMC114), on which no drug resistance data were available in the public domain. Compared with amprenavir, our analysis suggested that darunavir might be more potent to combat drug resistance. To quantitatively estimate binding affinities of drugs and study the contributions of protease residues to causing resistance, linear regression models were trained on MIECs using partial least squares (PLS). The MIEC-PLS models also achieved satisfactory prediction accuracy. Analysis of the fitting coefficients of MIECs in the regression model revealed the important resistance mutations and shed light into understanding the mechanisms of these mutations to cause resistance. Our study demonstrated the advantages of characterizing the protease-drug interaction using MIECs. We believe that MIEC-SVM and MIEC-PLS can help design new agents or combination of therapeutic regimens to counter HIV-1 protease resistant strains. PMID:18704937

  14. Declining prevalence of HIV-1 drug resistance in antiretroviral treatment-exposed individuals in Western Europe.

    PubMed

    De Luca, Andrea; Dunn, David; Zazzi, Maurizio; Camacho, Ricardo; Torti, Carlo; Fanti, Iuri; Kaiser, Rolf; Sönnerborg, Anders; Codoñer, Francisco M; Van Laethem, Kristel; Vandamme, Anne-Mieke; Bansi, Loveleen; Ghisetti, Valeria; van de Vijver, David A M C; Asboe, David; Prosperi, Mattia C F; Di Giambenedetto, Simona

    2013-04-15

    HIV-1 drug resistance represents a major obstacle to infection and disease control. This retrospective study analyzes trends and determinants of resistance in antiretroviral treatment (ART)-exposed individuals across 7 countries in Europe. Of 20 323 cases, 80% carried at least one resistance mutation: these declined from 81% in 1997 to 71% in 2008. Predicted extensive 3-class resistance was rare (3.2% considering the cumulative genotype) and peaked at 4.5% in 2005, decreasing thereafter. The proportion of cases exhausting available drug options dropped from 32% in 2000 to 1% in 2008. Reduced risk of resistance over calendar years was confirmed by multivariable analysis. PMID:23315324

  15. Change in the Prevalence of HIV-1 and the Rate of Transmitted Drug-Resistant HIV-1 in Haiphong, Northern Vietnam.

    PubMed

    Pham, Hung Viet; Ishizaki, Azumi; Nguyen, Cuong Hung; Saina, Matilda Chelimo; Hoang, Huyen Thi Thanh; Tran, Vuong Thi; Bi, Xiuqiong; Pham, Thuc Van; Ichimura, Hiroshi

    2015-07-01

    We previously reported a significant decrease in HIV-1 prevalence, with no increase in drug-resistant HIV-1 among injecting drug users (IDU), female sex workers (FSW), and blood donors (BD), in Haiphong, Vietnam, from 2007 to 2009. In 2012, 388 IDU, 51 FSW, and 200 BD were recruited for further analysis. None had a history of antiretroviral treatment. From 2007 to 2012, HIV-1 prevalence was reduced from 35.9% to 18.6% (p<0.001), 23.1% to 9.8% (p<0.05), and 2.9% to 1% (p=0.29) in IDU, FSW, and BD, respectively. Of 79 anti-HIV-1 antibody-positive samples, 61 were successfully analyzed for the pol-reverse transcriptase (RT) region. All HIV-1 strains were CRF01_AE. Nonnucleoside RT inhibitor-resistant mutations, Y181C/I, were detected in three subjects; one had the nucleoside RT inhibitor-resistant mutations L74V and M184V and one had E138K. The prevalence of transmitted drug-resistant HIV-1 in Haiphong increased slightly from 1.8% in 2007 to 6.6% in 2012 (p=0.06). PMID:25970090

  16. Evolving patterns of HIV-1 transmitted drug resistance in Poland in the years 2000-2008.

    PubMed

    Stańczak, Grzegorz P; Stańczak, Janusz J; Marczyńska, Magdalena; Firlag-Burkacka, Ewa; Wiercińska-Drapało, Alicja; Leszczyszyn-Pynka, Magdalena; Jabłonowska, Elzbieta; Małolepsza, Ewa; Dyda, Tomasz; Zabek, Piotr; Horban, Andrzej

    2010-07-01

    The aim of the study was to determine the rate of transmission of drug resistant human immunodeficiency virus-1 (HIV-1) variants among therapy-naïve HIV positive patients in Poland in the year 2008, to compare the data with the results from the years 2000 to 2007 and to monitor patterns of HIV-1 subtypes present in Polish population and their evolution. Complete protease and part of reverse transcriptase regions were sequenced from the sera of patients directed to the laboratory for drug resistance testing. The Stanford's HIVdb program was used for the interpretation of results and subtyping. The variants scoring at least "intermediate resistance" for at least one drug were considered as resistant. The results obtained were compared to those obtained in the years 2000-2007. A total of 95 patients were enrolled in the 2008 study. Homosexual transmission of infection was documented in more than 55% of all cases. The overall prevalence of transmitted drug resistance (TDR) was 5.3% (3.9% in 2007, 5.8% in 2006, and 14.1% in the years 2002-2005). The study from the years 2000 to 2001 revealed 28.7% prevalence. Preliminary analysis of the first half of 2009 shows the ratio of 7.8%. In four (4.2%) cases drug resistance was associated with protease inhibitors class, in one case (1.1%) with resistance to non-nucleoside reverse transcriptase inhibitors class. In four cases (4.2%) non-B subtype was identified (C, G, CRF01_AE, CRF02_AG). An increase of percentage of drug resistant mutants-from 3.9% (2007) to 5.3% (2008)-was recognized. In this study, TDR was limited to single classes of antiretroviral drugs. HIV-1 subtype B prevails in Poland. PMID:20513098

  17. HIV-1 Transmitted Drug Resistance Mutations in Newly Diagnosed Antiretroviral-Naive Patients in Turkey.

    PubMed

    Sayan, Murat; Sargin, Fatma; Inan, Dilara; Sevgi, Dilek Y; Celikbas, Aysel K; Yasar, Kadriye; Kaptan, Figen; Kutlu, Selda; Fisgin, Nuriye T; Inci, Ayse; Ceran, Nurgul; Karaoglan, Ilkay; Cagatay, Atahan; Celen, Mustafa K; Koruk, Suda T; Ceylan, Bahadir; Yildirmak, Taner; Akalın, Halis; Korten, Volkan; Willke, Ayse

    2016-01-01

    HIV-1 replication is rapid and highly error-prone. Transmission of a drug-resistant HIV-1 strain is possible and occurs within the HIV-1-infected population. In this study, we aimed to determine the prevalence of transmitted drug resistance mutations (TDRMs) in 1,306 newly diagnosed untreated HIV-1-infected patients from 21 cities across six regions of Turkey between 2010 and 2015. TDRMs were identified according to the criteria provided by the World Health Organization's 2009 list of surveillance drug resistance mutations. The HIV-1 TDRM prevalence was 10.1% (133/1,306) in Turkey. Primary drug resistance mutations (K65R, M184V) and thymidine analogue-associated mutations (TAMs) were evaluated together as nucleos(t)ide reverse transcriptase inhibitor (NRTI) mutations. NRTI TDRMs were found in 8.1% (107/1,306) of patients. However, TAMs were divided into three categories and M41L, L210W, and T215Y mutations were found for TAM1 in 97 (7.4%) patients, D67N, K70R, K219E/Q/N/R, T215F, and T215C/D/S mutations were detected for TAM2 in 52 (3.9%) patients, and M41L + K219N and M41L + T215C/D/S mutations were detected for the TAM1 + TAM2 profile in 22 (1.7%) patients, respectively. Nonnucleoside reverse transcriptase inhibitor-associated TDRMs were detected in 3.3% (44/1,306) of patients (L100I, K101E/P, K103N/S, V179F, Y188H/L/M, Y181I/C, and G190A/E/S) and TDRMs to protease inhibitors were detected in 2.3% (30/1,306) of patients (M46L, I50V, I54V, Q58E, L76V, V82A/C/L/T, N83D, I84V, and L90M). In conclusion, long-term and large-scale monitoring of regional levels of HIV-1 TDRMs informs treatment guidelines and provides feedback on the success of HIV-1 prevention and treatment efforts. PMID:26414663

  18. Multiple drug resistant mechanisms against darunavir, amprenavir, and nelfinavir of HIV-1 PR

    NASA Astrophysics Data System (ADS)

    Liu, Xiaoqing; Dai, Qi; Xiu, Zhilong

    2013-02-01

    Acquired immune deficiency syndrome (AIDS) is a disease of the human immune system caused by the human immunodeficiency virus (HIV), which is infecting more humans and is expanding faster in the world. The illness interferes with the immune system, making people with AIDS much more likely to get infections, including opportunistic infections and tumors that do not affect people with working immune systems. HIV-1 PR is one of the major targets of anti-AIDS drug discovery. It is, therefore, necessary to develop some inhibitors against HIV-1 PR. In this work, we executed molecular dynamics (MDs) simulation of HIV-1 PR with drugs darunavir (DRV), amprenavir (APV), nelfinavir (NFV), and examined the resistant mechanism of L10I, G48V, I54V, and L90M mutations of this PR, aiming at designing promising drugs. The comparative analysis suggests that the existences of dodecahydroisoquinoline ring at P1' subsite, 4-aminophenylsulfonamide at P2' subsite, and bis-tetrahydrofuranylurethane at P2 subsite are helpful for maintaining the high affinity of the inhibitor for the protease and exhibiting high potency against multiple drug resistance (MDR) mutant protease.

  19. HIV-1 Genetic Diversity and Drug Resistance among Senegalese Patients in the Public Health System

    PubMed Central

    Thiam, Moussa; Diop-Ndiaye, Halimatou; Diouf, Aminata Diaw; Vidal, Nicole; Ndiaye, Ousseynou; Ndiaye, Ibrahima; Ngom-Gueye, Ndeye Fatou; Diallo, Sada; Diongue, Oumy Diop; Camara, Makhtar; Seck, Abdoulaye; Mboup, Souleymane

    2013-01-01

    In this study, we investigated the prevalence of human immunodeficiency virus type 1 (HIV-1) drug resistance mutations and genetic variability among Senegalese patients undergoing highly active antiretroviral therapy (ART) in the public health system. We conducted a cross-sectional study of 72 patients with suspected therapeutic failure. HIV-1 genotyping was performed with Viroseq HIV-1 Genotyping System v2.0 or the procedure developed by the ANRS AC11 resistance study group, and a phylogenetic analysis was performed. The median follow-up visit was at 40 (range, 12 to 123) months, and the median viral load was 4.67 (range, 3.13 to 6.94) log10 copies/ml. The first-line therapeutic regimen was nucleoside reverse transcriptase inhibitors (NRTIs) plus efavirenz (EFV) or NRTIs plus nevirapine (NVP) (54/72 patients; 75%), and the second-line therapy was NRTIs plus a protease inhibitor (PI/r) (18/72; 25%). Fifty-five patients (55/72; 76.39%) had at least one drug resistance mutation. The drug resistance rates were 72.22 and 88.89% for the first-line and second-line ARTs, respectively. In NRTI mutations, thymidine analog mutations (TAMs) were found in 50.79% and the M184V mutation was found in 34.92% of the samples. For non-NRTI resistance, we noted a predominance of the K103N mutation (46.27%). For PI/r, several cases of mutations were found with a predominance of M46I and L76V/F at 24% each. The phylogenetic analysis revealed CRF02_AG as the predominant circulating recombinant form (43/72; 59.72%). We found a high prevalence of resistance mutations and a high rate of TAMs among Senegalese patients in the public health system. These findings emphasize the need to improve virological monitoring in resource-limited settings. PMID:23241378

  20. Computational study of protein specificity: The molecular basis of HIV-1 protease drug resistance

    PubMed Central

    Wang, Wei; Kollman, Peter A.

    2001-01-01

    Drug resistance has sharply limited the effectiveness of HIV-1 protease inhibitors in AIDS therapy. It is critically important to understand the basis of this resistance for designing new drugs. We have evaluated the free energy contribution of each residue in the HIV protease in binding to one of its substrates and to the five FDA-approved protease drugs. Analysis of these free energy profiles and the variability at each sequence position suggests: (i) single drug resistance mutations are likely to occur at not well conserved residues if they interact more favorably with drugs than with the substrate; and (ii) resistance-evading drugs should have a free energy profile similar to the substrate and interact most favorably with well conserved residues. We also propose an empirical parameter, called the free energy/variability value, which combines free energy calculation and sequence analysis to suggest possible drug resistance mutations on the protease. The free energy/variability value is defined as the product of one residue's contribution to the binding free energy and the variability of that residue. This parameter can assist in designing resistance-evading drugs for any target. PMID:11752442

  1. A cell-free enzymatic activity assay for the evaluation of HIV-1 drug resistance to protease inhibitors

    PubMed Central

    Matsunaga, Satoko; Masaoka, Takashi; Sawasaki, Tatsuya; Morishita, Ryo; Iwatani, Yasumasa; Tatsumi, Masashi; Endo, Yaeta; Yamamoto, Naoki; Sugiura, Wataru; Ryo, Akihide

    2015-01-01

    Due to their high frequency of genomic mutations, human retroviruses often develop resistance to antiretroviral drugs. The emergence of drug-resistant human immunodeficiency virus type 1 (HIV-1) is a significant obstacle to the effective long-term treatment of HIV infection. The development of a rapid and versatile drug-susceptibility assay would enable acquisition of phenotypic information and facilitate determination of the appropriate choice of antiretroviral agents. In this study, we developed a novel in vitro method, termed the Cell-free drug susceptibility assay (CFDSA), for monitoring phenotypic information regarding the drug resistance of HIV-1 protease (PR). The CFDSA utilizes a wheat germ cell-free protein production system to synthesize enzymatically active HIV-1 PRs directly from PCR products amplified from HIV-1 molecular clones or clinical isolates in a rapid one-step procedure. Enzymatic activity of PRs can be readily measured by AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) in the presence or absence of clinically used protease inhibitors (PIs). CFDSA measurement of drug resistance was based on the fold resistance to the half-maximal inhibitory concentration (IC50) of various PIs. The CFDSA could serve as a non-infectious, rapid, accessible, and reliable alternative to infectious cell-based phenotypic assays for evaluation of PI-resistant HIV-1. PMID:26583013

  2. HIV-1 Integrase Strand Transfer Inhibitors with Reduced Susceptibility to Drug Resistant Mutant Integrases.

    PubMed

    Zhao, Xue Zhi; Smith, Steven J; Maskell, Daniel P; Metifiot, Mathieu; Pye, Valerie E; Fesen, Katherine; Marchand, Christophe; Pommier, Yves; Cherepanov, Peter; Hughes, Stephen H; Burke, Terrence R

    2016-04-15

    HIV integrase (IN) strand transfer inhibitors (INSTIs) are among the newest anti-AIDS drugs; however, mutant forms of IN can confer resistance. We developed noncytotoxic naphthyridine-containing INSTIs that retain low nanomolar IC50 values against HIV-1 variants harboring all of the major INSTI-resistant mutations. We found by analyzing crystal structures of inhibitors bound to the IN from the prototype foamy virus (PFV) that the most successful inhibitors show striking mimicry of the bound viral DNA prior to 3'-processing and the bound host DNA prior to strand transfer. Using this concept of "bi-substrate mimicry," we developed a new broadly effective inhibitor that not only mimics aspects of both the bound target and viral DNA but also more completely fills the space they would normally occupy. Maximizing shape complementarity and recapitulating structural components encompassing both of the IN DNA substrates could serve as a guiding principle for the development of new INSTIs. PMID:26808478

  3. Viraemia and HIV-1 drug resistance mutations among patients receiving antiretroviral treatment in Mozambique.

    PubMed

    Maldonado, F; Biot, M; Roman, F; Masquelier, C; Anapenge, M; Bastos, R; Chuquela, H C; Arendt, V; Schmit, J C; Zachariah, R

    2009-06-01

    This study was conducted among individuals taking first-line antiretroviral treatment (ART) for at least 12 months under programme conditions in Maputo, Mozambique in order to report on the level of detectable viraemia and the proportion and types of drug resistance mutations among those with detectable viral loads. HIV-1 RNA viral load levels (lower detection limit <50 copies/ml) were measured, and resistance mutations were sequenced. One hundred and forty-nine consecutive patients (69% females, median age 36 years) were included after a mean follow-up time of 23 months. One hundred and seven (72%; 95% CI 64-79) had undetectable viral load, while in 42 (28%, 95% CI 21-36) viral load was detectable (range 50-58884 copies/ml). From 15 patients with viral load >1000 copies/ml, 12 viruses were sequenced: eight were C subtypes and four were circulating recombinant forms (CRF08). Eight (5%; 95% CI 2-9) patients with detectable viral load had one or more major resistance mutations. Nucleoside reverse transcriptase inhibitor (NRTI) and non-NRTI mutations were observed. There were no major mutations for resistance to protease inhibitors. In Maputo, the level of detectable viraemia is reassuringly low. While embarking on ART scale-up, wider surveillance is warranted to monitor programme quality and limit the development of drug resistance, which remains a major potential challenge for the future of ART in Africa. PMID:18804251

  4. Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance.

    PubMed

    Wang, Chunlin; Mitsuya, Yumi; Gharizadeh, Baback; Ronaghi, Mostafa; Shafer, Robert W

    2007-08-01

    The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in > or =3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations. PMID:17600086

  5. Structure-Based Design of Novel HIV-1 Protease Inhibitors to Combat Drug Resistance

    SciTech Connect

    Ghosh,A.; Sridhar, P.; Leshchenko, S.; Hussain, A.; Li, J.; Kovalevsky, A.; Walters, D.; Wedelind, J.; Grum-Tokars, V.; et al.

    2006-01-01

    Structure-based design and synthesis of novel HIV protease inhibitors are described. The inhibitors are designed specifically to interact with the backbone of HIV protease active site to combat drug resistance. Inhibitor 3 has exhibited exceedingly potent enzyme inhibitory and antiviral potency. Furthermore, this inhibitor maintains impressive potency against a wide spectrum of HIV including a variety of multi-PI-resistant clinical strains. The inhibitors incorporated a stereochemically defined 5-hexahydrocyclopenta[b]furanyl urethane as the P2-ligand into the (R)-(hydroxyethylamino)sulfonamide isostere. Optically active (3aS,5R,6aR)-5-hydroxy-hexahydrocyclopenta[b]furan was prepared by an enzymatic asymmetrization of meso-diacetate with acetyl cholinesterase, radical cyclization, and Lewis acid-catalyzed anomeric reduction as the key steps. A protein-ligand X-ray crystal structure of inhibitor 3-bound HIV-1 protease (1.35 Angstroms resolution) revealed extensive interactions in the HIV protease active site including strong hydrogen bonding interactions with the backbone. This design strategy may lead to novel inhibitors that can combat drug resistance.

  6. Stability of HIV-1 Nucleic Acids in Dried Blood Spot Samples for HIV-1 Drug Resistance Genotyping

    PubMed Central

    Aitken, Susan C.; Wallis, Carole L.; Stevens, Wendy; de Wit, Tobias Rinke; Schuurman, Rob

    2015-01-01

    Dried blood spots (DBS) are an easy to collect sample-type that can stabilize biological material at ambient temperature for transport and storage, making them ideal for use in resource-limited settings (RLS). We investigated the effect of storage temperature and duration on ability to detect mixed HIV-1 viral RNA populations, and subsequently viral RNA populations in a background of proviral DNA. Part one of the study used DBS samples of whole blood spiked with specific quantities of HIV-1 subtype-B and -C RNA to study mixed virus population detection. Part two used DBS comprising of HIV-1 subtype-B proviral DNA containing U1 cells combined with HIV-1 subtype-C RNA to mimic HIV-1 infected clinical samples as a model system to study the relative stability of HIV-1 RNA and DNA in DBS. Prepared DBS were stored at -20°C and +30°C for periods of one day, one, two, and four weeks. Samples were genotyped to determine changes in the detection of mixtures in the sample over time. From two weeks onwards, storage at +30°C resulted in gradual, time-related reduction in the detection of mixed virus population at log10 VL 4.0 but not at log10 5.0. Proviral DNA and viral RNA were both stable for at least 52 weeks when stored at -20°C, compared to progressive RNA decay over time at +30°C. DBS storage conditions and duration had a significant effect on HIV-1 RNA amplification. Our results demonstrate that DBS storage at ambient temperature (+30°C) should not exceed two weeks, with long-term storage at -20°C or lower. PMID:26147689

  7. Stability of HIV-1 Nucleic Acids in Dried Blood Spot Samples for HIV-1 Drug Resistance Genotyping.

    PubMed

    Aitken, Susan C; Wallis, Carole L; Stevens, Wendy; de Wit, Tobias Rinke; Schuurman, Rob

    2015-01-01

    Dried blood spots (DBS) are an easy to collect sample-type that can stabilize biological material at ambient temperature for transport and storage, making them ideal for use in resource-limited settings (RLS). We investigated the effect of storage temperature and duration on ability to detect mixed HIV-1 viral RNA populations, and subsequently viral RNA populations in a background of proviral DNA. Part one of the study used DBS samples of whole blood spiked with specific quantities of HIV-1 subtype-B and -C RNA to study mixed virus population detection. Part two used DBS comprising of HIV-1 subtype-B proviral DNA containing U1 cells combined with HIV-1 subtype-C RNA to mimic HIV-1 infected clinical samples as a model system to study the relative stability of HIV-1 RNA and DNA in DBS. Prepared DBS were stored at -20 °C and +30 °C for periods of one day, one, two, and four weeks. Samples were genotyped to determine changes in the detection of mixtures in the sample over time. From two weeks onwards, storage at +30 °C resulted in gradual, time-related reduction in the detection of mixed virus population at log10 VL 4.0 but not at log10 5.0. Proviral DNA and viral RNA were both stable for at least 52 weeks when stored at -20 °C, compared to progressive RNA decay over time at +30 °C. DBS storage conditions and duration had a significant effect on HIV-1 RNA amplification. Our results demonstrate that DBS storage at ambient temperature (+30 °C) should not exceed two weeks, with long-term storage at -20 °C or lower. PMID:26147689

  8. Transmitted Drug Resistance Mutations in Antiretroviral-Naïve Injection Drug Users with Chronic HIV-1 Infection in Iran

    PubMed Central

    Memarnejadian, Arash; Menbari, Shahoo; Vahabpour, Rouhollah; Aghasadeghi, Mohammad Reza; Mostafavi, Ehsan; Abdi, Mohammad

    2015-01-01

    The growing incidence and transmission of drug resistant HIV-1 strains due to widespread use of antiretroviral therapy (ART) can jeopardize the success of first-line ART. While there is a known moderate prevalence of transmitted drug resistance (TDR) among newly infected Iranians, no data exist about the rate of these primary resistance mutations among the ART-naïve, chronically infected individuals who are, in fact, the main candidates for ART initiation. To address this issue, we collected blood samples from 40 ART-naïve injection drug-users (IDUs) with chronic HIV-1 infection (seroconversion time ranging from 2 to 9 years) living in Sanandaj, Iran, followed by sequencing of the protease and reverse-transcriptase regions from their HIV-1 genome. Phylogenetic analyses of the sequenced regions revealed that all samples were CRF35_AD. Transmitted resistance mutations were interpreted as surveillance drug-resistant mutations (SDRMs) based on the world health organization (WHO) algorithm. The frequency of SDRMs to any class of antiretroviral drugs was 15%, which included mutations to nucleoside reverse transcriptase inhibitors (NRTIs, 10%), with M41L and M184V as the most common (5%), and non-nucleoside reverse transcriptase inhibitors (NNRTIs, 5%), with K103N as the only detected mutation (5%). Although not in the WHO SDRMs list, several minor protease inhibitor resistant mutations listed in the International Antiviral Society-USA panel were identified, of which M36I, H69K, L89M/V/I (each one 100%) and K20R/T (92.5%) can be considered as polymorphic signatures for CRF35_AD.The relatively high rate of TDR mutations in our study raises concerns about the risk of treatment failure in chronically infected IDUs of Sanandaj city. These results suggest that routine resistance testing should be considered before the therapy initiation in this area. Additional surveillance studies are required to generalize this deduction to other cities of Iran. PMID:25962088

  9. Transmitted Drug Resistance Mutations in Antiretroviral-Naïve Injection Drug Users with Chronic HIV-1 Infection in Iran.

    PubMed

    Memarnejadian, Arash; Menbari, Shahoo; Mansouri, Seyed Ali; Sadeghi, Leila; Vahabpour, Rouhollah; Aghasadeghi, Mohammad Reza; Mostafavi, Ehsan; Abdi, Mohammad

    2015-01-01

    The growing incidence and transmission of drug resistant HIV-1 strains due to widespread use of antiretroviral therapy (ART) can jeopardize the success of first-line ART. While there is a known moderate prevalence of transmitted drug resistance (TDR) among newly infected Iranians, no data exist about the rate of these primary resistance mutations among the ART-naïve, chronically infected individuals who are, in fact, the main candidates for ART initiation. To address this issue, we collected blood samples from 40 ART-naïve injection drug-users (IDUs) with chronic HIV-1 infection (seroconversion time ranging from 2 to 9 years) living in Sanandaj, Iran, followed by sequencing of the protease and reverse-transcriptase regions from their HIV-1 genome. Phylogenetic analyses of the sequenced regions revealed that all samples were CRF35_AD. Transmitted resistance mutations were interpreted as surveillance drug-resistant mutations (SDRMs) based on the world health organization (WHO) algorithm. The frequency of SDRMs to any class of antiretroviral drugs was 15%, which included mutations to nucleoside reverse transcriptase inhibitors (NRTIs, 10%), with M41L and M184V as the most common (5%), and non-nucleoside reverse transcriptase inhibitors (NNRTIs, 5%), with K103N as the only detected mutation (5%). Although not in the WHO SDRMs list, several minor protease inhibitor resistant mutations listed in the International Antiviral Society-USA panel were identified, of which M36I, H69K, L89M/V/I (each one 100%) and K20R/T (92.5%) can be considered as polymorphic signatures for CRF35_AD.The relatively high rate of TDR mutations in our study raises concerns about the risk of treatment failure in chronically infected IDUs of Sanandaj city. These results suggest that routine resistance testing should be considered before the therapy initiation in this area. Additional surveillance studies are required to generalize this deduction to other cities of Iran. PMID:25962088

  10. Cytotoxic T cell recognition of an HIV-1 reverse transcriptase variant peptide incorporating the K103N drug resistance mutation

    PubMed Central

    Mahnke, Lisa; Clifford, David

    2006-01-01

    During HIV-1 infection, cytotoxic T cell (CTL) responses exert strong selective pressure on the replicating virus population. Here we report evidence for T cell activity against the drug resistant K103N region of viral reverse transcriptase in three HIV-1 infected patients exposed to NNRTI antiretroviral drugs. We further characterize the response in one patient by ELISPOT analysis. A nine amino acid peptide incorporating 103N was recognized by patient T cells whereas the wild type was not. The RT K103N mutation is selected by the NNRTI class of HIV drugs. We hypothesize that, in certain individuals, CTL responses against 103N-containing epitopes may protect against NNRTI drug resistance. Characterizing such responses in the context of HLA subtypes could lead to tailored HIV drug therapy or to the design of therapeutic vaccines. PMID:16970827

  11. Neutralizing antibody and anti-retroviral drug sensitivities of HIV-1 isolates resistant to small molecule CCR5 inhibitors

    SciTech Connect

    Pugach, Pavel; Ketas, Thomas J.; Michael, Elizabeth; Moore, John P.

    2008-08-01

    The small molecule CCR5 inhibitors are a new class of drugs for treating infection by human immunodeficiency virus type 1 (HIV-1). They act by binding to the CCR5 co-receptor and preventing its use during HIV-1-cell fusion. Escape mutants can be raised against CCR5 inhibitors in vitro and will arise when these drugs are used clinically. Here, we have assessed the responses of CCR5 inhibitor-resistant viruses to other anti-retroviral drugs that act by different mechanisms, and their sensitivities to neutralizing antibodies (NAbs). The rationale for the latter study is that the resistance pathway for CCR5 inhibitors involves changes in the HIV-1 envelope glycoproteins (Env), which are also targets for NAbs. The escape mutants CC101.19 and D1/85.16 were selected for resistance to AD101 and vicriviroc (VVC), respectively, from the primary R5 HIV-1 isolate CC1/85. Each escape mutant was cross-resistant to other small molecule CCR5 inhibitors (aplaviroc, maraviroc, VVC, AD101 and CMPD 167), but sensitive to protein ligands of CCR5: the modified chemokine PSC-RANTES and the humanized MAb PRO-140. The resistant viruses also retained wild-type sensitivity to the nucleoside reverse transcriptase inhibitor (RTI) zidovudine, the non-nucleoside RTI nevirapine, the protease inhibitor atazanavir and other attachment and fusion inhibitors that act independently of CCR5 (BMS-806, PRO-542 and enfuvirtide). Of note is that the escape mutants were more sensitive than the parental CC1/85 isolate to a subset of neutralizing monoclonal antibodies and to some sera from HIV-1-infected people, implying that sequence changes in Env that confer resistance to CCR5 inhibitors can increase the accessibility of some NAb epitopes. The need to preserve NAb resistance may therefore be a constraint upon how escape from CCR5 inhibitors occurs in vivo.

  12. HIV-1 Drug Resistance Mutations Among Antiretroviral-Naïve HIV-1–Infected Patients in Asia: Results From the TREAT Asia Studies to Evaluate Resistance-Monitoring Study

    PubMed Central

    Oyomopito, Rebecca; Sirivichayakul, Sunee; Sirisanthana, Thira; Kantipong, Pacharee; Lee, Christopher K. C.; Kamarulzaman, Adeeba; Messerschmidt, Liesl; Law, Matthew G.; Phanuphak, Praphan

    2011-01-01

    (See editorial commentary by Jordan on pages 1058–1060.) Of 682 antiretroviral-naïve patients initiating antiretroviral therapy in a prospective, multicenter human immunodeficiency virus type 1 (HIV-1) drug resistance monitoring study involving 8 sites in Hong Kong, Malaysia, and Thailand, the prevalence of patients with ≥1 drug resistance mutation was 13.8%. Primary HIV drug resistance is emerging after rapid scaling-up of antiretroviral therapy use in Asia. PMID:21460324

  13. Comparisons of Primary HIV-1 Drug Resistance between Recent and Chronic HIV-1 Infection within a Sub-Regional Cohort of Asian Patients

    PubMed Central

    Kiertiburanakul, Sasisopin; Chaiwarith, Romanee; Sirivichayakul, Sunee; Ditangco, Rossana; Jiamsakul, Awachana; Li, Patrick C. K.; Kantipong, Pacharee; Lee, Christopher; Ratanasuwan, Winai; Kamarulzaman, Adeeba; Sohn, Annette H.; Sungkanuparph, Somnuek

    2013-01-01

    Background The emergence and transmission of HIV-1 drug resistance (HIVDR) has raised concerns after rapid global antiretroviral therapy (ART) scale-up. There are limited data on the epidemiology of primary HIVDR in resource-limited settings in Asia. We aimed to determine the prevalence and compare the distribution of HIVDR in a cohort of ART-naïve Asian patients with recent and chronic HIV-1 infection. Methods Multicenter prospective study was conducted in ART-naïve patients between 2007 and 2010. Resistance-associated mutations (RAMs) were assessed using the World Health Organization 2009 list for surveillance of primary HIVDR. Results A total of 458 patients with recent and 1,340 patients with chronic HIV-1 infection were included in the analysis. The overall prevalence of primary HIVDR was 4.6%. Recently infected patients had a higher prevalence of primary HIVDR (6.1% vs. 4.0%, p = 0.065) and frequencies of RAMs to protease inhibitors (PIs; 3.9% vs. 1.0%, p<0.001). Among those with recent infection, the most common RAMs to nucleoside reverse transcriptase inhibitors (NRTIs) were M184I/V and T215D/E/F/I/S/Y (1.1%), to non-NRTIs was Y181C (1.3%), and to PIs was M46I (1.5%). Of patients with chronic infection, T215D/E/F/I/S/Y (0.8%; NRTI), Y181C (0.5%; non-NRTI), and M46I (0.4%; PI) were the most common RAMs. K70R (p = 0.016) and M46I (p = 0.026) were found more frequently among recently infected patients. In multivariate logistic regression analysis in patients with chronic infection, heterosexual contact as a risk factor for HIV-1 infection was less likely to be associated with primary HIVDR compared to other risk categories (odds ratio 0.34, 95% confidence interval 0.20–0.59, p<0.001). Conclusions The prevalence of primary HIVDR was higher among patients with recent than chronic HIV-1 infection in our cohort, but of borderline statistical significance. Chronically infected patients with non-heterosexual risks for HIV were more likely to have

  14. An allosteric modulator of HIV-1 protease shows equipotent inhibition of wild-type and drug-resistant proteases.

    PubMed

    Ung, Peter M-U; Dunbar, James B; Gestwicki, Jason E; Carlson, Heather A

    2014-08-14

    NMR and MD simulations have demonstrated that the flaps of HIV-1 protease (HIV-1p) adopt a range of conformations that are coupled with its enzymatic activity. Previously, a model was created for an allosteric site located between the flap and the core of HIV-1p, called the Eye site (Biopolymers 2008, 89, 643-652). Here, results from our first study were combined with a ligand-based, lead-hopping method to identify a novel compound (NIT). NIT inhibits HIV-1p, independent of the presence of an active-site inhibitor such as pepstatin A. Assays showed that NIT acts on an allosteric site other than the dimerization interface. MD simulations of the ligand-protein complex show that NIT stably binds in the Eye site and restricts the flaps. That bound state of NIT is consistent with a crystal structure of similar fragments bound in the Eye site (Chem. Biol. Drug Des. 2010, 75, 257-268). Most importantly, NIT is equally potent against wild-type and a multidrug-resistant mutant of HIV-1p, which highlights the promise of allosteric inhibitors circumventing existing clinical resistance. PMID:25062388

  15. Human APOBEC3G drives HIV-1 evolution and the development of drug resistance

    SciTech Connect

    Bhattacharya, Tamoy; Kim, Eun - Young; Koning, Fransje; Malim, Michael; Wolinsky, Steven M

    2008-01-01

    Human APOBEC3G (hA3G) is an innate virus restriction factor that induces deamination of specific cytidine residues in single-stranded human immunodeficiency virus type 1 (HIV-1) DNA. Whereas destructive hA3G editing leads to a profound loss of HIV-1 infectivity, more limited editing could be a source of adaptation and diversification. Here we show that the presence of hA3G in T-cells can drive the development of diversity in HIV-1 populations and that under selection pressure imposed by the nucleotide analog reverse transcriptase inhibitor 3TC ((-)2',3'-dideoxy-3'-thiacytidine), a single point mutation that confers 3TC resistance, methionine 184 to isoleucine (M1841), emerges rapidly and reaches fixation. These results provide strong evidence that mutation by hA3G is an important source of genetic variation on which natural selection acts to shape the structure of the viral population and drive the tempo of HIV-1 evolution.

  16. More effective drugs lead to harder selective sweeps in the evolution of drug resistance in HIV-1

    PubMed Central

    Feder, Alison F; Rhee, Soo-Yon; Holmes, Susan P; Shafer, Robert W; Petrov, Dmitri A; Pennings, Pleuni S

    2016-01-01

    In the early days of HIV treatment, drug resistance occurred rapidly and predictably in all patients, but under modern treatments, resistance arises slowly, if at all. The probability of resistance should be controlled by the rate of generation of resistance mutations. If many adaptive mutations arise simultaneously, then adaptation proceeds by soft selective sweeps in which multiple adaptive mutations spread concomitantly, but if adaptive mutations occur rarely in the population, then a single adaptive mutation should spread alone in a hard selective sweep. Here, we use 6717 HIV-1 consensus sequences from patients treated with first-line therapies between 1989 and 2013 to confirm that the transition from fast to slow evolution of drug resistance was indeed accompanied with the expected transition from soft to hard selective sweeps. This suggests more generally that evolution proceeds via hard sweeps if resistance is unlikely and via soft sweeps if it is likely. DOI: http://dx.doi.org/10.7554/eLife.10670.001 PMID:26882502

  17. Time trends in drug resistant HIV-1 infections in the United Kingdom up to 2009: multicentre observational study

    PubMed Central

    2012-01-01

    Objective To evaluate whether the prevalence of HIV-1 transmitted drug resistance has continued to decline in infections probably acquired within the United Kingdom. Design Multicentre observational study. Setting All UK public laboratories conducting tests for genotypic HIV resistance as a part of routine care. Participants 14 584 patients infected with HIV-1 subtype B virus, who were first tested for resistance before receiving antiretroviral therapy between January 2002 and December 2009. Main outcome measure Prevalence of transmitted drug resistance, defined as one or more resistance mutations from the surveillance list recommended by the World Health Organization. Results 1654 (11.3%, 95% confidence interval 10.8% to 11.9%) patients had one or more mutations associated with transmitted HIV-1 drug resistance; prevalence was found to decline from 15.5% in 2002 to 9.6% in 2007, followed by a slight increase to 10.9% in 2009 (P=0.21). This later rise was mainly a result of increases in resistance to nucleos(t)ide reverse transcriptase inhibitors (from 5.4% in 2007 to 6.6% in 2009, P=0.24) and protease inhibitors (1.5% to 2.1%, P=0.12). Thymidine analogue mutations, including T215 revertants, remained the most frequent mutations associated with nucleos(t)ide reverse transcriptase inhibitors, despite a considerable fall in stavudine and zidovudine use between 2002 and 2009 (from 29.4% of drug regimens in 2002 to 0.8% in 2009, from 47.9% to 8.8%, respectively). Conclusions The previously observed decline in the prevalence of transmitted drug resistance in HIV-1 infections probably acquired in the UK seems to have stabilised. The continued high prevalence of thymidine analogue mutations suggests that the source of this resistance may be increasingly from patients who have not undergone antiretroviral therapy and who harbour resistant viruses. Testing of all newly diagnosed HIV-1 positive people should be continued. PMID:22915687

  18. Effects of drug-resistant mutations on the dynamic properties of HIV-1 protease and inhibition by Amprenavir and Darunavir

    PubMed Central

    Yu, Yuqi; Wang, Jinan; Shao, Qiang; Shi, Jiye; Zhu, Weiliang

    2015-01-01

    Molecular dynamics simulations are performed to investigate the dynamic properties of wild-type HIV-1 protease and its two multi-drug-resistant variants (Flap + (L10I/G48V/I54V/V82A) and Act (V82T/I84V)) as well as their binding with APV and DRV inhibitors. The hydrophobic interactions between flap and 80 s (80’s) loop residues (mainly I50-I84’ and I50’-I84) play an important role in maintaining the closed conformation of HIV-1 protease. The double mutation in Act variant weakens the hydrophobic interactions, leading to the transition from closed to semi-open conformation of apo Act. APV or DRV binds with HIV-1 protease via both hydrophobic and hydrogen bonding interactions. The hydrophobic interactions from the inhibitor is aimed to the residues of I50 (I50’), I84 (I84’), and V82 (V82’) which create hydrophobic core clusters to further stabilize the closed conformation of flaps, and the hydrogen bonding interactions are mainly focused with the active site of HIV-1 protease. The combined change in the two kinds of protease-inhibitor interactions is correlated with the observed resistance mutations. The present study sheds light on the microscopic mechanism underlying the mutation effects on the dynamics of HIV-1 protease and the inhibition by APV and DRV, providing useful information to the design of more potent and effective HIV-1 protease inhibitors. PMID:26012849

  19. Influence of Drug Resistance Mutations on the Activity of HIV-1 Subtypes A and B Integrases: a Comparative Study

    PubMed Central

    Shadrina, O. A.; Zatsepin, T. S.; Agapkina, Yu. Yu.; Isaguliants, M. G.; Gottikh, M. B.

    2015-01-01

    Integration of human immunodeficiency virus (HIV-1) DNA into the genome of an infected cell is one of the key steps in the viral replication cycle. The viral enzyme integrase (IN), which catalyzes the integration, is an attractive target for the development of new antiviral drugs. However, the HIV-1 therapy often results in the IN gene mutations inducing viral resistance to integration inhibitors. To assess the impact of drug resistance mutations on the activity of IN of HIV-1 subtype A strain FSU-A, which is dominant in Russia, variants of the consensus IN of this subtype containing the primary resistance mutations G118R and Q148K and secondary compensatory substitutions E138K and G140S were prepared and characterized. Comparative study of these enzymes with the corresponding mutants of IN of HIV-1 subtype B strains HXB-2 was performed. The mutation Q148K almost equally reduced the activity of integrases of both subtypes. Its negative effect was partially compensated by the secondary mutations E138K and G140S. Primary substitution G118R had different influence on the activity of proteins of the subtypes A and B, and the compensatory effect of the secondary substitution E138K also depended on the viral subtype. Comparison of the mutants resistance to the known strand transfer inhibitors raltegravir and elvitegravir, and a new inhibitor XZ-259 (a dihydro-1H-isoindol derivative), showed that integrases of both subtypes with the Q148K mutation were insensitive to raltegravir and elvitegravir but were effectively inhibited by XZ-259. The substitution G118R slightly reduced the efficiency of IN inhibition by raltegravir and elvitegravir and caused no resistance to XZ_259. PMID:25927004

  20. HIV-1 Phenotypic Reverse Transcriptase Inhibitor Drug Resistance Test Interpretation Is Not Dependent on the Subtype of the Virus Backbone

    PubMed Central

    Bronze, Michelle; Steegen, Kim; Wallis, Carole L.; De Wolf, Hans; Papathanasopoulos, Maria A.; Van Houtte, Margriet; Stevens, Wendy S.; de Wit, Tobias Rinke; Stuyver, Lieven J.

    2012-01-01

    To date, the majority of HIV-1 phenotypic resistance testing has been performed with subtype B virus backbones (e.g. HXB2). However, the relevance of using this backbone to determine resistance in non-subtype B HIV-1 viruses still needs to be assessed. From 114 HIV-1 subtype C clinical samples (36 ARV-naïve, 78 ARV-exposed), pol amplicons were produced and analyzed for phenotypic resistance using both a subtype B- and C-backbone in which the pol fragment was deleted. Phenotypic resistance was assessed in resulting recombinant virus stocks (RVS) for a series of antiretroviral drugs (ARV's) and expressed as fold change (FC), yielding 1660 FC comparisons. These Antivirogram® derived FC values were categorized as having resistant or sensitive susceptibility based on biological cut-off values (BCOs). The concordance between resistance calls obtained for the same clinical sample but derived from two different backbones (i.e. B and C) accounted for 86.1% (1429/1660) of the FC comparisons. However, when taking the assay variability into account, 95.8% (1590/1660) of the phenotypic data could be considered as being concordant with respect to their resistance call. No difference in the capacity to detect resistance associated with M184V, K103N and V106M mutations was noted between the two backbones. The following was concluded: (i) A high level of concordance was shown between the two backbone phenotypic resistance profiles; (ii) Assay variability is largely responsible for discordant results (i.e. for FC values close to BCO); (iii) Confidence intervals should be given around the BCO's, when assessing resistance in HIV-1 subtype C; (iv) No systematic resistance under- or overcalling of subtype C amplicons in the B-backbone was observed; (v) Virus backbone subtype sequence variability outside the pol region does not contribute to phenotypic FC values. In conclusion the HXB2 virus backbone remains an acceptable vector for phenotyping HIV-1 subtype C pol amplicons. PMID

  1. High frequency of antiviral drug resistance and non-b subtypes in HIV-1 patients failing antiviral therapy in Cuba

    PubMed Central

    Kouri, Vivian; Alemán, Yoan; Pérez, Lissette; Pérez, Jorge; Fonseca, Carlos; Correa, Consuelo; Aragonés, Carlos; Campos, Jorge; Álvarez, Delmis; Schrooten, Yoeri; Vinken, Lore; Limia, Celia; Soto, Yudira; Vandamme, Anne-Mieke; Van Laethem, Kristel

    2014-01-01

    Introduction Emergence of HIV-1 drug resistance may limit the sustained benefits of antiretroviral therapy (ART) in settings with limited laboratory monitoring and drug options. The objective is to implement the surveillance of drug resistance and subtypes in HIV-1 patients failing ART in Cuba. Methods This study compiled clinical and genotypic drug resistance data 588 ART-experienced HIV-1 patients attending a clinical center in Havana in 2009–2013. Drug resistance testing was performed as part of routine clinical care. Drug resistance mutations and levels were determined using Rega version 8.0.2. Results Eighty-three percent received solely ART containing at least three drugs. Patients from 2009 to 2010 were longer treated (median: 4.9 vs 2.7 years) and exposed to more ART regimens (median: 4 vs 2 regimens) compared to patients from 2011–2013. Nucleoside reverse transcriptase inhibitor (NRTI), non-nucleoside RTI (NNRTI) and PI mutations were present in 83.5, 77.4 and 52.0%. Full-class resistance (FCR) to NRTI, NNRTI, PI and multidrug resistance (MDR) were detected in 25.0, 33.7, 11.4 and 6.3%. FCR to NRTI, NNRTI, PI and MDR were present in 12.8, 28.7, 0 and 0% after first-line failure (164 patients) and in 23.1, 34.6, 3.8 and 3.1% after second-line failure (130 patients). Subtype B (32.5%), BG recombinants (19.6%) and CRF19_cpx (16.2%) were the most prevalent genetic forms. Subtype distribution did not change significantly between 2009–2010 and 2011–2013, except for BG recombinants that increased from 12.2 to 21.3% (p=0.002). Conclusions Our study found a high prevalence of drug resistance and supports the need for appropriate laboratory monitoring in clinical practice and access to drug options in case of virological failure. PMID:25397499

  2. Prevalence of Transmitted Drug Resistance Mutations in HIV-1-Infected Drug-Naive Patients from Urban and Suburban Regions of Kenya.

    PubMed

    Onsongo, Simon; Abidi, Syed Hani; Khamadi, Samoel; Shah, Reena; Kageha, Sheila; Ojwang, Peter; Ali, Syed; Okinda, Nancy

    2016-03-01

    HIV was first described in Kenya in 1984-1985. Currently, Kenya has an estimated HIV-1 prevalence of 6.2%. With the introduction of antiretroviral drugs, the survival of most HIV patients has been prolonged markedly. However, this is greatly threatened by increasing rates of antiretroviral dug resistance, which may eventually lead to suboptimal treatment outcomes. The objective of this study was to characterize currently occurring antiretroviral drug resistance mutations among drug-naive patients visiting two referral hospitals in Kenya. Using polymerase chain reaction, the HIV protease gene was amplified from blood samples of 63 study participants. The sequences were used to determine HIV-1 subtype and presence/prevalence of mutations associated with resistance to protease inhibitors. Finally, the protease gene was variably measured using Shannon entropy analysis. Analysis of frequency of HIV-1 subtypes revealed subtype A to be the predominant subtype, while the analysis of drug resistance mutations revealed the presence of four minor drug resistance mutations associated weakly with resistance to protease inhibitors. Among these mutations, L33I was the most prevalent mutation. Shannon entropy analysis revealed high genomic variability, especially in region spanning nucleotides 1-55, 113-170, and 205-240. This study warrants the need for dedicated efforts to improve compliance to antiretroviral therapy and reduce transmitted resistance rates, which will greatly ensure the therapeutic efficacy of antiretroviral drugs. PMID:26401720

  3. Treatment of human immunodeficiency virus type 1 (HIV-1)-infected cells with combinations of HIV-1-specific inhibitors results in a different resistance pattern than does treatment with single-drug therapy.

    PubMed Central

    Balzarini, J; Karlsson, A; Pérez-Pérez, M J; Camarasa, M J; Tarpley, W G; De Clercq, E

    1993-01-01

    Human immunodeficiency virus type 1 (HIV-1)-infected CEM cells were treated by the HIV-1-specific inhibitors bis-heteroarylpiperazine (BHAP), 4,5,6,7-tetrahydro-5-methylimidazo[4,5,1-jk][1,4]benzodiazepin-2(1 H)-on e (TIBO) R82913, nevirapine, and the N3-methylthymine derivative of [2',5'-bis-O-(tert-butyldimethylsilyl)-beta-D-ribofuranosyl]-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide) (TSAO-m3T), as single agents or in combination, at escalating concentrations. When used individually, the compounds led to the emergence of drug-resistant virus strains within two to five subcultivations. The resulting strains were designated HIV-1/BHAP, HIV-1/TIBO, HIV-1/Nev, and HIV-1/TSAO-m3T, respectively. The mutant viruses showed the following amino acid substitutions in their reverse transcriptase (RT): Leu-100-->Ile for HIV-1/BHAP; Lys-103-->Asn for HIV-1/TIBO; Val-106-->Ala for HIV-1/Nev; and Glu-138-->Lys for HIV-1/TSAO-m3T. Both the Tyr-181-->Cys and Val-106-->Ala mutations were found in another mutant emerging following treatment with nevirapine at escalating concentrations. The BHAP-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and TSAO-m3T, whereas the TSAO-m3T-resistant virus remained fully sensitive to the inhibitory effects of nevirapine and BHAP. When different pairs of nonnucleoside RT inhibitors (i.e., BHAP plus TSAO-m3T, nevirapine plus TSAO-m3T, TIBO plus TSAO-m3T, nevirapine plus TIBO, and BHAP plus nevirapine) were used, resistant virus emerged as fast as with single-drug therapy. In all cases the Tyr-181-->Cys mutation appeared; the virus showed markedly reduced sensitivity to all HIV-1-specific inhibitors but retained sensitivity to 2',3'-dideoxynucleoside analogs such as zidovudine, ddC, and ddI. Our findings argue against simultaneous combination of two different nonnucleoside RT inhibitors that are unable to inhibit HIV-1 mutant strains containing the Tyr-181-->Cys mutation when administered as single

  4. HIV-1 Genetic Characteristics and Transmitted Drug Resistance among Men Who Have Sex with Men in Kunming, China

    PubMed Central

    Chen, Min; Ma, Yanling; Su, Yingzhen; Yang, Li; Zhang, Renzhong; Yang, Chaojun; Chen, Huichao; Yan, Wenyun; Shi, Yuhua; Dong, Lijuan; Chen, Ling; Jia, Manhong; Lu, Lin

    2014-01-01

    Background Yunnan has been severely affected by HIV/AIDS in China. Recently, the reported prevalence of HIV-1 among men who have sex with men (MSM) in Yunnan was high in China. To monitor dynamic HIV-1 epidemic among Yunnan MSM, HIV-1 genetic characteristics and transmitted drug resistance (TDR) were investigated. Methods Blood samples from 131 newly HIV-1 diagnosed MSM were continuously collected at fixed sites from January 2010 to December 2012 in Kunming City, Yunnan Province. Partial gag, pol and env genes were sequenced. Phylogenetic, evolutionary and genotypic drug resistance analyses were performed. Results Multiple genotypes were identified among MSM in Kunming, including CRF01_AE (64.9%), CRF07_BC (25.2%), unique recombinant forms (URFs, 5.3%), subtype B (3.1%) and CRF08_BC (1.5%). CRF01_AE and CRF07_BC were the predominant strains. The mean of genetic distance within CRF01_AE were larger than that within CRF07_BC. The estimated introducing time of CRF01_AE in Yunnan MSM (1996.9) is earlier than that of CRF07_BC (2002.8). In this study, subtype B was first identified in Yunnan MSM. CRF08_BC seems to be the distinctive strain in Yunnan MSM, which was seldom found among MSM outside Yunnan. The proportion of URFs increased, which further contributed to genetic diversity among MSM. Strikingly, genetic relatedness was found among these strains with MSM isolates from multiple provinces, which suggested that a nationwide transmission network may exist. TDR-associated mutations were identified in 4.6% individuals. The multivariate analysis revealed that non-native MSM and divorced/widowed MSM were independently associated with a higher TDR rate. Conclusion This work revealed diverse HIV-1 genetics, national transmission networks and a baseline level of TDR in MSM. These findings enhance our understanding of the distribution and evolution of HIV-1 in MSM, and are valuable for developing HIV prevention strategies for MSM. PMID:24489829

  5. Insights into the molecular mechanism of inhibition and drug resistance for HIV-1 RT with carbovir triphosphate.

    PubMed

    Ray, Adrian S; Yang, Zhenjun; Shi, Junxing; Hobbs, Ann; Schinazi, Raymond F; Chu, Chung K; Anderson, Karen S

    2002-04-23

    understanding of the molecular mechanism of inhibition and drug resistance led to the discovery of a novel prodrug of D4G. This compound shows promise as a potent antiviral especially with the drug resistant M184V HIV-1 RT that is so often encountered in a clinical setting. PMID:11955063

  6. Mechanism of Drug Resistance Revealed by the Crystal Structure of the Unliganded HIV-1 Protease with F53L Mutation

    SciTech Connect

    Liu, Fengling; Kovalevsky, Andrey Y.; Louis, John M.; Boross, Peter I.; Wang, Yuan-Fang; Harrison, Robert W.; Weber, Irene T.

    2010-12-03

    Mutations in HIV-1 protease (PR) that produce resistance to antiviral PR inhibitors are a major problem in AIDS therapy. The mutation F53L arising from antiretroviral therapy was introduced into the flexible flap region of the wild-type PR to study its effect and potential role in developing drug resistance. Compared to wild-type PR, PR{sub F53L} showed lower (15%) catalytic efficiency, 20-fold weaker inhibition by the clinical drug indinavir, and reduced dimer stability, while the inhibition constants of two peptide analog inhibitors were slightly lower than those for PR. The crystal structure of PR{sub F53L} was determined in the unliganded form at 1.35 {angstrom} resolution in space group P4{sub 1}2{sub 1}2. The tips of the flaps in PR{sub F53L} had a wider separation than in unliganded wild-type PR, probably due to the absence of hydrophobic interactions of the side-chains of Phe53 and Ile50{prime}. The changes in interactions between the flaps agreed with the reduced stability of PR{sub F53L} relative to wild-type PR. The altered flap interactions in the unliganded form of PR{sub F53L} suggest a distinct mechanism for drug resistance, which has not been observed in other common drug-resistant mutants.

  7. HIV-1 Genetic Diversity and Transmitted Drug Resistance Among Recently Infected Individuals at Men Who Have Sex with Men Sentinel Surveillance Points in Hebei Province, China.

    PubMed

    Lu, Xinli; Kang, Xianjiang; Chen, Suliang; Zhao, Hongru; Liu, Yongjian; Zhao, Cuiying; Zhang, Yuqi; Li, Jingyun; Cui, Ze; Wang, Xianfeng

    2015-10-01

    For this study, 50 HIV-1 plasma samples of recently infected men who have sex with men (MSM) were amplified and sequenced. Multiple subtypes were identified by phylogenetic analyses of HIV-1 gag, env, and pol gene regions, including CRF01_AE (56.0%), CRF07_BC (30.0%), subtype B (12.0%), and unique recombinant forms (URFs, 6.0%). CRF01_AE was the most frequent genotype in the epidemic. Three recombination patterns of URFs were identified: 01BC, 01B, and 01C. The rate of HIV-1 transmitted drug resistance (TDR) mutation (M46L) was 2.08% (1/48). URFs and TDR first identified in this study suggest that HIV-1 prevalence is more and more complicated, and HIV-1 drug-resistant strains have begun to spread among at risk populations in Hebei. Our findings can provide vital information for an efficient surveillance system and strategic HIV prevention and control measures in China by revealing the evolutionary status and HIV-1 TDR of HIV-1 strains among recently infected MSM in Hebei Province. PMID:26200883

  8. Use of Dried Plasma Spots for HIV-1 Viral Load Determination and Drug Resistance Genotyping in Mexican Patients

    PubMed Central

    Rodriguez-Auad, Juan Pablo; Rojas-Montes, Othon; Maldonado-Rodriguez, Angelica; Alvarez-Muñoz, Ma. Teresa; Muñoz, Onofre; Torres-Ibarra, Rocio; Vazquez-Rosales, Guillermo

    2015-01-01

    Monitoring antiretroviral therapy using measurements of viral load (VL) and the genotyping of resistance mutations is not routinely performed in low- to middle-income countries because of the high costs of the commercial assays that are used. The analysis of dried plasma spot (DPS) samples on filter paper may represent an alternative for resource-limited settings. Therefore, we evaluated the usefulness of analyzing DPS samples to determine VL and identify drug resistance mutations (DRM) in a group of HIV-1 patients. The VL was measured from 22 paired plasma and DPS samples. In these samples, the average VL was 4.7 log10 copies/mL in liquid plasma and 4.1 log10 copies/mL in DPS, with a correlation coefficient of R = 0.83. A 1.1 kb fragment of HIV pol could be amplified in 14/22 (63.6%) of the DPS samples and the same value was amplified in plasma samples. A collection of ten paired DPS and liquid plasma samples was evaluated for the presence of DRM; an excellent correlation was found in the identification of DRM between the paired samples. All HIV-1 pol sequences that were obtained corresponded to HIV subtype B. The analysis of DPS samples offers an attractive alternative for monitoring ARV therapy in resource-limited settings. PMID:26779533

  9. Pretreatment HIV-1 drug resistance is strongly associated with virologic failure in HIV-infected patients receiving partly active antiretroviral regimens

    PubMed Central

    Parikh, Urvi M; Mellors, John W

    2016-01-01

    The scale-up of antiretroviral therapy in sub-Saharan Africa has significantly reduced mortality from AIDS. Hamers et al. explores the impact of pretreatment HIV-1 drug resistance on virologic failure, immunologic response, and acquisition of drug resistance after 1 year of first-line antiretroviral therapy by longitudinally evaluating 2579 participants with pretreatment drug resistance results from the PharmAccess African Studies to Evaluate Resistance Monitoring (PASER-M) cohort. Participants with pretreatment drug resistance who were given a treatment regimen with reduced activity to at least one prescribed drug had a significantly greater risk of virologic failure and acquired drug resistance compared with both participants without pretreatment drug resistance, and participants with pretreatment drug resistance who were prescribed fully active regimens. This paper by Hamers et al. validates the need for at least three fully active antiretroviral drugs to prevent the acquisition of drug resistance and to optimize treatment success in resource limited settings. PMID:22913352

  10. HIV-1 protease inhibitors from inverse design in the substrate envelope exhibit subnanomolar binding to drug-resistant variants.

    PubMed

    Altman, Michael D; Ali, Akbar; Reddy, G S Kiran Kumar; Nalam, Madhavi N L; Anjum, Saima Ghafoor; Cao, Hong; Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X; Gilson, Michael K; Schiffer, Celia A; Rana, Tariq M; Tidor, Bruce

    2008-05-14

    The acquisition of drug-resistant mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from a Ki of 30-50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6-13-fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors: robust binders (maximum affinity loss of 14-16-fold), moderate binders (35-80-fold), and susceptible binders (greater than 100-fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance. PMID:18412349

  11. HIV-1 Protease Inhibitors from Inverse Design in the Substrate Envelope Exhibit Subnanomolar Binding to Drug-Resistant Variants

    PubMed Central

    Altman, Michael D.; Ali, Akbar; Reddy, G. S. Kiran Kumar; Nalam, Madhavi N. L.; Anjum, Saima Ghafoor; Cao, Hong; Chellappan, Sripriya; Kairys, Visvaldas; Fernandes, Miguel X.; Gilson, Michael K.; Schiffer, Celia A.; Rana, Tariq M.; Tidor, Bruce

    2010-01-01

    The acquisition of drug-resistance mutations by infectious pathogens remains a pressing health concern, and the development of strategies to combat this threat is a priority. Here we have applied a general strategy, inverse design using the substrate envelope, to develop inhibitors of HIV-1 protease. Structure-based computation was used to design inhibitors predicted to stay within a consensus substrate volume in the binding site. Two rounds of design, synthesis, experimental testing, and structural analysis were carried out, resulting in a total of 51 compounds. Improvements in design methodology led to a roughly 1000-fold affinity enhancement to a wild-type protease for the best binders, from Ki of 30–50 nM in round one to below 100 pM in round two. Crystal structures of a subset of complexes revealed a binding mode similar to each design that respected the substrate envelope in nearly all cases. All four best binders from round one exhibited broad specificity against a clinically relevant panel of drug-resistant HIV-1 protease variants, losing no more than 6–13 fold affinity relative to wild type. Testing a subset of second-round compounds against the panel of resistant variants revealed three classes of inhibitors — robust binders (maximum affinity loss of 14–16 fold), moderate binders (35–80 fold), and susceptible binders (greater than 100 fold). Although for especially high-affinity inhibitors additional factors may also be important, overall, these results suggest that designing inhibitors using the substrate envelope may be a useful strategy in the development of therapeutics with low susceptibility to resistance. PMID:18412349

  12. Transmitted drug resistance in women with intrapartum HIV-1 diagnosis: a pilot epidemiological survey in Buenos Aires, Argentina

    PubMed Central

    Cecchini, Diego; Zapiola, Ines; Fernandez Giuliano, Silvina; Martinez, Marina; Rodriguez, Claudia; Belen Bouzas, Maria

    2014-01-01

    Introduction Surveillance of primary resistance to antiretroviral drugs is particularly important in pregnant population, in which infection by drug-resistant HIV has not only implications for maternal treatment, but could also jeopardize the efficacy of neonatal prophylaxis. We aim to describe the prevalence of resistance associated mutations (RAMs) in pregnant women with intrapartum HIV diagnosis in a public hospital of Buenos Aires, Argentina. Materials and Methods Prospective pilot study (period from 2008 to October 2013). Plasma samples were tested for viral load by Versant HIV-1 RNA 3.0 (bDNA) and sequenced using HIV-1 TRUGENE™Genotyping Kit (Siemens). The prevalence of RAMs was analyzed according to World Health Organization (WHO) criteria. Results Of 231 HIV-infected pregnant women assisted, 6% (n=14) had intrapartum diagnosis of HIV infection. 12 patients (85.7%) had previous pregnancies, 10 (71.4%) had inadequate prenatal care and 3 (23.1%) seroconverted during pregnancy. Maternal characteristics (expressed medians and ranges) were: age 25.5 (16–35) years; gestational age at birth: 39 (30–42) weeks; CD4 count: 500 (132–925) cells/µL; viral load: 9418 (1800–55299) copies/mL. No one had hepatitis B virus (HBV) or hepatitis C virus (HCV) coinfection; four (33.3%) had syphilis. Eight patients (57.1%) had vaginal delivery and six emergency C-section (42.9%). In six cases (46.2%), membrane rupture was spontaneous; four patients (28.6%) failed to receive intrapartum zidovudine (ZDV) infusion. In 12 patients a genotypic resistance test was performed: two (16.7%) had WHO RAMs corresponding to K103N mutation in both cases, conferring high-level resistance to nevirapine (NVP) and efavirenz. Two newborns (14.3%) were preterm. All received neonatal prophylaxis: ZDV in 1 case and combined prophylaxis (ZDV/3TC/NVP) in the remaining 13 (92.9%). All newborns were formula-fed. Two (14.3%) had congenital syphilis, one of whom died. One newborn was HIV

  13. HIV-1 Drug Resistance Mutations: an Updated Framework for the Second Decade of HAART

    PubMed Central

    Shafer, Robert W.; Schapiro, Jonathan M.

    2008-01-01

    More than 200 mutations are associated with antiretroviral resistance to drugs belonging to six licensed antiretroviral classes. More than 50 reverse transcriptase mutations are associated with nucleoside reverse transcriptase inhibitor resistance including M184V, thymidine analog mutations, mutations associated with non-thymidine analog containing regimens, multi-nucleoside resistance mutations, and several recently identified accessory mutations. More than 40 reverse transcriptase mutations are associated with nonnucleoside reverse transcriptase inhibitor resistance including major primary and secondary mutations, non-polymorphic minor mutations, and polymorphic accessory mutations. More than 60 mutations are associated with protease inhibitor resistance including major protease, accessory protease, and protease cleavage site mutations. More than 30 integrase mutations are associated with the licensed integrase inhibitor raltegravir and the investigational inhibitor elvitegravir. More than 15 gp41 mutations are associated with the fusion inhibitor enfuvirtide. CCR5 inhibitor resistance results from mutations that promote gp120 binding to an inhibitor-bound CCR5 receptor or CXCR4 tropism; however, the genotypic correlates of these processes are not yet well characterized. PMID:18615118

  14. A Novel Substrate-Based HIV-1 Protease Inhibitor Drug Resistance Mechanism

    PubMed Central

    Nijhuis, Monique; van Maarseveen, Noortje M; Lastere, Stephane; Schipper, Pauline; Coakley, Eoin; Glass, Bärbel; Rovenska, Mirka; de Jong, Dorien; Chappey, Colombe; Goedegebuure, Irma W; Heilek-Snyder, Gabrielle; Dulude, Dominic; Cammack, Nick; Brakier-Gingras, Lea; Konvalinka, Jan; Parkin, Neil; Kräusslich, Hans-Georg; Brun-Vezinet, Francoise; Boucher, Charles A. B

    2007-01-01

    Background HIV protease inhibitor (PI) therapy results in the rapid selection of drug resistant viral variants harbouring one or two substitutions in the viral protease. To combat PI resistance development, two approaches have been developed. The first is to increase the level of PI in the plasma of the patient, and the second is to develop novel PI with high potency against the known PI-resistant HIV protease variants. Both approaches share the requirement for a considerable increase in the number of protease mutations to lead to clinical resistance, thereby increasing the genetic barrier. We investigated whether HIV could yet again find a way to become less susceptible to these novel inhibitors. Methods and Findings We have performed in vitro selection experiments using a novel PI with an increased genetic barrier (RO033-4649) and demonstrated selection of three viruses 4- to 8-fold resistant to all PI compared to wild type. These PI-resistant viruses did not have a single substitution in the viral protease. Full genomic sequencing revealed the presence of NC/p1 cleavage site substitutions in the viral Gag polyprotein (K436E and/or I437T/V) in all three resistant viruses. These changes, when introduced in a reference strain, conferred PI resistance. The mechanism leading to PI resistance is enhancement of the processing efficiency of the altered substrate by wild-type protease. Analysis of genotypic and phenotypic resistance profiles of 28,000 clinical isolates demonstrated the presence of these NC/p1 cleavage site mutations in some clinical samples (codon 431 substitutions in 13%, codon 436 substitutions in 8%, and codon 437 substitutions in 10%). Moreover, these cleavage site substitutions were highly significantly associated with reduced susceptibility to PI in clinical isolates lacking primary protease mutations. Furthermore, we used data from a clinical trial (NARVAL, ANRS 088) to demonstrate that these NC/p1 cleavage site changes are associated with

  15. (Alkylamino) piperidine bis(heteroaryl)piperizine analogs are potent, broad-spectrum nonnucleoside reverse transcriptase inhibitors of drug-resistant isolates of human immunodeficiency virus type 1 (HIV-1) and select for drug-resistant variants of HIV-1IIIB with reduced replication phenotypes.

    PubMed Central

    Olmsted, R A; Slade, D E; Kopta, L A; Poppe, S M; Poel, T J; Newport, S W; Rank, K B; Biles, C; Morge, R A; Dueweke, T J; Yagi, Y; Romero, D L; Thomas, R C; Sharma, S K; Tarpley, W G

    1996-01-01

    The (alkylamino)piperidine bis(heteroaryl)piperizines (AAP-BHAPs) are a new class of human immunodeficiency virus type 1 (HIV-1)-specific inhibitors which were identified by targeted screening of recombinant reverse transcriptase (RT) enzymes carrying key nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance-conferring mutations and NNRTI-resistant variants of HIV-1. Phenotypic profiling of the two most potent AAP-BHAPs, U-95133 and U-104489, against in vitro-selected drug-resistant HIV-1 variants carrying the NNRTI resistance-conferring mutation (Tyr->Cys) at position 181 of the HIV-1 RT revealed submicromolar 90% inhibitory concentration estimates for these compounds. Moreover, U-104489 demonstrated potent activity against BHA-P-resistant HIV-1MF harboring the Pro-236->Leu RT substitution and significantly suppressed the replication of clinical isolates of HIV-1 resistant to both delavirdine (BHAP U-90152T) and zidovudine. Biochemical and phenotypic characterization of AAP-BHAPresistant HIV-1IIIB variants revealed that high-level resistance to the AAP-BHAPs was mediated by a Gly-190->Glu substitution in RT, which had a deleterious effect on the integrity and enzymatic activity of virion-associated RT heterodimers, as well as the replication capacity of these resistant viruses. PMID:8648704

  16. Prevalence of drug resistance mutations in HAART patients infected with HIV-1 CRF06_cpx in Estonia.

    PubMed

    Avi, Radko; Pauskar, Merit; Karki, Tõnis; Kallas, Eveli; Jõgeda, Ene-Ly; Margus, Tõnu; Huik, Kristi; Lutsar, Irja

    2016-03-01

    HIV-1 drug resistance mutations (DRMs) and substitutions were assessed after the failure of the first line non-nucleoside reverse transcriptase inhibitors (NNRTIs) + 2 nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) treatment regimens (efavirenz [EFV] + lamivudine[3TC] + zidovudine [ZDV] vs. EFV + 3TC + ddI) among the HIV-1 CRF06_cpx infected subjects in Estonia. HIV-1 genomic RNA was sequenced; DRMs and amino acid substitutions were compared in 44 treatment naïve and 45 first-line NNRTI + 2 NRTI treatment failed patients consisting of EFV + 3TC + ZDV (n = 17) and EFV + 3TC + didanosine[ddI] (n = 21) therapy failed sub-populations. At least one DRM was found in 78% of treatment experienced patients. The most common NRTI mutations were M184V (80%), L74V (31%), L74I (17%), K219E (9%), and M184I (9%), NNRTI mutations were K103N (83%), P225H (14%), L100I (11%), and Y188L (11%), reflecting generally the similar pattern of DRMs to that seen in treatment failed subtype B viruses. Sub-population analysis revealed that EFV + 3TC + ddI failed patients had more DRMs compared to EFV + 3TC + ZDV failed patients, especially the ddI DRM L74IV and several additional NNRTI DRMs. Additionally, CRF06_cpx specific mutation E179V and substitutions R32K, K122E, and V200AE were also detected in treatment experienced population. After the failure of the first-line EFV + 3TC + ddI therapy HIV-1 CRF06_cpx viruses develop additional NRTI and NNRTI mutations compared to EFV + 3TC + ZDV regimen. Therefore the usage of EFV + 3TC + ddI in this subtype decreases the options for next regimens containing abacavir, and NNRTI class agents. PMID:26291050

  17. Field Study of Dried Blood Spot Specimens for HIV-1 Drug Resistance Genotyping

    PubMed Central

    Parry, C. M.; Diallo, K.; Mwebaza, S.; Batamwita, R.; DeVos, J.; Bbosa, N.; Lyagoba, F.; Magambo, B.; Jordan, M. R.; Downing, R.; Zhang, G.; Kaleebu, P.; Bertagnolio, S.

    2014-01-01

    Dried blood spots (DBS) are an alternative specimen type for HIV drug resistance genotyping in resource-limited settings. Data relating to the impact of DBS storage and shipment conditions on genotyping efficiency under field conditions are limited. We compared the genotyping efficiencies and resistance profiles of DBS stored and shipped at different temperatures to those of plasma specimens collected in parallel from patients receiving antiretroviral therapy in Uganda. Plasma and four DBS cards from anti-coagulated venous blood and a fifth card from finger-prick blood were prepared from 103 HIV patients with a median viral load (VL) of 57,062 copies/ml (range, 1,081 to 2,964,191). DBS were stored at ambient temperature for 2 or 4 weeks or frozen at −80°C and shipped from Uganda to the United States at ambient temperature or frozen on dry ice for genotyping using a broadly sensitive in-house method. Plasma (97.1%) and DBS (98.1%) stored and shipped frozen had similar genotyping efficiencies. DBS stored frozen (97.1%) or at ambient temperature for 2 weeks (93.2%) and shipped at ambient temperature also had similar genotyping efficiencies. Genotyping efficiency was reduced for DBS stored at ambient temperature for 4 weeks (89.3%, P = 0.03) or prepared from finger-prick blood and stored at ambient temperature for 2 weeks (77.7%, P < 0.001) compared to DBS prepared from venous blood and handled similarly. Resistance profiles were similar between plasma and DBS specimens. This report delineates the optimal DBS collection, storage, and shipping conditions and opens a new avenue for cost-saving ambient-temperature DBS specimen shipments for HIV drug resistance (HIVDR) surveillances in resource-limited settings. PMID:24871219

  18. Prevalence of HIV-1 Subtypes and Drug Resistance-Associated Mutations in HIV-1-Positive Treatment-Naive Pregnant Women in Pointe Noire, Republic of the Congo (Kento-Mwana Project).

    PubMed

    Bruzzone, Bianca; Saladini, Francesco; Sticchi, Laura; Mayinda Mboungou, Franc A; Barresi, Renata; Caligiuri, Patrizia; Calzi, Anna; Zazzi, Maurizio; Icardi, Giancarlo; Viscoli, Claudio; Bisio, Francesca

    2015-08-01

    The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005-2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission. PMID:25970260

  19. Amprenavir complexes with HIV-1 protease and its drug-resistant mutants altering hydrophobic clusters

    SciTech Connect

    Shen, Chen-Hsiang; Wang, Yuan-Fang; Kovalevsky, Andrey Y.; Harrison, Robert W.; Weber, Irene T.

    2010-10-22

    The structural and kinetic effects of amprenavir (APV), a clinical HIV protease (PR) inhibitor, were analyzed with wild-type enzyme and mutants with single substitutions of V32I, I50V, I54V, I54M, I84V and L90M that are common in drug resistance. Crystal structures of the APV complexes at resolutions of 1.02-1.85 {angstrom} reveal the structural changes due to the mutations. Substitution of the larger side chains in PR{sub V32I}, PR{sub I54M} and PR{sub L90M} resulted in the formation of new hydrophobic contacts with flap residues, residues 79 and 80, and Asp25, respectively. Mutation to smaller side chains eliminated hydrophobic interactions in the PR{sub I50V} and PR{sub I54V} structures. The PR{sub I84V}-APV complex had lost hydrophobic contacts with APV, the PR{sub V32I}-APV complex showed increased hydrophobic contacts within the hydrophobic cluster and the PR{sub I50V} complex had weaker polar and hydrophobic interactions with APV. The observed structural changes in PR{sub I84V}-APV, PR{sub V32I}-APV and PR{sub I50V}-APV were related to their reduced inhibition by APV of six-, 10- and 30-fold, respectively, relative to wild-type PR. The APV complexes were compared with the corresponding saquinavir complexes. The PR dimers had distinct rearrangements of the flaps and 80's loops that adapt to the different P1{prime} groups of the inhibitors, while maintaining contacts within the hydrophobic cluster. These small changes in the loops and weak internal interactions produce the different patterns of resistant mutations for the two drugs.

  20. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay.

    PubMed

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John; Gao, Feng; Yang, Chunfu

    2016-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  1. Simultaneous Detection of Major Drug Resistance Mutations of HIV-1 Subtype B Viruses from Dried Blood Spot Specimens by Multiplex Allele-Specific Assay

    PubMed Central

    Zhang, Guoqing; Cai, Fangping; de Rivera, Ivette Lorenzana; Zhou, Zhiyong; Zhang, Jing; Nkengasong, John

    2015-01-01

    A multiplex allele-specific (MAS) assay has been developed for the detection of HIV-1 subtype C drug resistance mutations (DRMs). We have optimized the MAS assay to determine subtype B DRMs in dried blood spots (DBS) collected from patients on antiretroviral therapy. The new assay accurately detected DRMs, including low-abundance mutations that were often missed by Sanger sequencing. PMID:26560533

  2. Trends in Prevalence of HIV-1 Drug Resistance in a Public Clinic in Maputo, Mozambique

    PubMed Central

    Bila, Dulce Celina Adolfo; Boullosa, Lídia Teodoro; Abreu, Celina Monteiro; Jani, Ilesh Vinodrai; Tanuri, Amilcar

    2015-01-01

    Background An observational study was conducted in Maputo, Mozambique, to investigate trends in prevalence of HIV drug resistance (HIVDR) in antiretroviral (ART) naïve subjects initiating highly active antiretroviral treatment (HAART). Methodology/Principal Findings To evaluate the pattern of drug resistance mutations (DRMs) found in adults on ART failing first-line HAART [patients with detectable viral load (VL)]. Untreated subjects [Group 1 (G1; n=99)] and 274 treated subjects with variable length of exposure to ARV´s [6–12 months, Group 2 (G2;n=93); 12-24 months, Group 3 (G3;n=81); >24 months (G4;n=100)] were enrolled. Virological and immunological failure (VF and IF) were measured based on viral load (VL) and T lymphocyte CD4+ cells (TCD4+) count and genotypic resistance was also performed. Major subtype found was C (untreated: n=66, 97,06%; treated: n=36, 91.7%). Maximum virological suppression was observed in G3, and significant differences intragroup were observed between VF and IF in G4 (p=0.022). Intergroup differences were observed between G3 and G4 for VF (p=0.023) and IF between G2 and G4 (p=0.0018). Viral suppression (<50 copies/ml) ranged from 84.9% to 90.1%, and concordant VL and DRM ranged from 25% to 57%. WHO cut-off for determining VF as given by 2010 guidelines (>5000 copies/ml) identified 50% of subjects carrying DRM compared to 100% when lower VL cut-off was used (<50 copies/ml). Length of exposure to ARVs was directly proportional to the complexity of DRM patterns. In Mozambique, VL suppression was achieved in 76% of individuals after 24 months on HAART. This is in agreement with WHO target for HIVDR prevention target (70%). Conclusions We demonstrated that the best way to determine therapeutic failure is VL compared to CD4 counts. The rationalized use of VL testing is needed to ensure timely detection of treatment failures preventing the occurrence of TDR and new infections. PMID:26151752

  3. The HEPT Analogue WPR-6 Is Active against a Broad Spectrum of Nonnucleoside Reverse Transcriptase Drug-Resistant HIV-1 Strains of Different Serotypes.

    PubMed

    Xu, Weisi; Zhao, Jianxiong; Sun, Jianping; Yin, Qianqian; Wang, Yan; Jiao, Yang; Liu, Junyi; Jiang, Shibo; Shao, Yiming; Wang, Xiaowei; Ma, Liying

    2015-08-01

    Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are important components of the highly active antiretroviral therapy (HAART) used to treat human immunodeficiency type 1 virus (HIV-1). However, because of the emergence of drug resistance and the adverse effects of current anti-HIV drugs, it is essential to develop novel NNRTIs with an excellent safety profile, improved activity against NNRTI-resistant viruses, and enhanced activity against clinical isolates of different subtypes. Here, we have identified 1-[(benzyloxy)methyl]-6-(3,5-dimethylbenzyl)-5-iodopyrimidine-2,4(1H,3H)-dione (WPR-6), a novel NNRTI with a 50% effective concentration (EC50) of 2 to 4 nM against laboratory-adapted HIV-1 strain SF33 and an EC50 of 7 to 14 nM against nucleoside reverse transcriptase inhibitor-resistant HIV-1 strain 7391 with a therapeutic index of >1 × 10(4). A panel of five representative clinical virus isolates of different subtypes circulating predominantly in China was highly sensitive to WPR-6, with EC50s ranging from 1 to 6 nM. In addition, WPR-6 showed excellent antiviral potency against the most prevalent NNRTI-resistant viruses containing the K103N and Y181C mutations. To determine whether WPR-6 selects for novel resistant mutants, in vitro resistance selection was conducted with laboratory-adapted HIV-1 strain SF33 on MT-4 cells. The results demonstrated that V106I and Y188L were the two dominant NNRTI-associated resistance mutations detected in the breakthrough viruses. Taken together, these in vitro data indicate that WPR-6 has greater efficacy than the reference HEPT analogue TNK651 and the marketed drug nevirapine against HIV-1. However, to develop it as a new NNRTI, further improvement of its pharmacological properties is warranted. PMID:26055365

  4. Capacity building and predictors of success for HIV-1 drug resistance testing in the Asia-Pacific region and Africa

    PubMed Central

    Land, Sally; Zhou, Julian; Cunningham, Philip; Sohn, Annette H; Singtoroj, Thida; Katzenstein, David; Mann, Marita; Sayer, David; Kantor, Rami

    2013-01-01

    Background The TREAT Asia Quality Assessment Scheme (TAQAS) was developed as a quality assessment programme through expert education and training, for laboratories in the Asia-Pacific and Africa that perform HIV drug-resistance (HIVDR) genotyping. We evaluated the programme performance and factors associated with high-quality HIVDR genotyping. Methods Laboratories used their standard protocols to test panels of human immunodeficiency virus (HIV)-positive plasma samples or electropherograms. Protocols were documented and performance was evaluated according to a newly developed scoring system, agreement with panel-specific consensus sequence, and detection of drug-resistance mutations (DRMs) and mixtures of wild-type and resistant virus (mixtures). High-quality performance was defined as detection of ≥95% DRMs. Results Over 4.5 years, 23 participating laboratories in 13 countries tested 45 samples (30 HIV-1 subtype B; 15 non-B subtypes) in nine panels. Median detection of DRMs was 88–98% in plasma panels and 90–97% in electropherogram panels. Laboratories were supported to amend and improve their test outcomes as appropriate. Three laboratories that detected <80% DRMs in early panels demonstrated subsequent improvement. Sample complexity factors – number of DRMs (p<0.001) and number of DRMs as mixtures (p<0.001); and laboratory performance factors – detection of mixtures (p<0.001) and agreement with consensus sequence (p<0.001), were associated with high performance; sample format (plasma or electropherogram), subtype and genotyping protocol were not. Conclusion High-quality HIVDR genotyping was achieved in the TAQAS collaborative laboratory network. Sample complexity and detection of mixtures were associated with performance quality. Laboratories conducting HIVDR genotyping are encouraged to participate in quality assessment programmes. PMID:23845227

  5. Evaluating the substrate-envelope hypothesis: structural analysis of novel HIV-1 protease inhibitors designed to be robust against drug resistance.

    PubMed

    Nalam, Madhavi N L; Ali, Akbar; Altman, Michael D; Reddy, G S Kiran Kumar; Chellappan, Sripriya; Kairys, Visvaldas; Ozen, Aysegül; Cao, Hong; Gilson, Michael K; Tidor, Bruce; Rana, Tariq M; Schiffer, Celia A

    2010-05-01

    Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type protease ranging from 58 nM to 0.8 pM was chosen for crystallographic analysis. The inhibitor-protease complexes revealed that tightly binding inhibitors (at the picomolar level of affinity) appear to "lock" into the protease active site by forming hydrogen bonds to particular active-site residues. Both this hydrogen bonding pattern and subtle variations in protein-ligand van der Waals interactions distinguish nanomolar from picomolar inhibitors. In general, inhibitors that fit within the substrate envelope, regardless of whether they are picomolar or nanomolar, have flatter profiles with respect to drug-resistant protease variants than inhibitors that protrude beyond the substrate envelope; this provides a strong rationale for incorporating substrate-envelope constraints into structure-based design strategies to develop new HIV-1 protease inhibitors. PMID:20237088

  6. Evaluating the Substrate-Envelope Hypothesis: Structural Analysis of Novel HIV-1 Protease Inhibitors Designed To Be Robust against Drug Resistance

    PubMed Central

    Nalam, Madhavi N. L.; Ali, Akbar; Altman, Michael D.; Reddy, G. S. Kiran Kumar; Chellappan, Sripriya; Kairys, Visvaldas; Özen, Ayşegül; Cao, Hong; Gilson, Michael K.; Tidor, Bruce; Rana, Tariq M.; Schiffer, Celia A.

    2010-01-01

    Drug resistance mutations in HIV-1 protease selectively alter inhibitor binding without significantly affecting substrate recognition and cleavage. This alteration in molecular recognition led us to develop the substrate-envelope hypothesis which predicts that HIV-1 protease inhibitors that fit within the overlapping consensus volume of the substrates are less likely to be susceptible to drug-resistant mutations, as a mutation impacting such inhibitors would simultaneously impact the processing of substrates. To evaluate this hypothesis, over 130 HIV-1 protease inhibitors were designed and synthesized using three different approaches with and without substrate-envelope constraints. A subset of 16 representative inhibitors with binding affinities to wild-type protease ranging from 58 nM to 0.8 pM was chosen for crystallographic analysis. The inhibitor-protease complexes revealed that tightly binding inhibitors (at the picomolar level of affinity) appear to “lock” into the protease active site by forming hydrogen bonds to particular active-site residues. Both this hydrogen bonding pattern and subtle variations in protein-ligand van der Waals interactions distinguish nanomolar from picomolar inhibitors. In general, inhibitors that fit within the substrate envelope, regardless of whether they are picomolar or nanomolar, have flatter profiles with respect to drug-resistant protease variants than inhibitors that protrude beyond the substrate envelope; this provides a strong rationale for incorporating substrate-envelope constraints into structure-based design strategies to develop new HIV-1 protease inhibitors. PMID:20237088

  7. Pre-existing Minority Drug-Resistant HIV-1 Variants, Adherence, and Risk of Antiretroviral Treatment Failure

    PubMed Central

    Paredes, Roger; Lalama, Christina M.; Ribaudo, Heather J.; Schackman, Bruce R.; Shikuma, Cecilia; Giguel, Francoise; Meyer, William A.; Johnson, Victoria A.; Fiscus, Susan A.; D’Aquila, Richard T.; Gulick, Roy M.; Kuritzkes, Daniel R.

    2009-01-01

    Background The clinical relevance of detecting minority drug-resistant HIV-1 variants is uncertain. Methods To determine the effect of pre-existing minority non-nucleoside reverse transcriptase inhibitor (NNRTI)-resistant variants on the risk of virologic failure (VF), we reanalyzed a case-cohort substudy of efavirenz recipients in ACTG A5095. Minority K103N or Y181C populations were determined by allele-specific PCR (ASPCR) in subjects without NNRTI resistance by population sequencing. Weighted Cox proportional hazards models adjusted for recent adherence estimated the relative risk of VF in the presence of NNRTI-resistant minority variants. Results The evaluable case-cohort sample included 195 subjects from the randomly selected subcohort (51 with VF, 144 without failure [NF]), plus 127 of the remaining subjects with VF. Presence of minority K103N or Y181C mutations, or both, was detected in 8 (4.4%), 54 (29.5%) and 11 (6%), respectively, of 183 evaluable subjects in the random subcohort. Detection of minority Y181C mutants was associated with an increased risk of VF in the setting of recent adherence (HR=3.45, CI=1.90, 6.26), but not in non-adherent subjects (HR=1.39, CI=0.58, 3.29). Of note, 70% of subjects with minority Y181C achieved long-term viral suppression. Conclusions In adherent patients, pre-existing minority Y181C mutants more than tripled the risk of VF of first-line efavirenz-based ART. PMID:20102271

  8. Increasing HIV-1 pretreatment drug resistance among antiretroviral-naïve adults initiating treatment between 2006 and 2014 in Nairobi, Kenya.

    PubMed

    Chung, Michael H; Silverman, Rachel; Beck, Ingrid A; Yatich, Nelly; Dross, Sandra; McKernan-Mullin, Jennifer; Bii, Stephen; Tapia, Kenneth; Stern, Joshua; Chohan, Bhavna; Sakr, Samah R; Kiarie, James N; Frenkel, Lisa M

    2016-06-19

    Antiretroviral-naïve adults initiating antiretroviral therapy in Nairobi, Kenya were tested for HIV-1 drug resistance at codons K103N, Y181C, G190A, M184V, and K65R using an oligonucleotide ligation assay. Prevalence of pretreatment drug resistance increased from 3.89% in 2006 to 10.93% in 2014 (P < 0.001), and 95% of those with resistance had at least one nonnucleoside reverse transcriptase inhibitor mutation. Resistance to tenofovir (K65R) was found in 2014 but not in 2006. PMID:27058353

  9. Exploring the drug resistance of V32I and M46L mutant HIV-1 protease to inhibitor TMC114: flap dynamics and binding mechanism.

    PubMed

    Meher, Biswa Ranjan; Wang, Yixuan

    2015-03-01

    Inhibitors of HIV-1 protease (HIV-1-pr) generally only bind to the active site of the protease. However, for some mutants such as V32I and M46L the TMC114 can bind not only to the active cavity but also to the groove of the flexible flaps. Although the second binding site suggests the higher efficiency of the drug against HIV-1-pr, the drug resistance in HIV-1-pr due to mutations cannot be ignored, which prompts us to investigate the molecular mechanisms of drug resistance and behavior of double bound TMC114 (2T) to HIV-1-pr. The conformational dynamics of HIV-1-pr and the binding of TMC114 to the WT, V32I and M46L mutants were investigated with all-atom molecular dynamic (MD) simulation. The 20 ns MD simulation shows many fascinating effects of the inhibitor binding to the WT and mutant proteases. MM-PBSA calculations explain the binding free energies unfavorable for the M46L and V32I mutants as compared to the WT. For the single binding (1T) the less binding affinity can be attributed to the entropic loss for both V32I-1T and M46L-1T. Although the second binding of TMC114 with flap does increase binding energy for the mutants (V32I-2T and M46L-2T), the considerable entropy loss results in the lower binding Gibbs free energies. Thus, binding of TMC114 in the flap region does not help much in the total gain in binding affinity of the system, which was verified from this study and thereby validating experiments. PMID:25562662

  10. Viral Tropism and Antiretroviral Drug Resistance in HIV-1 Subtype C-Infected Patients Failing Highly Active Antiretroviral Therapy in Johannesburg, South Africa

    PubMed Central

    Ketseoglou, Irene; Lukhwareni, Azwidowi; Steegen, Kim; Carmona, Sergio; Stevens, Wendy S.

    2014-01-01

    Abstract Reports show that up to 30% of antiretroviral drug-naive patients in Johannesburg have CXCR4-utilizing HIV-1 subtype C. We assessed whether HIV-1 subtype C-infected individuals failing highly active antiretroviral therapy (HAART) have a higher proportion of CXCR4-utilizing viruses compared to antiretroviral drug-naive patients. The V3 loop was sequenced from plasma from 100 randomly selected HAART-failing patients, and tropism was established using predictive algorithms. All patients harbored HIV-1 subtype C with at least one antiretroviral drug resistance mutation. Viral tropism prediction in individuals failing HAART revealed similar proportions (29%) of X4-utilizing viruses compared to antiretroviral drug-naive patients (30%). Findings are in contrast to reports from Durban in which 60% of HAART-failing subjects harbored X4/dual/mixed-tropic viruses. Despite differences in proportions of X4-tropism within South Africa, the high proportion of thymidine analogue mutations (TAMs) and CXCR4-utilizing HIV-1 highlights the need for intensified monitoring of HAART patients and the predicament of diminishing drug options, including CCR5 antagonists, for patients failing therapy. PMID:24224886

  11. Profile of the HIV epidemic in Cape Verde: molecular epidemiology and drug resistance mutations among HIV-1 and HIV-2 infected patients from distinct islands of the archipelago.

    PubMed

    de Pina-Araujo, Isabel Inês M; Guimarães, Monick L; Bello, Gonzalo; Vicente, Ana Carolina P; Morgado, Mariza G

    2014-01-01

    HIV-1 and HIV-2 have been detected in Cape Verde since 1987, but little is known regarding the genetic diversity of these viruses in this archipelago, located near the West African coast. In this study, we characterized the molecular epidemiology of HIV-1 and HIV-2 and described the occurrence of drug resistance mutations (DRM) among antiretroviral therapy naïve (ARTn) patients and patients under treatment (ARTexp) from different Cape Verde islands. Blood samples, socio-demographic and clinical-laboratory data were obtained from 221 HIV-positive individuals during 2010-2011. Phylogenetic and bootscan analyses of the pol region (1300 bp) were performed for viral subtyping. HIV-1 and HIV-2 DRM were evaluated for ARTn and ARTexp patients using the Stanford HIV Database and HIV-GRADE e.V. Algorithm Homepage, respectively. Among the 221 patients (169 [76.5%] HIV-1, 43 [19.5%] HIV-2 and 9 [4.1%] HIV-1/HIV-2 co-infections), 67% were female. The median ages were 34 (IQR = 1-75) and 47 (IQR = 12-84) for HIV-1 and HIV-2, respectively. HIV-1 infections were due to subtypes G (36.6%), CRF02_AG (30.6%), F1 (9.7%), URFs (10.4%), B (5.2%), CRF05_DF (3.0%), C (2.2%), CRF06_cpx (0.7%), CRF25_cpx (0.7%) and CRF49_cpx (0.7%), whereas all HIV-2 infections belonged to group A. Transmitted DRM (TDRM) was observed in 3.4% (2/58) of ARTn HIV-1-infected patients (1.7% NRTI, 1.7% NNRTI), but not among those with HIV-2. Among ARTexp patients, DRM was observed in 47.8% (33/69) of HIV-1 (37.7% NRTI, 37.7% NNRTI, 7.4% PI, 33.3% for two classes) and 17.6% (3/17) of HIV-2-infections (17.6% NRTI, 11.8% PI, 11.8% both). This study indicates that Cape Verde has a complex and unique HIV-1 molecular epidemiological scenario dominated by HIV-1 subtypes G, CRF02_AG and F1 and HIV-2 subtype A. The occurrence of TDRM and the relatively high level of DRM among treated patients are of concern. Continuous monitoring of patients on ART, including genotyping, are public policies to be implemented

  12. Profile of the HIV Epidemic in Cape Verde: Molecular Epidemiology and Drug Resistance Mutations among HIV-1 and HIV-2 Infected Patients from Distinct Islands of the Archipelago

    PubMed Central

    de Pina-Araujo, Isabel Inês M.; Guimarães, Monick L.; Bello, Gonzalo; Vicente, Ana Carolina P.; Morgado, Mariza G.

    2014-01-01

    HIV-1 and HIV-2 have been detected in Cape Verde since 1987, but little is known regarding the genetic diversity of these viruses in this archipelago, located near the West African coast. In this study, we characterized the molecular epidemiology of HIV-1 and HIV-2 and described the occurrence of drug resistance mutations (DRM) among antiretroviral therapy naïve (ARTn) patients and patients under treatment (ARTexp) from different Cape Verde islands. Blood samples, socio-demographic and clinical-laboratory data were obtained from 221 HIV-positive individuals during 2010–2011. Phylogenetic and bootscan analyses of the pol region (1300 bp) were performed for viral subtyping. HIV-1 and HIV-2 DRM were evaluated for ARTn and ARTexp patients using the Stanford HIV Database and HIV-GRADE e.V. Algorithm Homepage, respectively. Among the 221 patients (169 [76.5%] HIV-1, 43 [19.5%] HIV-2 and 9 [4.1%] HIV-1/HIV-2 co-infections), 67% were female. The median ages were 34 (IQR = 1–75) and 47 (IQR = 12–84) for HIV-1 and HIV-2, respectively. HIV-1 infections were due to subtypes G (36.6%), CRF02_AG (30.6%), F1 (9.7%), URFs (10.4%), B (5.2%), CRF05_DF (3.0%), C (2.2%), CRF06_cpx (0.7%), CRF25_cpx (0.7%) and CRF49_cpx (0.7%), whereas all HIV-2 infections belonged to group A. Transmitted DRM (TDRM) was observed in 3.4% (2/58) of ARTn HIV-1-infected patients (1.7% NRTI, 1.7% NNRTI), but not among those with HIV-2. Among ARTexp patients, DRM was observed in 47.8% (33/69) of HIV-1 (37.7% NRTI, 37.7% NNRTI, 7.4% PI, 33.3% for two classes) and 17.6% (3/17) of HIV-2-infections (17.6% NRTI, 11.8% PI, 11.8% both). This study indicates that Cape Verde has a complex and unique HIV-1 molecular epidemiological scenario dominated by HIV-1 subtypes G, CRF02_AG and F1 and HIV-2 subtype A. The occurrence of TDRM and the relatively high level of DRM among treated patients are of concern. Continuous monitoring of patients on ART, including genotyping, are public policies to be

  13. Genetic diversity of HIV-1 and transmitted drug resistance among newly diagnosed individuals with HIV infection in Hangzhou, China.

    PubMed

    Zhang, Jiafeng; Guo, Zhihong; Yang, Jiezhe; Pan, Xiaohong; Jiang, Jun; Ding, Xiaobei; Zhang, Wenjun; Xia, Yan; Xu, Yun; Huang, Jingjing

    2015-10-01

    HIV transmitted drug resistance (TDR) can compromise antiretroviral therapy (ART) in resource-limited countries like China where ART has been scaled up and thus leads to an important public health concern. The aim of the study was to elucidate the HIV-1 genetic characteristics and TDR in Hangzhou, China. Two-hundred eleven ART-naive, newly diagnosed individuals were enrolled during January and August 2013. Specimens were classified as recent or chronic infections using the BED capture enzyme immunoassay (BED-CEIA). The pol fragment covering the entire protease and the first 300 codons of the reverse transcriptase gene was amplified by RT-PCR and nested PCR. Genotypic drug resistance (DR) and phylogenetic analysis were performed on the 200 obtained sequences. Multiple genotypes were identified, including CRF01_AE (62.0%), CRF07_BC (31.0%), subtype B (2.0%), CRF08_BC (1.5%), CRF55_01B (1.0%), CRF18_cpx (0.5%), and unique recombinant forms (URFs, 2.0%). All the four URFs were found in men who have sex with men, consisting of a recombination of CRF01_AE with subtype B or CRF07_BC. The prevalence of primary DR in newly diagnosed individuals in Hangzhou was low (4.0%). The proportion of DR mutation to protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) was 1.5%, 1.5%, and 1.0%, respectively. BED-CEIA revealed that 21.8% (45/211) of the specimens were associated with recent infections. The prevalence of TDR in recent infections was moderate (6.5%). High HIV diversity and relatively high prevalence of TDR in new infections has been found in Hangzhou, indicating an increasing challenge for future HIV prevention and treatment. PMID:25899877

  14. HIV-1 infection and pregnancy in young women in Brazil: socioeconomic and drug resistance profiles in a cross-sectional study

    PubMed Central

    Lima, Yanna Andressa Ramos; Reis, Mônica Nogueira Guarda; Cardoso, Ludimila Paula Vaz; Stefani, Mariane Martins Araújo

    2016-01-01

    Objectives To describe socioeconomic and antiretroviral (ARV) drug resistance profiles among young pregnant women infected with HIV-1. Setting A public health antenatal programme responsible for screening ∼90 000 pregnant women per year for nine different infectious diseases in Central Western Brazil. Participants 96 young pregnant women (15–24 years) infected with HIV-1. Primary and secondary outcome measures Standard interviews and blood samples were taken at the time of recruitment, at the first medical appointment after confirmation of diagnosis of HIV-1 infection, and before ARV prophylaxis initiation. Clinical and laboratory data were retrieved from medical files. HIV-1 pol gene sequences (entire protease/PR, partial reverse transcriptase/RT) were obtained from plasma RNA. ARV resistance mutations (CPR/Stanford HIV-1; International AIDS Society-USA databases) were identified. Results The median age was 21 years; most reported <8 years education; 73% were recently diagnosed. Approximately 20% (19/96) presented late for antenatal care (after 26 gestational weeks), while 49% reported ≥2 previous pregnancies. Possible heterosexual transmission by an HIV-1 infected partner (17%) and commercial sex work (2%) were reported. The median of CD4 cell count was 526 cells/mm3; the median viral load was: 10 056 copies/mL in ARV-naïve (48/96) patients and 5881 copies/mL in ARV-exposed (48/96) patients. Two probable seroconversion cases during pregnancy were identified in adolescents. One mother-to-child transmission case (1.0%) was observed. Transmitted drug resistance among ARV-naïve patients was 9.3% (CI 95% 3.3% to 19.6%); secondary drug resistance among ARV-exposed patients was 12.5% (CI 95% 4.7% to 25.6%). Conclusions Despite high access to antenatal care, the low socioeconomic-educational profiles seen in these young HIV-1-infected women highlight the necessity of improved public health educational and preventive strategies regarding HIV infection

  15. HIV-1 Drug Resistance and Second-line Treatment in Children Randomized to Switch at Low versus Higher RNA Thresholds

    PubMed Central

    Harrison, Linda; Melvin, Ann; Fiscus, Susan; Saidi, Yacine; Nastouli, Eleni; Harper, Lynda; Compagnucci, Alexandra; Babiker, Abdel; McKinney, Ross; Gibb, Diana; Tudor-Williams, Gareth

    2015-01-01

    Background The PENPACT-1 trial compared virologic thresholds to determine when to switch to second-line antiretroviral therapy (ART). Using PENPACT-1 data, we aimed to describe HIV-1 drug resistance accumulation on first-line ART by virologic threshold. Methods PENPACT-1 had a 2x2 factorial design, randomizing HIV-infected children to start protease inhibitor (PI) versus non-nucleoside reverse transcriptase inhibitor (NNRTI) based ART, and switch at a 1000c/ml versus 30000c/ml threshold. Switch-criteria were: not achieving the threshold by week 24, confirmed rebound above the threshold thereafter, or CDC-C event. Resistance tests were performed on samples ≥1000c/ml before switch, re-suppression and at 4-year/trial-end. Results Sixty-seven children started PI-based ART and were randomized to switch at 1000c/ml (PI-1000), 64 PIs and 30000c/ml (PI-30000), 67 NNRTIs and 1000c/ml (NNRTI-1000), and 65 NNRTI and 30000c/ml (NNRTI-30000). Ninety-four (36%) children reached the 1000c/ml switch-criteria during 5 years follow-up. In 30000c/ml threshold arms, median time from 1000c/ml to 30000c/ml switch-criteria was 58 (PI) versus 80 (NNRTI) weeks (P=0.81). In NNRTI-30000 more NRTI resistance mutations accumulated than other groups. NNRTI mutations were selected before switching at 1000c/ml (23% NNRTI-1000, 27% NNRTI-30000). Sixty-two children started abacavir+lamivudine, 166 lamivudine+zidovudine or stavudine, and 35 other NRTIs. The abacavir+lamivudine group acquired fewest NRTI mutations. Of 60 switched to second-line, 79% PI-1000, 63% PI-30000, 64% NNRTI-1000 and 100% NNRTI-30000 were <400c/ml 24 weeks later. Conclusion Children on first-line NNRTI-based ART who were randomized to switch at a higher virologic threshold developed the most resistance, yet re-suppressed on second-line. An abacavir+lamivudine NRTI combination seemed protective against development of NRTI resistance. PMID:26322666

  16. Design, Synthesis, and Biological and Structural Evaluations of Novel HIV-1 Protease Inhibitors To Combat Drug Resistance

    SciTech Connect

    Parai, Maloy Kumar; Huggins, David J.; Cao, Hong; Nalam, Madhavi N.L.; Ali, Akbar; Schiffer, Celia A.; Tidor, Bruce; Rana, Tariq M.

    2012-09-11

    A series of new HIV-1 protease inhibitors (PIs) were designed using a general strategy that combines computational structure-based design with substrate-envelope constraints. The PIs incorporate various alcohol-derived P2 carbamates with acyclic and cyclic heteroatomic functionalities into the (R)-hydroxyethylamine isostere. Most of the new PIs show potent binding affinities against wild-type HIV-1 protease and three multidrug resistant (MDR) variants. In particular, inhibitors containing the 2,2-dichloroacetamide, pyrrolidinone, imidazolidinone, and oxazolidinone moieties at P2 are the most potent with Ki values in the picomolar range. Several new PIs exhibit nanomolar antiviral potencies against patient-derived wild-type viruses from HIV-1 clades A, B, and C and two MDR variants. Crystal structure analyses of four potent inhibitors revealed that carbonyl groups of the new P2 moieties promote extensive hydrogen bond interactions with the invariant Asp29 residue of the protease. These structure-activity relationship findings can be utilized to design new PIs with enhanced enzyme inhibitory and antiviral potencies.

  17. The prevalence and determinants of drug-resistance-associated mutations in the HIV-1-infected MSM population of Henan Province in China.

    PubMed

    Hou, Li-Juan; Wang, Hong-Wei; Duan, Shu-Peng; Zhuo, Ya; Zhou, Yan-Cai; Wu, Hong-Jie; Shen, Bao-Sheng

    2015-08-01

    To estimate the prevalence of human immunodeficiency virus (HIV) drug resistance (DR) in a population of men who have sex with men (MSM) from Henan Province of China and to identify the DR-associated HIV-1 mutations in these MSM. The HIV-positive status of the MSM subjects in this study was confirmed using ELISA and Western blotting. The MSM subjects were classified into non-treatment group (n = 106) and treatment group (n = 313). CD4(+) T-lymphocyte counts were obtained by flow cytometry, and viral load was measured by branched DNA (bDNA) signal amplification assay. HIV-1 genotypic resistance tests were performed by sequence analysis of the HIV-1 protease and reverse transcriptase genes. In the non-treatment group, 15 patients (14.2 %) displayed DR to non-nucleoside reverse transcriptase inhibitor (NNRTI). In the treatment group, the failure rate of viral suppression was 38.33 % and the DR rate was 33.2 %, which was higher than the rate observed in the non-treatment group (P < 0.05). The incidence of mutations corresponding to NNRTI resistance was significantly higher than the incidence of mutations corresponding to nucleoside reverse transcriptase inhibitor (NRTI) resistance (32.9 % vs. 26.5 %) in the cohort. After antiretroviral therapy (ART), the frequencies of K103N, G190A, Y181C, and V106A mutations were highly elevated. Logistic regression analysis results showed that duration of treatment, poor treatment compliance, drug abuse and homosexual orientation are the major risk factors for DR in this MSM population (all P < 0.05). Our results showed that DR-associated mutations in the HIV-1-infected MSM population increased significantly after ART. Furthermore, duration of treatment, poor treatment compliance, drug abuse and homosexual orientation were identified as the risk factors for DR in the MSM population from Henan Province in China. PMID:26077516

  18. Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase

    PubMed Central

    Melody, Kevin; McBeth, Sarah; Kline, Christopher; Kashuba, Angela D. M.; Mellors, John W.

    2015-01-01

    Preexposure prophylaxis (PrEP) using antiretroviral drugs is effective in reducing the risk of human immunodeficiency virus type 1 (HIV-1) infection, but adherence to the PrEP regimen is needed. To improve adherence, a long-acting injectable formulation of the nonnucleoside reverse transcriptase (RT) inhibitor rilpivirine (RPV LA) has been developed. However, there are concerns that PrEP may select for drug-resistant mutations during preexisting or breakthrough infections, which could promote the spread of drug resistance and limit options for antiretroviral therapy. To address this concern, we administered RPV LA to macaques infected with simian immunodeficiency virus containing HIV-1 RT (RT-SHIV). Peak plasma RPV levels were equivalent to those reported in human trials and waned over time after dosing. RPV LA resulted in a 2-log decrease in plasma viremia, and the therapeutic effect was maintained for 15 weeks, until plasma drug concentrations dropped below 25 ng/ml. RT mutations E138G and E138Q were detected in single clones from plasma virus in separate animals only at one time point, and no resistance mutations were detected in viral RNA isolated from tissues. Wild-type and E138Q RT-SHIV displayed similar RPV susceptibilities in vitro, whereas E138G conferred 2-fold resistance to RPV. Overall, selection of RPV-resistant variants was rare in an RT-SHIV macaque model despite prolonged exposure to slowly decreasing RPV concentrations following injection of RPV LA. PMID:26438501

  19. Low Frequency of Drug-Resistant Variants Selected by Long-Acting Rilpivirine in Macaques Infected with Simian Immunodeficiency Virus Containing HIV-1 Reverse Transcriptase.

    PubMed

    Melody, Kevin; McBeth, Sarah; Kline, Christopher; Kashuba, Angela D M; Mellors, John W; Ambrose, Zandrea

    2015-12-01

    Preexposure prophylaxis (PrEP) using antiretroviral drugs is effective in reducing the risk of human immunodeficiency virus type 1 (HIV-1) infection, but adherence to the PrEP regimen is needed. To improve adherence, a long-acting injectable formulation of the nonnucleoside reverse transcriptase (RT) inhibitor rilpivirine (RPV LA) has been developed. However, there are concerns that PrEP may select for drug-resistant mutations during preexisting or breakthrough infections, which could promote the spread of drug resistance and limit options for antiretroviral therapy. To address this concern, we administered RPV LA to macaques infected with simian immunodeficiency virus containing HIV-1 RT (RT-SHIV). Peak plasma RPV levels were equivalent to those reported in human trials and waned over time after dosing. RPV LA resulted in a 2-log decrease in plasma viremia, and the therapeutic effect was maintained for 15 weeks, until plasma drug concentrations dropped below 25 ng/ml. RT mutations E138G and E138Q were detected in single clones from plasma virus in separate animals only at one time point, and no resistance mutations were detected in viral RNA isolated from tissues. Wild-type and E138Q RT-SHIV displayed similar RPV susceptibilities in vitro, whereas E138G conferred 2-fold resistance to RPV. Overall, selection of RPV-resistant variants was rare in an RT-SHIV macaque model despite prolonged exposure to slowly decreasing RPV concentrations following injection of RPV LA. PMID:26438501

  20. High rates of virological failure and drug resistance in perinatally HIV-1-infected children and adolescents receiving lifelong antiretroviral therapy in routine clinics in Togo

    PubMed Central

    Salou, Mounerou; Dagnra, Anoumou Y; Butel, Christelle; Vidal, Nicole; Serrano, Laetitia; Takassi, Elom; Konou, Abla A; Houndenou, Spero; Dapam, Nina; Singo-Tokofaï, Assetina; Pitche, Palokinam; Atakouma, Yao; Prince-David, Mireille; Delaporte, Eric; Peeters, Martine

    2016-01-01

    Introduction Antiretroviral treatment (ART) has been scaled up over the last decade but compared to adults, children living with HIV are less likely to receive ART. Moreover, children and adolescents are more vulnerable than adults to virological failure (VF) and emergence of drug resistance. In this study we determined virological outcome in perinatally HIV-1-infected children and adolescents receiving ART in Togo. Methods HIV viral load (VL) testing was consecutively proposed to all children and adolescents who were on ART for at least 12 months when attending HIV healthcare services for their routine follow-up visit (June to September 2014). Plasma HIV-1 VL was measured using the m2000 RealTime HIV-1 assay (Abbott Molecular, Des Plaines, IL, USA). Genotypic drug resistance was done for all samples with VL>1000 copies/ml. Results and discussion Among 283 perinatally HIV-1-infected children and adolescents included, 167 (59%) were adolescents and 116 (41%) were children. The median duration on ART was 48 months (interquartile range: 28 to 68 months). For 228 (80.6%), the current ART combination consisted of two nucleoside reverse transcriptase inhibitors (NRTIs) (zidovudine and lamivudine) and one non-nucleoside reverse transcriptase inhibitor (NNRTI) (nevirapine or efavirenz). Only 28 (9.9%) were on a protease inhibitor (PI)-based regimen. VL was below the detection limit (i.e. 40 copies/ml) for 102 (36%), between 40 and 1000 copies/ml for 35 (12.4%) and above 1000 copies/ml for 146 (51.6%). Genotypic drug-resistance testing was successful for 125/146 (85.6%); 110/125 (88.0%) were resistant to both NRTIs and NNRTIs, 1/125 (0.8%) to NRTIs only, 4/125 (3.2%) to NNRTIs only and three harboured viruses resistant to reverse transcriptase and PIs. Overall, 86% (108/125) of children and adolescents experiencing VF and successfully genotyped, corresponding thus to at least 38% of the study population, had either no effective ART or had only a single effective drug in

  1. A Novel Drug-Resistant HIV-1 Circulating Recombinant Form CRF76_01B Identified by Near Full-Length Genome Analysis.

    PubMed

    Ogawa, Satoko; Hachiya, Atsuko; Hosaka, Masumi; Matsuda, Masakazu; Ode, Hirotaka; Shigemi, Urara; Okazaki, Reiko; Sadamasu, Kenji; Nagashima, Mami; Toyokawa, Takao; Tateyama, Masao; Tanaka, Yasuhito; Sugiura, Wataru; Yokomaku, Yoshiyuki; Iwatani, Yasumasa

    2016-03-01

    HIV-1 CRF01_AE and subtype B (B) have dominated and their different circulating recombinant forms (CRFs) have emerged in East and Southeast Asian countries. Here, we report a novel drug-resistant HIV-1 CRF. Five independent recombinant specimens exhibiting discordant subtype results for the gag, pol, and env sequences were isolated. These recombinants had the CRF01_AE (gag p17)/B (pol PR-RT and IN)/CRF01_AE (env C2-V3) pattern similar to CRF69_01B. Sequence analysis of four near full-length HIV-1 genomes revealed a unique phylogenetic cluster distinct from previously reported CRFs. Of the four recombinants, three shared an identical mosaic structure including seven breakpoints in the gag, pol, vif, and env regions, designated CRF76_01B. The one remaining recombinant had additional recombination breakpoints in the vpu region and exhibited another unique recombinant form composed of CRF76_01B and B. These findings provide important insight into the transmission dynamics of HIV-1 in Asia that may be important for its effective prevention. PMID:26528581

  2. Prevention of mother-to-child HIV-1 transmission in Burkina Faso: evaluation of vertical transmission by PCR, molecular characterization of subtypes and determination of antiretroviral drugs resistance

    PubMed Central

    Sagna, Tani; Bisseye, Cyrille; Compaore, Tegewende R.; Kagone, Therese S.; Djigma, Florencia W.; Ouermi, Djeneba; Pirkle, Catherine M.; Zeba, Moctar T. A.; Bazie, Valerie J. T.; Douamba, Zoenabo; Moret, Remy; Pietra, Virginio; Koama, Adjirita; Gnoula, Charlemagne; Sia, Joseph D.; Nikiema, Jean-Baptiste; Simpore, Jacques

    2015-01-01

    Background Vertical human immunodeficiency virus (HIV) transmission is a public health problem in Burkina Faso. The main objective of this study on the prevention of mother-to-child HIV-1 transmission was to determine the residual risk of HIV transmission in infants born to mothers receiving highly active antiretroviral therapy (HAART). Moreover, we detect HIV antiretroviral (ARV) drug resistance among mother–infant pairs and identify subtypes and circulating recombinant forms (CRF) in Burkina Faso. Design In this study, 3,215 samples of pregnant women were analyzed for HIV using rapid tests. Vertical transmission was estimated by polymerase chain reaction in 6-month-old infants born to women who tested HIV positive. HIV-1 resistance to ARV, subtypes, and CRFs was determined through ViroSeq kit using the ABI PRISM 3,130 sequencer. Results In this study, 12.26% (394/3,215) of the pregnant women were diagnosed HIV positive. There was 0.52% (2/388) overall vertical transmission of HIV, with rates of 1.75% (2/114) among mothers under prophylaxis and 0.00% (0/274) for those under HAART. Genetic mutations were also isolated that induce resistance to ARV such as M184V, Y115F, K103N, Y181C, V179E, and G190A. There were subtypes and CRF of HIV-1 present, the most common being: CRF06_CPX (58.8%), CRF02_AG (35.3%), and subtype G (5.9%). Conclusions ARV drugs reduce the residual rate of HIV vertical transmission. However, the virus has developed resistance to ARV, which could limit future therapeutic options when treatment is needed. Resistance to ARV therefore requires a permanent interaction between researchers, physicians, and pharmacists, to strengthen the network of monitoring and surveillance of drug resistance in Burkina Faso. PMID:25630709

  3. Characterization of HIV-1 Resistance to Tenofovir Alafenamide In Vitro.

    PubMed

    Margot, Nicolas A; Johnson, Audun; Miller, Michael D; Callebaut, Christian

    2015-10-01

    Tenofovir alafenamide (TAF) is an investigational prodrug of the HIV-1 nucleotide reverse transcriptase (RT) inhibitor (NtRTI) tenofovir (TFV), with improved potency and drug delivery properties over the current prodrug, tenofovir disoproxil fumarate (TDF). TAF is currently in phase 3 clinical studies for the treatment of HIV-1 infection, in combination with other antiretroviral agents. Phase 1 and 2 studies have shown that TAF was associated with increased peripheral blood mononuclear cell (PBMC) drug loading and increased suppression of HIV-1 replication compared to treatment with TDF. In this study, selection of in vitro resistance to both TAF and the parent compound, TFV, led to the emergence of HIV-1 with the K65R amino acid substitution in RT with 6.5-fold-reduced susceptibility to TAF. Although TAF is more potent than TFV in vitro, the antiviral susceptibilities to TAF and TFV of a large panel of nucleoside/nucleotide RT inhibitor (NRTI)-resistant mutants were highly correlated (R(2) = 0.97), indicating that the two compounds have virtually the same resistance profile when assessed as fold change from the wild type. TAF showed full antiviral activity in PBMCs against primary HIV-1 isolates with protease inhibitor, nonnucleoside RT inhibitor (NNRTI), or integrase strand transfer inhibitor resistance but reduced activity against isolates with extensive NRTI resistance amino acid substitutions. However, the increased cell loading of TFV with TAF versus TDF observed in vivo suggests that TAF may retain activity against TDF-resistant mutant viruses. PMID:26149983

  4. Epidemiological Surveillance of HIV-1 Transmitted Drug Resistance in Spain in 2004-2012: Relevance of Transmission Clusters in the Propagation of Resistance Mutations

    PubMed Central

    Vega, Yolanda; Delgado, Elena; Fernández-García, Aurora; Cuevas, Maria Teresa; Thomson, Michael M.; Montero, Vanessa; Sánchez, Monica; Sánchez, Ana Maria; Pérez-Álvarez, Lucia

    2015-01-01

    Our objectives were to carry out an epidemiological surveillance study on transmitted drug resistance (TDR) among individuals newly diagnosed of HIV-1 infection during a nine year period in Spain and to assess the role of transmission clusters (TC) in the propagation of resistant strains. An overall of 1614 newly diagnosed individuals were included in the study from January 2004 through December 2012. Individuals come from two different Spanish regions: Galicia and the Basque Country. Resistance mutations to reverse transcriptase inhibitors (RTI) and protease inhibitors (PI) were analyzed according to mutations included in the surveillance drug-resistance mutations list updated in 2009. TC were defined as those comprising viruses from five or more individuals whose sequences clustered in maximum likelihood phylogenetic trees with a bootstrap value ≥90%. The overall prevalence of TDR to any drug was 9.9%: 4.9% to nucleoside RTIs (NRTIs), 3.6% to non-nucleoside RTIs (NNRTIs), and 2.7% to PIs. A significant decrease of TDR to NRTIs over time was observed [from 10% in 2004 to 2% in 2012 (p=0.01)]. Sixty eight (42.2%) of 161 sequences with TDR were included in 25 TC composed of 5 or more individuals. Of them, 9 clusters harbored TDR associated with high level resistance to antiretroviral drugs. T215D revertant mutation was transmitted in a large cluster comprising 25 individuals. The impact of epidemiological networks on TDR frequency may explain its persistence in newly diagnosed individuals. The knowledge of the populations involved in TC would facilitate the design of prevention programs and public health interventions. PMID:26010948

  5. Primer ID Informs Next-Generation Sequencing Platforms and Reveals Preexisting Drug Resistance Mutations in the HIV-1 Reverse Transcriptase Coding Domain

    PubMed Central

    Keys, Jessica R.; Zhou, Shuntai; Anderson, Jeffrey A.; Eron, Joseph J.; Rackoff, Lauren A.; Jabara, Cassandra

    2015-01-01

    Abstract Sequencing of a bulk polymerase chain reaction (PCR) product to identify drug resistance mutations informs antiretroviral therapy selection but has limited sensitivity for minority variants. Alternatively, deep sequencing is capable of detecting minority variants but is subject to sequencing errors and PCR resampling due to low input templates. We screened for resistance mutations among 184 HIV-1-infected, therapy-naive subjects using the 454 sequencing platform to sequence two amplicons spanning HIV-1 reverse transcriptase codons 34–245. Samples from 19 subjects were also analyzed using the MiSeq sequencing platform for comparison. Errors and PCR resampling were addressed by tagging each HIV-1 RNA template copy (i.e., cDNA) with a unique sequence tag (Primer ID), allowing a consensus sequence to be constructed for each original template from resampled sequences. In control reactions, Primer ID reduced 454 and MiSeq errors from 71 to 2.6 and from 24 to 1.2 errors/10,000 nucleotides, respectively. MiSeq also allowed accurate sequencing of codon 65, an important drug resistance position embedded in a homopolymeric run that is poorly resolved by the 454 platform. Excluding homopolymeric positions, 14% of subjects had evidence of ≥1 resistance mutation among Primer ID consensus sequences, compared to 2.7% by bulk population sequencing. When calls were restricted to mutations that appeared twice among consensus sequence populations, 6% of subjects had detectable resistance mutations. The use of Primer ID revealed 5–15% template utilization on average, limiting the depth of deep sequencing sampling and revealing sampling variation due to low template utilization. Primer ID addresses important limitations of deep sequencing and produces less biased estimates of low-level resistance mutations in the viral population. PMID:25748056

  6. Primer ID Informs Next-Generation Sequencing Platforms and Reveals Preexisting Drug Resistance Mutations in the HIV-1 Reverse Transcriptase Coding Domain.

    PubMed

    Keys, Jessica R; Zhou, Shuntai; Anderson, Jeffrey A; Eron, Joseph J; Rackoff, Lauren A; Jabara, Cassandra; Swanstrom, Ronald

    2015-06-01

    Sequencing of a bulk polymerase chain reaction (PCR) product to identify drug resistance mutations informs antiretroviral therapy selection but has limited sensitivity for minority variants. Alternatively, deep sequencing is capable of detecting minority variants but is subject to sequencing errors and PCR resampling due to low input templates. We screened for resistance mutations among 184 HIV-1-infected, therapy-naive subjects using the 454 sequencing platform to sequence two amplicons spanning HIV-1 reverse transcriptase codons 34-245. Samples from 19 subjects were also analyzed using the MiSeq sequencing platform for comparison. Errors and PCR resampling were addressed by tagging each HIV-1 RNA template copy (i.e., cDNA) with a unique sequence tag (Primer ID), allowing a consensus sequence to be constructed for each original template from resampled sequences. In control reactions, Primer ID reduced 454 and MiSeq errors from 71 to 2.6 and from 24 to 1.2 errors/10,000 nucleotides, respectively. MiSeq also allowed accurate sequencing of codon 65, an important drug resistance position embedded in a homopolymeric run that is poorly resolved by the 454 platform. Excluding homopolymeric positions, 14% of subjects had evidence of ≥1 resistance mutation among Primer ID consensus sequences, compared to 2.7% by bulk population sequencing. When calls were restricted to mutations that appeared twice among consensus sequence populations, 6% of subjects had detectable resistance mutations. The use of Primer ID revealed 5-15% template utilization on average, limiting the depth of deep sequencing sampling and revealing sampling variation due to low template utilization. Primer ID addresses important limitations of deep sequencing and produces less biased estimates of low-level resistance mutations in the viral population. PMID:25748056

  7. Patterns of drug resistance among newly diagnosed HIV-1 infected patients in Greece during the last decade: the crucial role of transmission networks

    PubMed Central

    Paraskevis, Dimitrios; Zavitsanou, Assimina; Magiorkinis, Emmanouil; Gargalianos, Panagiotis; Xylomenos, Georgios; Lazanas, Marios; Chini, Maria; Skoutelis, Athanasios; Papastamopoulos, Vasileios; Antoniadou, Anastasia; Papadopoulos, Antonios; Psichogiou, Mina; Daikos, Georgios; Vassilakis, Alexis; Chrysos, Georgios; Paparizos, Vasilis; Kourkounti, Sofia; Sambatakou, Helen; Kordossis, Theodoros; Koratzanis, Georgios; Panagopoulos, Periklis; Maltezos, Evangelos; Drimis, Stylianos; Hatzakis, Angelos

    2014-01-01

    Introduction The prevalence of drug resistance is approximately 10% in Europe and North America among newly infected patients. We aim to investigate the temporal patterns of resistance among drug naive HIV-infected individuals in Greece and also to determine transmission networking among those with resistant strains. Materials and Methods Protease (PR) and partial reverse transcriptase (RT) sequences were determined from 2499 newly diagnosed HIV-1 patients, in Greece, during 2003–2013. Genotypic drug resistance was estimated using the HIVdb: Genotypic Resistance Interpretation Algorithm. We identified transmission clusters of resistant strains on the basis of a large collection of HIV-1 sequences from 4024 seropositives in Greece. Phylodynamic analysis was performed using a Bayesian method. Results We estimated drug resistance levels among naïve patients on the basis of all resistance mutations in PR and partial RT. The overall prevalence of resistance was 19.6% (490/2499). Resistance to NNRTIs was the most common (397/2499, 15.9%) followed by PIs (116/2499, 4.6%) and NRTIs (79/2499, 3.2%). We found a significant trend for decreasing resistance to NRTIs over time (6.7%–1.6%). There was no time trend for the overall PI and NNRTI resistance. The most frequently observed major resistant sites in PR were V82 (2.0%) and L90 (1.8%). In RT, we found E138 (58.6%), K103 (13.1%), V179 (8.4%) and T215 (7.1%), M41 (4.7%) associated with resistance to NNRTIs and NRTIs, respectively. The prevalence of K103N and E138Q were significantly increased during 2003–2013. Crucially, we found that both K103N, E138Q are associated with transmission networking within men having sex with men (MSM) and intravenous drug user (IDU) local networks. The K103N network included seropositives across Greece, while the latter only from the recent IDU outbreak in Athens metropolitan area (1). Phylodynamic analyses revealed that the exponential growth for K103N network started in 2009 (Figure 1

  8. Use of amplification refractory mutation system PCR assay as a simple and effective tool to detect HIV-1 drug resistance mutations.

    PubMed

    Nanfack, Aubin J; Agyingi, Lucy; Noubiap, Jean Jacques N; Ngai, Johnson N; Colizzi, Vittorio; Nyambi, Phillipe N

    2015-05-01

    Access to genotyping assays to determine successful antiretroviral treatment (ART) is limited in resource-constrained settings by high cost, suggesting the need for a cost-effective and simplified method to identify HIV-1 drug resistance (HIVDR) mutations. In this study, an amplification refractory mutation system (ARMS)-PCR assay was developed and used to investigate the most frequent HIVDR mutations affecting first-line ART in settings where WHO ART guidelines are applied. Seventy-five HIV-positive (HIV(+)) samples from Cameroon were used to assess the performance of this assay. Sequencing of HIV-1 reverse transcriptase was simultaneously performed for comparison, and discordant samples were tested with a Trugene HIV-1 genotyping kit. The ARMS-PCR assay was able to detect M184V, T215Y/F, K103N, and Y181C mutations with sensitivities of 96.8%, 85.7%, 91.3%, and 70%, respectively, and specificities of 90.6%, 95%, 100%, 96.9%, respectively, compared with data on sequencing. The results indicated the highest positive predictive value for K103N (100%) and the highest negative predictive value for M184V (97.5%). ARMS-PCR's limits of detection for mutations M184V, T215Y/F, K103N, and Y181C were <75 copies/ml, 143 copies/ml, 143 copies/ml, and 836 copies/ml, respectively. ARMS-PCR efficiently identified mutations in individuals harboring different HIV-1 clades (CRF02_AG and non-CRF02_AG). In addition, this approach was more cost-effective than other genotyping assays. The high throughput, the cost-effectiveness, and the simplicity of the ARMS-PCR assay make it a suitable tool to monitor HIVDR patterns in resource-constrained settings with broad HIV-1 genetic diversity. PMID:25788547

  9. Impact of Primary Elvitegravir Resistance-Associated Mutations in HIV-1 Integrase on Drug Susceptibility and Viral Replication Fitness

    PubMed Central

    Hluhanich, Rebecca M.; Goodman, Derrick D.; Andreatta, Kristen N.; Margot, Nicolas A.; Ye, Linda; Niedziela-Majka, Anita; Barnes, Tiffany L.; Novikov, Nikolai; Chen, Xiaowu; Svarovskaia, Evguenia S.; McColl, Damian J.; White, Kirsten L.; Miller, Michael D.

    2013-01-01

    Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG. PMID:23529738

  10. Impact of primary elvitegravir resistance-associated mutations in HIV-1 integrase on drug susceptibility and viral replication fitness.

    PubMed

    Abram, Michael E; Hluhanich, Rebecca M; Goodman, Derrick D; Andreatta, Kristen N; Margot, Nicolas A; Ye, Linda; Niedziela-Majka, Anita; Barnes, Tiffany L; Novikov, Nikolai; Chen, Xiaowu; Svarovskaia, Evguenia S; McColl, Damian J; White, Kirsten L; Miller, Michael D

    2013-06-01

    Elvitegravir (EVG) is an effective HIV-1 integrase (IN) strand transfer inhibitor (INSTI) in advanced clinical development. Primary INSTI resistance-associated mutations (RAMs) at six IN positions have been identified in HIV-1-infected patients failing EVG-containing regimens in clinical studies: T66I/A/K, E92Q/G, T97A, S147G, Q148R/H/K, and N155H. In this study, the effect of these primary IN mutations, alone and in combination, on susceptibility to the INSTIs EVG, raltegravir (RAL), and dolutegravir (DTG); IN enzyme activities; and viral replication fitness was characterized. Recombinant viruses containing the six most common mutations exhibited a range of reduced EVG susceptibility: 92-fold for Q148R, 30-fold for N155H, 26-fold for E92Q, 10-fold for T66I, 4-fold for S147G, and 2-fold for T97A. Less commonly observed primary IN mutations also showed a range of reduced EVG susceptibilities: 40- to 94-fold for T66K and Q148K and 5- to 10-fold for T66A, E92G, and Q148H. Some primary IN mutations exhibited broad cross-resistance between EVG and RAL (T66K, E92Q, Q148R/H/K, and N155H), while others retained susceptibility to RAL (T66I/A, E92G, T97A, and S147G). Dual combinations of primary IN mutations further reduced INSTI susceptibility, replication capacity, and viral fitness relative to either mutation alone. Susceptibility to DTG was retained by single primary IN mutations but reduced by dual mutation combinations with Q148R. Primary EVG RAMs also diminished IN enzymatic activities, concordant with their structural proximity to the active site. Greater reductions in viral fitness of dual mutation combinations may explain why some primary INSTI RAMs do not readily coexist on the same HIV-1 genome but rather establish independent pathways of resistance to EVG. PMID:23529738

  11. Prevalence of transmitted HIV-1 drug resistance among female sex workers and men who have sex with men in El Salvador, Central America.

    PubMed

    Murillo, Wendy; Lorenzana de Rivera, Ivette; Albert, Jan; Guardado, María Elena; Nieto, Ana Isabel; Paz-Bailey, Gabriela

    2012-10-01

    Transmitted drug resistance has important implications for the successful use and management of therapy among persons infected with HIV. We estimated the prevalence of transmitted drug resistance in 145 samples from female sex workers (n = 47) and men who have sex with men (n = 98) in El Salvador. Samples were collected during March to September 2008, using a respondent driven sampling. The HIV-1 pol gene was sequenced to identify drug resistance mutations and transmitted drug resistance was scored as recommended by World Health Organization. Specimens were classified as recent or established infections using the Immunoglobulin G-Capture BED-Enzyme Immunoassay. The overall prevalence of transmitted drug resistance was 9.4% (95% CI: 4.7-16.1%), and was 5.9% for non-nucleoside reverse transcriptase inhibitors, 4.2% for nucleoside reverse transcriptase inhibitors, and 0.8% for protease inhibitors. Transmitted drug resistance prevalence was 10.3% (95% CI: 2.8-24.2%) among female sex workers, and 9.0% (95% CI: 3.6-17.6%) among men who have sex with men. Nineteen patients were classified as having recent infection (16.2%, 95% CI: 10.1-24.2%), while 98 patients (83.8%, 95% CI: 75.8-89.9%) were classified as having established infections. Transmitted drug resistance among recent and established infections was similar at 10.5% and 9.2%, respectively. This study shows that the prevalence of transmitted drug resistance is moderate among female sex workers and men who have sex with men in El Salvador. These results highlight the importance of transmitted drug resistance surveillance in a representative sample of recently infected patients following the World Health Organization guidelines. PMID:22930496

  12. Characterizing the Diverse Mutational Pathways Associated with R5-Tropic Maraviroc Resistance: HIV-1 That Uses the Drug-Bound CCR5 Coreceptor

    PubMed Central

    Jiang, Xiaowei; Feyertag, Felix; Meehan, Conor J.; McCormack, Grace P.; Travers, Simon A.; Craig, Charles; Westby, Mike; Lewis, Marilyn

    2015-01-01

    ABSTRACT Entry inhibitors represent a potent class of antiretroviral drugs that target a host cell protein, CCR5, an HIV-1 entry coreceptor, and not viral protein. Lack of sensitivity can occur due to preexisting virus that uses the CXCR4 coreceptor, while true resistance occurs through viral adaptation to use a drug-bound CCR5 coreceptor. To understand this R5 resistance pathway, we analyzed >500 envelope protein sequences and phenotypes from viruses of 20 patients from the clinical trials MOTIVATE 1 and 2, in which treatment-experienced patients received maraviroc plus optimized background therapy. The resistant viral population was phylogenetically distinct and associated with a genetic bottleneck in each patient, consistent with de novo emergence of resistance. Recombination analysis showed that the C2-V3-C3 region tends to genotypically correspond to the recombinant's phenotype, indicating its primary importance in conferring resistance. Between patients, there was a notable lack of commonality in the specific sites conferring resistance, confirming the unusual nature of R5-tropic resistance. We used coevolutionary and positive-selection analyses to characterize the genotypic determinants of resistance and found that (i) there are complicated covariation networks, indicating frequent coevolutionary/compensatory changes in the context of protein structure; (ii) covarying sites under positive selection are enriched in resistant viruses; (iii) CD4 binding sites form part of a unique covariation network independent of the V3 loop; and (iv) the covariation network formed between the V3 loop and other regions of gp120 and gp41 intersects sites involved in glycosylation and protein secretion. These results demonstrate that while envelope sequence mutations are the key to conferring maraviroc resistance, the specific changes involved are context dependent and thus inherently unpredictable. IMPORTANCE The entry inhibitor drug maraviroc makes the cell coreceptor CCR5

  13. Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex

    SciTech Connect

    Prashar, Vishal; Bihani, Subhash C.; Das, Amit; Rao, D.R.; Hosur, M.V.

    2010-06-11

    The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

  14. Analysis of 454 sequencing error rate, error sources, and artifact recombination for detection of Low-frequency drug resistance mutations in HIV-1 DNA

    PubMed Central

    2013-01-01

    Background 454 sequencing technology is a promising approach for characterizing HIV-1 populations and for identifying low frequency mutations. The utility of 454 technology for determining allele frequencies and linkage associations in HIV infected individuals has not been extensively investigated. We evaluated the performance of 454 sequencing for characterizing HIV populations with defined allele frequencies. Results We constructed two HIV-1 RT clones. Clone A was a wild type sequence. Clone B was identical to clone A except it contained 13 introduced drug resistant mutations. The clones were mixed at ratios ranging from 1% to 50% and were amplified by standard PCR conditions and by PCR conditions aimed at reducing PCR-based recombination. The products were sequenced using 454 pyrosequencing. Sequence analysis from standard PCR amplification revealed that 14% of all sequencing reads from a sample with a 50:50 mixture of wild type and mutant DNA were recombinants. The majority of the recombinants were the result of a single crossover event which can happen during PCR when the DNA polymerase terminates synthesis prematurely. The incompletely extended template then competes for primer sites in subsequent rounds of PCR. Although less often, a spectrum of other distinct crossover patterns was also detected. In addition, we observed point mutation errors ranging from 0.01% to 1.0% per base as well as indel (insertion and deletion) errors ranging from 0.02% to nearly 50%. The point errors (single nucleotide substitution errors) were mainly introduced during PCR while indels were the result of pyrosequencing. We then used new PCR conditions designed to reduce PCR-based recombination. Using these new conditions, the frequency of recombination was reduced 27-fold. The new conditions had no effect on point mutation errors. We found that 454 pyrosequencing was capable of identifying minority HIV-1 mutations at frequencies down to 0.1% at some nucleotide positions. Conclusion

  15. Computational Studies of a Mechanism for Binding and Drug Resistance in the Wild Type and Four Mutations of HIV-1 Protease with a GRL-0519 Inhibitor

    PubMed Central

    Hu, Guodong; Ma, Aijing; Dou, Xianghua; Zhao, Liling; Wang, Jihua

    2016-01-01

    Drug resistance of mutations in HIV-1 protease (PR) is the most severe challenge to the long-term efficacy of HIV-1 PR inhibitor in highly active antiretroviral therapy. To elucidate the molecular mechanism of drug resistance associated with mutations (D30N, I50V, I54M, and V82A) and inhibitor (GRL-0519) complexes, we have performed five molecular dynamics (MD) simulations and calculated the binding free energies using the molecular mechanics Poisson–Boltzmann surface area (MM-PBSA) method. The ranking of calculated binding free energies is in accordance with the experimental data. The free energy spectra of each residue and inhibitor interaction for all complexes show a similar binding model. Analysis based on the MD trajectories and contribution of each residues show that groups R2 and R3 mainly contribute van der Waals energies, while groups R1 and R4 contribute electrostatic interaction by hydrogen bonds. The drug resistance of D30N can be attributed to the decline in binding affinity of residues 28 and 29. The size of Val50 is smaller than Ile50 causes the residue to move, especially in chain A. The stable hydrophobic core, including the side chain of Ile54 in the wild type (WT) complex, became unstable in I54M because the side chain of Met54 is flexible with two alternative conformations. The binding affinity of Ala82 in V82A decreases relative to Val82 in WT. The present study could provide important guidance for the design of a potent new drug resisting the mutation inhibitors. PMID:27240358

  16. Computational Studies of a Mechanism for Binding and Drug Resistance in the Wild Type and Four Mutations of HIV-1 Protease with a GRL-0519 Inhibitor.

    PubMed

    Hu, Guodong; Ma, Aijing; Dou, Xianghua; Zhao, Liling; Wang, Jihua

    2016-01-01

    Drug resistance of mutations in HIV-1 protease (PR) is the most severe challenge to the long-term efficacy of HIV-1 PR inhibitor in highly active antiretroviral therapy. To elucidate the molecular mechanism of drug resistance associated with mutations (D30N, I50V, I54M, and V82A) and inhibitor (GRL-0519) complexes, we have performed five molecular dynamics (MD) simulations and calculated the binding free energies using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method. The ranking of calculated binding free energies is in accordance with the experimental data. The free energy spectra of each residue and inhibitor interaction for all complexes show a similar binding model. Analysis based on the MD trajectories and contribution of each residues show that groups R2 and R3 mainly contribute van der Waals energies, while groups R1 and R4 contribute electrostatic interaction by hydrogen bonds. The drug resistance of D30N can be attributed to the decline in binding affinity of residues 28 and 29. The size of Val50 is smaller than Ile50 causes the residue to move, especially in chain A. The stable hydrophobic core, including the side chain of Ile54 in the wild type (WT) complex, became unstable in I54M because the side chain of Met54 is flexible with two alternative conformations. The binding affinity of Ala82 in V82A decreases relative to Val82 in WT. The present study could provide important guidance for the design of a potent new drug resisting the mutation inhibitors. PMID:27240358

  17. Transmitted Drug Resistance and Antiretroviral Treatment Outcomes in Non-Subtype B HIV1- Infected Patients in South East Asia

    PubMed Central

    Phanuphak, Praphan; Sirivichayakul, Sunee; Jiamsakul, Awachana; Sungkanuparph, Somnuek; Kumarasamy, Nagalingeswaran; Lee, Man Po; Sirisanthana, Thira; Kantipong, Pacharee; Lee, Christopher; Kamarulzaman, Adeeba; Mustafa, Mahiran; Ditangco, Rossana; Merati, Tuti; Ratanasuwan, Winai; Singtoroj, Thida; Kantor, Rami

    2014-01-01

    Background We compared treatment outcomes of transmitted drug resistance (TDR) in patients on fully or partially sensitive drug regimens. Methods Factors associated with survival and failure were analyzed using Cox proportional hazards and discrete time conditional logistic models. Results TDR, found in 60/1471 (4.1%) Asian treatment naïve patients, was one of the significant predictors of failure. Patients with TDR to >1 drug in their regimen were >3 times as likely to fail compared to no TDR. Conclusion TDR was associated with failure in the context of non-fully sensitive regimens. Efforts are needed to incorporate resistance testing into national treatment programs. PMID:24413039

  18. CRF01_AE/B/C, a Novel Drug-Resistant HIV-1 Recombinant in Men Who Have Sex with Men in Beijing, China.

    PubMed

    Yan, Jing; Xin, Ruolei; Li, Zheng; Feng, Yi; Lu, Hongyan; Liao, Lingjie; Ruan, Yuhua; Shao, Yiming; Xing, Hui

    2015-07-01

    We report a unique HIV-1 recombinant strain (URF) from an HIV-positive man who has sex with men (MSM) in Beijing, China. This virus genome has insertions and multiple drug-resistant mutations to both nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs), as well as a complex intersubtype recombinant structure with 11 breakpoints. Phylogenetic analysis of the near full-length genome (NFLG) shows that this URF is comprised of gene regions originating from three circulating viral strains: CRF01_AE, subtype B, and subtype C. The parental CRF01_AE regions of the recombinant cluster with a previously described cluster 4 sublineage of CRF01_AE. The B regions of the recombinant cluster within the B (United States-European origin) subtype and the three subtype C regions cluster with a strain detected in China in 1998. The detection and characterization of this complex drug-resistant URF indicate an ongoing generation of recombinant strains among MSM, and will help to provide insight into our understanding of the dynamics and complexity of the HIV-1 epidemic in China. PMID:25962473

  19. HIV-1 Antiretroviral Drug Resistance Mutations in Treatment Naïve and Experienced Panamanian Subjects: Impact on National Use of EFV-Based Schemes.

    PubMed

    Mendoza, Yaxelis; Castillo Mewa, Juan; Martínez, Alexander A; Zaldívar, Yamitzel; Sosa, Néstor; Arteaga, Griselda; Armién, Blas; Bautista, Christian T; García-Morales, Claudia; Tapia-Trejo, Daniela; Ávila-Ríos, Santiago; Reyes-Terán, Gustavo; Bello, Gonzalo; Pascale, Juan M

    2016-01-01

    The use of antiretroviral therapy in HIV infected subjects prevents AIDS-related illness and delayed occurrence of death. In Panama, rollout of ART started in 1999 and national coverage has reached 62.8% since then. The objective of this study was to determine the level and patterns of acquired drug resistance mutations of clinical relevance (ADR-CRM) and surveillance drug resistance mutations (SDRMs) from 717 HIV-1 pol gene sequences obtained from 467 ARV drug-experienced and 250 ARV drug-naïve HIV-1 subtypes B infected subjects during 2007-2013, respectively. The overall prevalence of SDRM and of ADR-CRM during the study period was 9.2% and 87.6%, respectively. The majority of subjects with ADR-CRM had a pattern of mutations that confer resistance to at least two classes of ARV inhibitors. The non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations K103N and P225H were more prevalent in both ARV drug-naïve and ARV drug-experienced subjects. The nucleoside reverse transcriptase inhibitor (NRTI) mutation M184V was more frequent in ARV drug-experienced individuals, while T215YFrev and M41L were more frequent in ARV drug-naïve subjects. Prevalence of mutations associated to protease inhibitors (PI) was lower than 4.1% in both types of subjects. Therefore, there is a high level of resistance (>73%) to Efavirenz/Nevirapine, Lamivudine and Azidothymidine in ARV drug-experienced subjects, and an intermediate to high level of resistance (5-10%) to Efavirenz/Nevirapine in ARV drug-naïve subjects. During the study period, we observed an increasing trend in the prevalence of ADR-CRM in subjects under first-line schemes, but not significant changes in the prevalence of SDRM. These results reinforce the paramount importance of a national surveillance system of ADR-CRM and SDRM for national management policies of subjects living with HIV. PMID:27119150

  20. HIV-1 Antiretroviral Drug Resistance Mutations in Treatment Naïve and Experienced Panamanian Subjects: Impact on National Use of EFV-Based Schemes

    PubMed Central

    Mendoza, Yaxelis; Castillo Mewa, Juan; Martínez, Alexander A.; Zaldívar, Yamitzel; Sosa, Néstor; Arteaga, Griselda; Armién, Blas; Bautista, Christian T.; García-Morales, Claudia; Tapia-Trejo, Daniela; Ávila-Ríos, Santiago; Reyes-Terán, Gustavo; Bello, Gonzalo; Pascale, Juan M.

    2016-01-01

    The use of antiretroviral therapy in HIV infected subjects prevents AIDS-related illness and delayed occurrence of death. In Panama, rollout of ART started in 1999 and national coverage has reached 62.8% since then. The objective of this study was to determine the level and patterns of acquired drug resistance mutations of clinical relevance (ADR-CRM) and surveillance drug resistance mutations (SDRMs) from 717 HIV-1 pol gene sequences obtained from 467 ARV drug-experienced and 250 ARV drug-naïve HIV-1 subtypes B infected subjects during 2007–2013, respectively. The overall prevalence of SDRM and of ADR-CRM during the study period was 9.2% and 87.6%, respectively. The majority of subjects with ADR-CRM had a pattern of mutations that confer resistance to at least two classes of ARV inhibitors. The non-nucleoside reverse transcriptase inhibitor (NNRTI) mutations K103N and P225H were more prevalent in both ARV drug-naïve and ARV drug-experienced subjects. The nucleoside reverse transcriptase inhibitor (NRTI) mutation M184V was more frequent in ARV drug-experienced individuals, while T215YFrev and M41L were more frequent in ARV drug-naïve subjects. Prevalence of mutations associated to protease inhibitors (PI) was lower than 4.1% in both types of subjects. Therefore, there is a high level of resistance (>73%) to Efavirenz/Nevirapine, Lamivudine and Azidothymidine in ARV drug-experienced subjects, and an intermediate to high level of resistance (5–10%) to Efavirenz/Nevirapine in ARV drug-naïve subjects. During the study period, we observed an increasing trend in the prevalence of ADR-CRM in subjects under first-line schemes, but not significant changes in the prevalence of SDRM. These results reinforce the paramount importance of a national surveillance system of ADR-CRM and SDRM for national management policies of subjects living with HIV. PMID:27119150

  1. Multi-drug resistance profile of PR20 HIV-1 protease is attributed to distorted conformational and drug binding landscape: molecular dynamics insights.

    PubMed

    Chetty, Sarentha; Bhakat, Soumendranath; Martin, Alberto J M; Soliman, Mahmoud E S

    2016-01-01

    The PR20 HIV-1 protease, a variant with 20 mutations, exhibits high levels of multi-drug resistance; however, to date, there has been no report detailing the impact of these 20 mutations on the conformational and drug binding landscape at a molecular level. In this report, we demonstrate the first account of a comprehensive study designed to elaborate on the impact of these mutations on the dynamic features as well as drug binding and resistance profile, using extensive molecular dynamics analyses. Comparative MD simulations for the wild-type and PR20 HIV proteases, starting from bound and unbound conformations in each case, were performed. Results showed that the apo conformation of the PR20 variant of the HIV protease displayed a tendency to remain in the open conformation for a longer period of time when compared to the wild type. This led to a phenomena in which the inhibitor seated at the active site of PR20 tends to diffuse away from the binding site leading to a significant change in inhibitor-protein association. Calculating the per-residue fluctuation (RMSF) and radius of gyration, further validated these findings. MM/GBSA showed that the occurrence of 20 mutations led to a drop in the calculated binding free energies (ΔGbind) by ~25.17 kcal/mol and ~5 kcal/mol for p2-NC, a natural peptide substrate, and darunavir, respectively, when compared to wild type. Furthermore, the residue interaction network showed a diminished inter-residue hydrogen bond network and changes in inter-residue connections as a result of these mutations. The increased conformational flexibility in PR20 as a result of loss of intra- and inter-molecular hydrogen bond interactions and other prominent binding forces led to a loss of protease grip on ligand. It is interesting to note that the difference in conformational flexibility between PR20 and WT conformations was much higher in the case of substrate-bound conformation as compared to DRV. Thus, developing analogues of DRV by

  2. HIV-1 drug resistance in recently HIV-infected pregnant mother’s naïve to antiretroviral therapy in Dodoma urban, Tanzania

    PubMed Central

    2013-01-01

    Background HIV resistance affects virological response to therapy and efficacy of prophylaxis in mother-to-child-transmission. The study aims to assess the prevalence of HIV primary resistance in pregnant women naïve to antiretrovirals. Methods Cross sectional baseline analysis of a cohort of HIV + pregnant women (HPW) enrolled in the study entitled Antiretroviral Management of Antenatal and Natal HIV Infection (AMANI, peace in Kiswahili language). The AMANI study began in May 2010 in Dodoma, Tanzania. In this observational cohort, antiretroviral treatment was provided to all women from the 28th week of gestation until the end of the breastfeeding period. Baseline CD4 cell count, viral load and HIV drug-resistance genotype were collected. Results Drug-resistance analysis was performed on 97 naïve infected-mothers. The prevalence of all primary drug resistance and primary non-nucleoside reverse-transcriptase inhibitors resistance was 11.9% and 7.5%, respectively. K103S was found in two women with no M184V detection. HIV-1 subtype A was the most commonly identified, with a high prevalence of subtype A1, followed by C, D, C/D recombinant, A/C recombinant and A/D recombinant. HIV drug- resistance mutations were detected in A1 and C subtypes. Conclusion Our study reports an 11.9% prevalence rate of primary drug resistance in naïve HIV-infected pregnant women from a remote area of Tanzania. Considering that the non-nucleoside reverse-transcriptase inhibitors are part of the first-line antiretroviral regimen in Tanzania and all of Africa, resistance surveys should be prioritized in settings where antiretroviral therapy programs are scaled up. PMID:24053581

  3. Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors

    PubMed Central

    Schneider, Anna; Corona, Angela; Spöring, Imke; Jordan, Mareike; Buchholz, Bernd; Maccioni, Elias; Di Santo, Roberto; Bodem, Jochen; Tramontano, Enzo; Wöhrl, Birgitta M.

    2016-01-01

    We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs. PMID:26850643

  4. Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors.

    PubMed

    Schneider, Anna; Corona, Angela; Spöring, Imke; Jordan, Mareike; Buchholz, Bernd; Maccioni, Elias; Di Santo, Roberto; Bodem, Jochen; Tramontano, Enzo; Wöhrl, Birgitta M

    2016-03-18

    We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs. PMID:26850643

  5. The Roles of HIV-1 Proteins and Antiretroviral Drug Therapy in HIV-1-Associated Endothelial Dysfunction

    PubMed Central

    Kline, Erik R.; Sutliff, Roy L.

    2008-01-01

    Since the emergence of highly active antiretroviral therapy (HAART), human immunodeficiency virus-1 (HIV-1)-infected patients have demonstrated dramatic decreases in viral burden and opportunistic infections, and an overall increase in life expectancy. Despite these positive HAART-associated outcomes, it has become increasingly clear that HIV-1 patients have an enhanced risk of developing cardiovascular disease over time. Clinical studies are instrumental in our understanding of vascular dysfunction in the context of HIV-1 infection. However, most clinical studies often do not distinguish whether HIV-1 proteins, HAART, or a combination of these 2 factors cause cardiovascular complications. This review seeks to address the roles of both HIV-1 proteins and antiretroviral drugs in the development of endothelial dysfunction because endothelial dysfunction is the hallmark initial step of many cardiovascular diseases. We analyze recent in vitro and in vivo studies examining endothelial toxicity in response to HIV-1 proteins or in response to the various classes of antiretroviral drugs. Furthermore, we discuss the multiple mechanisms by which HIV-1 proteins and HAART injure the vascular endothelium in HIV-1 patients. By understanding the molecular mechanisms of HIV-1 protein- and antiretroviral-induced cardiovascular disease, we may ultimately improve the quality of life of HIV-1 patients through better drug design and the discovery of new pharmacological targets. PMID:18525451

  6. A Follow-Up of the Multicenter Collaborative Study on HIV-1 Drug Resistance and Tropism Testing Using 454 Ultra Deep Pyrosequencing

    PubMed Central

    St. John, Elizabeth P.; Simen, Birgitte B.; Turenchalk, Gregory S.; Braverman, Michael S.; Abbate, Isabella; Aerssens, Jeroen; Bouchez, Olivier; Gabriel, Christian; Izopet, Jacques; Meixenberger, Karolin; Di Giallonardo, Francesca; Schlapbach, Ralph; Paredes, Roger; Sakwa, James; Schmitz-Agheguian, Gudrun G.; Thielen, Alexander; Victor, Martin

    2016-01-01

    Background Ultra deep sequencing is of increasing use not only in research but also in diagnostics. For implementation of ultra deep sequencing assays in clinical laboratories for routine diagnostics, intra- and inter-laboratory testing are of the utmost importance. Methods A multicenter study was conducted to validate an updated assay design for 454 Life Sciences’ GS FLX Titanium system targeting protease/reverse transcriptase (RTP) and env (V3) regions to identify HIV-1 drug-resistance mutations and determine co-receptor use with high sensitivity. The study included 30 HIV-1 subtype B and 6 subtype non-B samples with viral titers (VT) of 3,940–447,400 copies/mL, two dilution series (52,129–1,340 and 25,130–734 copies/mL), and triplicate samples. Amplicons spanning PR codons 10–99, RT codons 1–251 and the entire V3 region were generated using barcoded primers. Analysis was performed using the GS Amplicon Variant Analyzer and geno2pheno for tropism. For comparison, population sequencing was performed using the ViroSeq HIV-1 genotyping system. Results The median sequencing depth across the 11 sites was 1,829 reads per position for RTP (IQR 592–3,488) and 2,410 for V3 (IQR 786–3,695). 10 preselected drug resistant variants were measured across sites and showed high inter-laboratory correlation across all sites with data (P<0.001). The triplicate samples of a plasmid mixture confirmed the high inter-laboratory consistency (mean% ± stdev: 4.6 ±0.5, 4.8 ±0.4, 4.9 ±0.3) and revealed good intra-laboratory consistency (mean% range ± stdev range: 4.2–5.2 ± 0.04–0.65). In the two dilutions series, no variants >20% were missed, variants 2–10% were detected at most sites (even at low VT), and variants 1–2% were detected by some sites. All mutations detected by population sequencing were also detected by UDS. Conclusions This assay design results in an accurate and reproducible approach to analyze HIV-1 mutant spectra, even at variant frequencies

  7. Potent Antiviral HIV-1 Protease Inhibitor GRL-02031 Adapts to the Structures of Drug Resistant Mutants with Its P1;#8242;-Pyrrolidinone Ring

    SciTech Connect

    Chang, Yu-Chung E.; Yu, XiaXia; Zhang, Ying; Tie, Yunfeng; Wang, Yuan-Fang; Yashchuk, Sofiya; Ghosh, Arun K.; Harrison, Robert W.; Weber, Irene T.

    2012-11-14

    GRL-02031 (1) is an HIV-1 protease (PR) inhibitor containing a novel P1' (R)-aminomethyl-2-pyrrolidinone group. Crystal structures at resolutions of 1.25-1.55 {angstrom} were analyzed for complexes of 1 with the PR containing major drug resistant mutations, PR{sub I47V}, PR{sub L76V}, PR{sub V82A}, and PR{sub N88D}. Mutations of I47V and V82A alter residues in the inhibitor-binding site, while L76V and N88D are distal mutations having no direct contact with the inhibitor. Substitution of a smaller amino acid in PR{sub I47V} and PR{sub L76V} and the altered charge of PR{sub N88D} are associated with significant local structural changes compared to the wild-type PR{sub WT}, while substitution of alanine in PR{sub V82A} increases the size of the S1' subsite. The P1' pyrrolidinone group of 1 accommodates to these local changes by assuming two different conformations. Overall, the conformation and interactions of 1 with PR mutants resemble those of PR{sub WT} with similar inhibition constants in good agreement with the antiviral potency on multidrug resistant HIV-1.

  8. Global epidemiology of drug resistance after failure of WHO recommended first-line regimens for adult HIV-1 infection: a multicentre retrospective cohort study

    PubMed Central

    2016-01-01

    Summary Background Antiretroviral therapy (ART) is crucial for controlling HIV-1 infection through wide-scale treatment as prevention and pre-exposure prophylaxis (PrEP). Potent tenofovir disoproxil fumarate-containing regimens are increasingly used to treat and prevent HIV, although few data exist for frequency and risk factors of acquired drug resistance in regions hardest hit by the HIV pandemic. We aimed to do a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART. Methods The TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials of HIV drug resistance testing in Europe, Latin and North America, sub-Saharan Africa, and Asia. We extracted and harmonised data for patients undergoing genotypic resistance testing after virological failure with a first-line regimen containing tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleotide reverse-transcriptase inhibitor (NNRTI; efavirenz or nevirapine). We used an individual participant-level meta-analysis and multiple logistic regression to identify covariates associated with drug resistance. Our primary outcome was tenofovir resistance, defined as presence of K65R/N or K70E/G/Q mutations in the reverse transcriptase (RT) gene. Findings We included 1926 patients from 36 countries with treatment failure between 1998 and 2015. Prevalence of tenofovir resistance was highest in sub-Saharan Africa (370/654 [57%]). Pre-ART CD4 cell count was the covariate most strongly associated with the development of tenofovir resistance (odds ratio [OR] 1·50, 95% CI 1·27–1·77 for CD4 cell count <100 cells per μL). Use of lamivudine versus emtricitabine increased the risk of tenofovir resistance across regions (OR 1·48, 95% CI 1·20–1·82). Of 700 individuals with tenofovir resistance, 578 (83%) had cytosine analogue resistance (M184V/I mutation), 543 (78%) had major NNRTI resistance, and 457 (65%) had both. The mean plasma

  9. The Need for Development of New HIV-1 Reverse Transcriptase and Integrase Inhibitors in the Aftermath of Antiviral Drug Resistance

    PubMed Central

    Wainberg, Mark A.

    2012-01-01

    The use of highly active antiretroviral therapy (HAART) involves combinations of drugs to achieve maximal virological response and reduce the potential for the emergence of antiviral resistance. There are two broad classes of reverse transcriptase inhibitors, the nucleoside reverse transcriptase inhibitors (NRTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Since the first classes of such compounds were developed, viral resistance against them has necessitated the continuous development of novel compounds within each class. This paper considers the NRTIs and NNRTIs currently in both preclinical and clinical development or approved for second line therapy and describes the patterns of resistance associated with their use, as well as the underlying mechanisms that have been described. Due to reasons of both affordability and availability, some reverse transcriptase inhibitors with low genetic barrier are more commonly used in resource-limited settings. Their use results to the emergence of specific patterns of antiviral resistance and so may require specific actions to preserve therapeutic options for patients in such settings. More recently, the advent of integrase strand transfer inhibitors represents another major step forward toward control of HIV infection, but these compounds are also susceptible to problems of HIV drug resistance. PMID:24278679

  10. HIV-1 RNA Levels and Antiretroviral Drug Resistance in Blood and Non-Blood Compartments from HIV-1–Infected Men and Women enrolled in AIDS Clinical Trials Group Study A5077

    PubMed Central

    Kantor, Rami; Bettendorf, Daniel; Bosch, Ronald J.; Mann, Marita; Katzenstein, David; Cu-Uvin, Susan; D’Aquila, Richard; Frenkel, Lisa; Fiscus, Susan; Coombs, Robert

    2014-01-01

    Background Detectable HIV-1 in body compartments can lead to transmission and antiretroviral resistance. Although sex differences in viral shedding have been demonstrated, mechanisms and magnitude are unclear. We compared RNA levels in blood, genital-secretions and saliva; and drug resistance in plasma and genital-secretions of men and women starting/changing antiretroviral therapy (ART) in the AIDS Clinical Trials Group (ACTG) 5077 study. Methods Blood, saliva and genital-secretions (compartment fluids) were collected from HIV-infected adults (≥13 years) at 14 United-States sites, who were initiating or changing ART with plasma viral load (VL) ≥2,000 copies/mL. VL testing was performed on all compartment fluids and HIV resistance genotyping on plasma and genital-secretions. Spearman rank correlations were used to evaluate concordance and Fisher’s and McNemar’s exact tests to compare VL between sexes and among compartments. Results Samples were available for 143 subjects; 36% treated (23 men, 29 women) and 64% ‘untreated’ (40 men, 51 women). RNA detection was significantly more frequent in plasma (100%) than genital-secretions (57%) and saliva (64%) (P<0.001). A higher proportion of men had genital shedding versus women (78% versus 41%), and RNA detection was more frequent in saliva versus genital-secretions in women when adjusted for censoring at the limit of assay detection. Inter-compartment fluid VL concordance was low in both sexes. In 22 (13 men, 9 women) paired plasma-genital-secretion genotypes from treated subjects, most had detectable resistance in both plasma (77%) and genital-secretions (68%). Resistance discordance was observed between compartments in 14% of subjects. Conclusions HIV shedding and drug resistance detection prior to initiation/change of ART in ACTG 5077 subjects differed among tissues and between sexes, making the gold standard blood-plasma compartment assessment not fully representative of HIV at other tissue sites

  11. Biochemical analysis of the role of G118R-linked dolutegravir drug resistance substitutions in HIV-1 integrase.

    PubMed

    Quashie, Peter K; Mesplède, Thibault; Han, Ying-Shan; Veres, Tamar; Osman, Nathan; Hassounah, Said; Sloan, Richard D; Xu, Hong-Tao; Wainberg, Mark A

    2013-12-01

    Drug resistance mutations (DRMs) have been reported for all currently approved anti-HIV drugs, including the latest integrase strand transfer inhibitors (INSTIs). We previously used the new INSTI dolutegravir (DTG) to select a G118R integrase resistance substitution in tissue culture and also showed that secondary substitutions emerged at positions H51Y and E138K. Now, we have characterized the impact of the G118R substitution, alone or in combination with either H51Y or E138K, on 3' processing and integrase strand transfer activity. The results show that G118R primarily impacted the strand transfer step of integration by diminishing the ability of integrase-long terminal repeat (LTR) complexes to bind target DNA. The addition of H51Y and E138K to G118R partially restored strand transfer activity by modulating the formation of integrase-LTR complexes through increasing LTR DNA affinity and total DNA binding, respectively. This unique mechanism, in which one function of HIV integrase partially compensates for the defect in another function, has not been previously reported. The G118R substitution resulted in low-level resistance to DTG, raltegravir (RAL), and elvitegravir (EVG). The addition of either of H51Y or E138K to G118R did not enhance resistance to DTG, RAL, or EVG. Homology modeling provided insight into the mechanism of resistance conferred by G118R as well as the effects of H51Y or E138K on enzyme activity. The G118R substitution therefore represents a potential avenue for resistance to DTG, similar to that previously described for the R263K substitution. For both pathways, secondary substitutions can lead to either diminished integrase activity and/or increased INSTI susceptibility. PMID:24080645

  12. CCR5 antibodies HGS004 and HGS101 preferentially inhibit drug-bound CCR5 infection and restore drug sensitivity of Maraviroc-resistant HIV-1 in primary cells

    SciTech Connect

    Latinovic, Olga; Reitz, Marvin; Le, Nhut M.; Foulke, James S.; Faetkenheuer, Gerd; Lehmann, Clara; Redfield, Robert R.; Heredia, Alonso

    2011-03-01

    R5 HIV-1 strains resistant to the CCR5 antagonist Maraviroc (MVC) can use drug-bound CCR5. We demonstrate that MVC-resistant HIV-1 exhibits delayed kinetics of coreceptor engagement and fusion during drug-bound versus free CCR5 infection of cell lines. Antibodies directed against the second extracellular loop (ECL2) of CCR5 had greater antiviral activity against MVC-bound compared to MVC-free CCR5 infection. However, in PBMCs, only ECL2 CCR5 antibodies HGS004 and HGS101, but not 2D7, inhibited infection by MVC resistant HIV-1 more potently with MVC-bound than with free CCR5. In addition, HGS004 and HGS101, but not 2D7, restored the antiviral activity of MVC against resistant virus in PBMCs. In flow cytometric studies, CCR5 binding by the HGS mAbs, but not by 2D7, was increased when PBMCs were treated with MVC, suggesting MVC increases exposure of the relevant epitope. Thus, HGS004 and HGS101 have antiviral mechanisms distinct from 2D7 and could help overcome MVC resistance.

  13. Therapy-Emergent Drug Resistance to Integrase Strand Transfer Inhibitors in HIV-1 Patients: A Subgroup Meta-Analysis of Clinical Trials

    PubMed Central

    Wang, Hongren; Huang, Xiaojun; Qin, Zhen; Deng, Zhaomin; Luo, Jun; Wang, Baoning; Li, Mingyuan

    2016-01-01

    Background Integrase strand transfer inhibitors (INSTIs) are a novel class of anti-HIV agents that show high activity in inhibiting HIV-1 replication. Currently, licensed INSTIs include raltegravir (RAL), elvitegravir (EVG) and dolutegravir (DTG); these drugs have played a critical role in AIDS therapy, serving as additional weapons in the arsenal for treating patients infected with HIV-1. To date, long-term data regarding clinical experience with INSTI use and the emergence of resistance remain scarce. However, the literature is likely now sufficiently comprehensive to warrant a meta-analysis of resistance to INSTIs. Methods Our team implemented a manuscript retrieval protocol using Medical Subject Headings (MeSH) via the Web of Science, MEDLINE, EMBASE, and Cochrane Central Register of Controlled Trials databases. We screened the literature based on inclusion and exclusion criteria and then performed a quality analysis and evaluation using RevMan software, Stata software, and the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE). We also performed a subgroup analysis. Finally, we calculated resistance rates and risk ratios (RRs) for the three types of drugs. Results We identified 26 references via the database search. A meta-analysis of the RAL data revealed that the resistance rate was 3.9% (95% CI = 2.9%-4.9%) for the selected randomized controlled trials (RCTs). However, the RAL resistance rate reached 40.9% (95% CI = 8.8%-72.9%) for the selected observational studies (OBSs). The rates of resistance to RAL that were associated with HIV subtypes A, B, and C as well as with more complex subtypes were 0.1% (95% CI = -0.7%-0.9%), 2.5% (95% CI = 0.5%-4.5%), 4.6% (95% CI = 2.7%-6.6%) and 2.2% (95% CI = 0.7%-3.7%), respectively. The rates of resistance to EVG and DTG were 1.2% (95% CI = 0.2%-2.2%) and 0.1% (95% CI = -0.2%-0.5%), respectively. Furthermore, we found that the RRs for antiviral resistance were 0.414 (95% CI = 0.210–0

  14. Comparable Long-Term Efficacy of Lopinavir/Ritonavir and Similar Drug-Resistance Profiles in Different HIV-1 Subtypes

    PubMed Central

    Grossman, Zehava; Schapiro, Jonathan M.; Levy, Itzchak; Elbirt, Daniel; Chowers, Michal; Riesenberg, Klaris; Olstein-Pops, Karen; Shahar, Eduardo; Istomin, Valery; Asher, Ilan; Gottessman, Bat-Sheva; Shemer, Yonat; Elinav, Hila; Hassoun, Gamal; Rosenberg, Shira; Averbuch, Diana; Machleb-Guri, Keren; Kra-Oz, Zipi; Radian-Sade, Sara; Rudich, Hagit; Ram, Daniela; Maayan, Shlomo; Agmon-Levin, Nancy; Sthoeger, Zev

    2014-01-01

    Background Analysis of potentially different impact of Lopinavir/Ritonavir (LPV/r) on non-B subtypes is confounded by dissimilarities in the conditions existing in different countries. We retrospectively compared its impact on populations infected with subtypes B and C in Israel, where patients infected with different subtypes receive the same treatment. Methods Clinical and demographic data were reported by physicians. Resistance was tested after treatment failure. Statistical analyses were conducted using SPSS. Results 607 LPV/r treated patients (365 male) were included. 139 had HIV subtype B, 391 C, and 77 other subtypes. At study end 429 (71%) were receiving LPV/r. No significant differences in PI treatment history and in median viral-load (VL) at treatment initiation and termination existed between subtypes. MSM discontinued LPV/r more often than others even when the virologic outcome was good (p = 0.001). VL was below detection level in 81% of patients for whom LPV/r was first PI and in 67% when it was second (P = 0.001). Median VL decrease from baseline was 1.9±0.1 logs and was not significantly associated with subtype. Median CD4 increase was: 162 and 92cells/µl, respectively, for patients receiving LPV/r as first and second PI (P = 0.001), and 175 and 98, respectively, for subtypes B and C (P<0.001). Only 52 (22%) of 237 patients genotyped while under LPV/r were fully resistant to the drug; 12(5%) were partially resistant. In48%, population sequencing did not reveal resistance to any drug notwithstanding the virologic failure. No difference was found in the rates of resistance development between B and C (p = 0.16). Conclusions Treatment with LPV/r appeared efficient and tolerable in both subtypes, B and C, but CD4 recovery was significantly better in virologically suppressed subtype-B patients. In both subtypes, LPV/r was more beneficial when given as first PI. Mostly, reasons other than resistance development caused discontinuation of

  15. Deep Sequencing of HIV-1 RNA and DNA in Newly Diagnosed Patients with Baseline Drug Resistance Showed No Indications for Hidden Resistance and Is Biased by Strong Interference of Hypermutation.

    PubMed

    Dauwe, Kenny; Staelens, Delfien; Vancoillie, Leen; Mortier, Virginie; Verhofstede, Chris

    2016-06-01

    Deep sequencing of plasma RNA or proviral DNA may be an interesting alternative to population sequencing for the detection of baseline transmitted HIV-1 drug resistance. Using a Roche 454 GS Junior HIV-1 prototype kit, we performed deep sequencing of the HIV-1 protease and reverse transcriptase genes on paired plasma and buffy coat samples from newly diagnosed HIV-1-positive individuals. Selection was based on the outcome of population sequencing and included 12 patients with either a revertant amino acid at codon 215 of the reverse transcriptase or a singleton resistance mutation, 4 patients with multiple resistance mutations, and 4 patients with wild-type virus. Deep sequencing of RNA and DNA detected 6 and 43 mutations, respectively, that were not identified by population sequencing. A subsequently performed hypermutation analysis, however, revealed hypermutation in 61.19% of 3,188 DNA reads with a resistance mutation. The removal of hypermutated reads dropped the number of additional mutations in DNA from 43 to 17. No hypermutation evidence was found in the RNA reads. Five of the 6 additional RNA mutations and all additional DNA mutations, after full exclusion of hypermutation bias, were observed in the 3 individuals with multiple resistance mutations detected by population sequencing. Despite focused selection of patients with T215 revertants or singleton mutations, deep sequencing failed to identify the resistant T215Y/F or M184V or any other resistance mutation, indicating that in most of these cases there is no hidden resistance and that the virus detected at diagnosis by population sequencing is the original infecting variant. PMID:27076656

  16. Pretreatment HIV Drug Resistance and HIV-1 Subtype C Are Independently Associated With Virologic Failure: Results From the Multinational PEARLS (ACTG A5175) Clinical Trial

    PubMed Central

    Kantor, Rami; Smeaton, Laura; Vardhanabhuti, Saran; Hudelson, Sarah E.; Wallis, Carol L.; Tripathy, Srikanth; Morgado, Mariza G.; Saravanan, Shanmugham; Balakrishnan, Pachamuthu; Reitsma, Marissa; Hart, Stephen; Mellors, John W.; Halvas, Elias; Grinsztejn, Beatriz; Hosseinipour, Mina C.; Kumwenda, Johnstone; La Rosa, Alberto; Lalloo, Umesh G.; Lama, Javier R.; Rassool, Mohammed; Santos, Breno R.; Supparatpinyo, Khuanchai; Hakim, James; Flanigan, Timothy; Kumarasamy, Nagalingeswaran; Campbell, Thomas B.; Eshleman, Susan H.

    2015-01-01

    Background. Evaluation of pretreatment HIV genotyping is needed globally to guide treatment programs. We examined the association of pretreatment (baseline) drug resistance and subtype with virologic failure in a multinational, randomized clinical trial that evaluated 3 antiretroviral treatment (ART) regimens and included resource-limited setting sites. Methods. Pol genotyping was performed in a nested case-cohort study including 270 randomly sampled participants (subcohort), and 218 additional participants failing ART (case group). Failure was defined as confirmed viral load (VL) >1000 copies/mL. Cox proportional hazards models estimated resistance–failure association. Results. In the representative subcohort (261/270 participants with genotypes; 44% women; median age, 35 years; median CD4 cell count, 151 cells/µL; median VL, 5.0 log10 copies/mL; 58% non-B subtypes), baseline resistance occurred in 4.2%, evenly distributed among treatment arms and subtypes. In the subcohort and case groups combined (466/488 participants with genotypes), used to examine the association between resistance and treatment failure, baseline resistance occurred in 7.1% (9.4% with failure, 4.3% without). Baseline resistance was significantly associated with shorter time to virologic failure (hazard ratio [HR], 2.03; P = .035), and after adjusting for sex, treatment arm, sex–treatment arm interaction, pretreatment CD4 cell count, baseline VL, and subtype, was still independently associated (HR, 2.1; P = .05). Compared with subtype B, subtype C infection was associated with higher failure risk (HR, 1.57; 95% confidence interval [CI], 1.04–2.35), whereas non-B/C subtype infection was associated with longer time to failure (HR, 0.47; 95% CI, .22–.98). Conclusions. In this global clinical trial, pretreatment resistance and HIV-1 subtype were independently associated with virologic failure. Pretreatment genotyping should be considered whenever feasible. Clinical Trials

  17. Predictive Value of HIV-1 Genotypic Resistance Test Interpretation Algorithms

    PubMed Central

    Rhee, Soo-Yon; Fessel, W. Jeffrey; Liu, Tommy F.; Marlowe, Natalia M.; Rowland, Charles M.; Rode, Richard A.; Vandamme, Anne-Mieke; Laethem, Kristel Van; Brun-Vezinet, Francçoise; Calvez, Vincent; Taylor, Jonathan; Hurley, Leo; Horberg, Michael; Shafer, Robert W.

    2016-01-01

    Background Interpreting human immunodeficienc virus type 1 (HIV-1) genotypic drug-resistance test results is challenging for clinicians treating HIV-1–infected patients. Multiple drug-resistance interpretation algorithms have been developed, but their predictive value has rarely been evaluated using contemporary clinical data sets. Methods We examined the predictive value of 4 algorithms at predicting virologic response (VR) during 734 treatment-change episodes (TCEs). VR was define as attaining plasma HIV-1 RNA levels below the limit of quantification Drug-specifi genotypic susceptibility scores (GSSs) were calculated by applying each algorithm to the baseline genotype. Weighted GSSs were calculated by multiplying drug-specifi GSSs by antiretroviral (ARV) potency factors. Regimen-specifi GSSs (rGSSs) were calculated by adding unweighted or weighted drug-specif c GSSs for each salvage therapy ARV. The predictive value of rGSSs were estimated by use of multivariate logistic regression. Results Of 734 TCEs, 475 (65%) were associated with VR. The rGSSs for the 4 algorithms were the variables most strongly predictive of VR. The adjusted rGSS odds ratios ranged from 1.6 to 2.2 (P < .001). Using 10-fold cross-validation, the averaged area under the receiver operating characteristic curve for all algorithms increased from 0.76 with unweighted rGSSs to 0.80 with weighted rGSSs. Conclusions Unweighted and weighted rGSSs of 4 genotypic resistance algorithms were the strongest independent predictors of VR. Optimizing ARV weighting may further improve VR predictions. PMID:19552527

  18. Regulatory Variation in HIV-1 Dependency Factor ZNRD1 Associates with Host Resistance to HIV-1 Acquisition

    PubMed Central

    An, Ping; Goedert, James J.; Donfield, Sharyne; Buchbinder, Susan; Kirk, Gregory D.; Detels, Roger; Winkler, Cheryl A.

    2014-01-01

    Background. ZNRD1 was identified as a host protein required for the completion of the human immunodeficiency virus (HIV) lifecycle in a genome-wide screen using small interfering RNA gene silencing. Subsequently, a genome-wide association study (GWAS) of host determinants for HIV-1 disease identified an association of single nucleotide polymorphisms (SNPs) in the ZNRD1 region with CD4+ T-cell depletion. Methods. We investigated the effects of SNPs in the ZNRD1 region on human immunodeficiency virus type 1 (HIV-1) infection and progression to clinical outcomes in 5 US-based HIV-1 longitudinal cohorts consisting of men who have sex with men, males with hemophilia, and injection drug users (IDUs) (n = 1865). SNP function was evaluated by electrophoretic mobility shift assay and promoter luciferase assay. Results. A haplotype in the ZNRD1 gene showed significant association with a 35% decreased risk of HIV-1 acquisition (OR = 0.65, 95% CI, .47–.89), independent of HLA-C rs9264942, in European Americans. The SNP rs3132130 tagging this haplotype, located in the ZNRD1 5′ upstream region, caused a loss of nuclear factor binding and decrease in ZNRD1 promoter activity. ZNRD1 variants also affected HIV-1 disease progression in European- and African-American cohorts. Conclusions. This study provides novel evidence that ZNRD1 polymorphism may confer host resistance to HIV-1 acquisition. PMID:24842830

  19. Silent mutations at codons 65 and 66 in reverse transcriptase alleviate indel formation and restore fitness in subtype B HIV-1 containing D67N and K70R drug resistance mutations

    PubMed Central

    Telwatte, Sushama; Hearps, Anna C.; Johnson, Adam; Latham, Catherine F.; Moore, Katie; Agius, Paul; Tachedjian, Mary; Sonza, Secondo; Sluis-Cremer, Nicolas; Harrigan, P. Richard; Tachedjian, Gilda

    2015-01-01

    Resistance to combined antiretroviral therapy (cART) in HIV-1-infected individuals is typically due to nonsynonymous mutations that change the protein sequence; however, the selection of synonymous or ‘silent’ mutations in the HIV-1 genome with cART has been reported. These silent K65K and K66K mutations in the HIV-1 reverse transcriptase (RT) occur in over 35% of drug-experienced individuals and are highly associated with the thymidine analog mutations D67N and K70R, which confer decreased susceptibility to most nucleoside and nucleotide RT inhibitors. However, the basis for selection of these silent mutations under selective drug pressure is unknown. Using Illumina next-generation sequencing, we demonstrate that the D67N/K70R substitutions in HIV-1 RT increase indel frequency by 100-fold at RT codons 65–67, consequently impairing viral fitness. Introduction of either K65K or K66K into HIV-1 containing D67N/K70R reversed the error-prone DNA synthesis at codons 65–67 in RT and improved viral replication fitness, but did not impact RT inhibitor drug susceptibility. These data provide new mechanistic insights into the role of silent mutations selected during antiretroviral therapy and have broader implications for the relevance of silent mutations in the evolution and fitness of RNA viruses. PMID:25765644

  20. Short Communication: Population-Based Surveillance of HIV-1 Drug Resistance in Cameroonian Adults Initiating Antiretroviral Therapy According to the World Health Organization Guidelines.

    PubMed

    Fokam, Joseph; Takou, Désiré; Santoro, Maria Mercedes; Akonie, Haniel Ze; Kouanfack, Charles; Ceccherini-Silberstein, Francesca; Colizzi, Vittorio; Perno, Carlo-Federico; Ndjolo, Alexis

    2016-04-01

    With ongoing earlier enrollment on and rapid scale-up of antiretroviral therapy (ART) in Cameroon, there are increasing risks of transmitted HIV drug resistance (HIVDR) at population levels. We, therefore, evaluated the threshold of HIVDR in a population initiating ART, to inform on the effectiveness of first-line regimens, considering HIV-1 diversity, plasma viral load (PVL), and CD4-based disease progression. A total of 53 adults [median (interquartile range, IQR) CD4: 162 cell/mm(3) (48-284); median (IQR) PVL: 5.34 log10 RNA (4.17-6.42) copies/ml] initiating ART in 2014 at the Yaoundé Central Hospital were enrolled for HIV-1 protease-reverse transcriptase sequencing. Drug resistance mutations (DRMs) were interpreted using the 2009 World Health Organization (WHO) list versus the Stanford HIVdb algorithm version 7.0. Level of DRMs was low (3.77%) versus moderate (7.55%), respectively, following the WHO list (T69D, K103N) versus Stanford HIVdb (T69D, A98G, K103N, K238T), respectively. Prevailing clade was CRF02_AG (71.70%). Based on Stanford HIVdb, a slightly higher proportion of patients with DRMs were found among ones infected with CRF02_AG than in those non-CRF02_AG infected (7.89% vs. 6.67%, p = 1.000), with lower PVL (7.69% <5.5 vs. 0% ≥5.5 log10 RNA copies/ml, p = .488) and with higher CD4 counts (9.52% CD4 ≥200 vs. 3.33% CD4 <200 cells/mm(3), p = .749). Thresholds of DRMs suggest that standard first-line regimens currently used in Cameroon may remain effective at population levels, despite scale-up of ART in the country, pending adherence, and closed virological monitoring. With an intent-to-diagnose approach, the discrepant levels of DRMs support using Stanford HIVdb to evaluate initial ART, while revising the WHO list for surveillance. PMID:26602836

  1. Field Evaluation of Dried Blood Spots for Routine HIV-1 Viral Load and Drug Resistance Monitoring in Patients Receiving Antiretroviral Therapy in Africa and Asia

    PubMed Central

    Monleau, Marjorie; Eymard-Duvernay, Sabrina; Dagnra, Anoumou; Kania, Dramane; Ngo-Giang-Huong, Nicole; Touré-Kane, Coumba; Truong, Lien X. T.; Chaix, Marie-Laure; Delaporte, Eric; Ayouba, Ahidjo; Peeters, Martine

    2014-01-01

    Dried blood spots (DBS) can be used in developing countries to alleviate the logistic constraints of using blood plasma specimens for viral load (VL) and HIV drug resistance (HIVDR) testing, but they should be assessed under field conditions. Between 2009 and 2011, we collected paired plasma-DBS samples from treatment-experienced HIV-1-infected adults in Burkina Faso, Cameroon, Senegal, Togo, Thailand, and Vietnam. The DBS were stored at an ambient temperature for 2 to 4 weeks and subsequently at −20°C before testing. VL testing was performed on the plasma samples and DBS using locally available methods: the Abbott m2000rt HIV-1 test, generic G2 real-time PCR, or the NucliSENS EasyQ version 1.2 test. In the case of virological failure (VF), i.e., a plasma VL of ≥1,000 copies/ml, HIVDR genotyping was performed on paired plasma-DBS samples. Overall, we compared 382 plasma-DBS sample pairs for DBS VL testing accuracy. The sensitivities of the different assays in different laboratories for detecting VF using DBS varied from 75% to 100% for the m2000rt test in labs B, C, and D, 91% to 93% for generic G2 real-time PCR in labs A and F, and 85% for the NucliSENS test in lab E. The specificities varied from 82% to 97% for the m2000rt and NucliSENS tests and reached only 60% for the generic G2 test. The NucliSENS test showed good agreement between plasma and DBS VL but underestimated the DBS VL. The lowest agreement was observed for the generic G2 test. Genotyping was successful for 96/124 (77%) DBS tested, and 75/96 (78%) plasma-DBS pairs had identical HIVDR mutations. Significant discrepancies in resistance interpretations were observed in 9 cases, 6 of which were from the same laboratory. DBS can be successfully used as an alternative to blood plasma samples for routine VL and HIVDR monitoring in African and Asian settings. However, the selection of an adequate VL measurement method and the definition of the VF threshold should be considered, and laboratory

  2. Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study

    PubMed Central

    Porter, Danielle P.; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D.; White, Kirsten L.

    2015-01-01

    At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study. PMID:26690199

  3. Emergent HIV-1 Drug Resistance Mutations Were Not Present at Low-Frequency at Baseline in Non-Nucleoside Reverse Transcriptase Inhibitor-Treated Subjects in the STaR Study.

    PubMed

    Porter, Danielle P; Daeumer, Martin; Thielen, Alexander; Chang, Silvia; Martin, Ross; Cohen, Cal; Miller, Michael D; White, Kirsten L

    2015-12-01

    At Week 96 of the Single-Tablet Regimen (STaR) study, more treatment-naïve subjects that received rilpivirine/emtricitabine/tenofovir DF (RPV/FTC/TDF) developed resistance mutations compared to those treated with efavirenz (EFV)/FTC/TDF by population sequencing. Furthermore, more RPV/FTC/TDF-treated subjects with baseline HIV-1 RNA >100,000 copies/mL developed resistance compared to subjects with baseline HIV-1 RNA ≤100,000 copies/mL. Here, deep sequencing was utilized to assess the presence of pre-existing low-frequency variants in subjects with and without resistance development in the STaR study. Deep sequencing (Illumina MiSeq) was performed on baseline and virologic failure samples for all subjects analyzed for resistance by population sequencing during the clinical study (n = 33), as well as baseline samples from control subjects with virologic response (n = 118). Primary NRTI or NNRTI drug resistance mutations present at low frequency (≥2% to 20%) were detected in 6.6% of baseline samples by deep sequencing, all of which occurred in control subjects. Deep sequencing results were generally consistent with population sequencing but detected additional primary NNRTI and NRTI resistance mutations at virologic failure in seven samples. HIV-1 drug resistance mutations emerging while on RPV/FTC/TDF or EFV/FTC/TDF treatment were not present at low frequency at baseline in the STaR study. PMID:26690199

  4. The L76V Drug Resistance Mutation Decreases the Dimer Stability and Rate of Autoprocessing of HIV-1 Protease by Reducing Internal Hydrophobic Contacts

    SciTech Connect

    Louis, John M.; Zhang, Ying; Sayer, Jane M.; Wang, Yuan-Fang; Harrison, Robert W.; Weber, Irene T.

    2011-09-06

    The mature HIV-1 protease (PR) bearing the L76V drug resistance mutation (PR{sub L76V}) is significantly less stable, with a >7-fold higher dimer dissociation constant (K{sub d}) of 71 {+-} 24 nM and twice the sensitivity to urea denaturation (UC{sub 50} = 0.85 M) relative to those of PR. Differential scanning calorimetry showed decreases in T{sub m} of 12 C for PR{sub L76V} in the absence of inhibitors and 5-7 C in the presence of inhibitors darunavir (DRV), saquinavir (SQV), and lopinavir (LPV), relative to that of PR. Isothermal titration calorimetry gave a ligand dissociation constant of 0.8 nM for DRV, {approx}160-fold higher than that of PR, consistent with DRV resistance. Crystal structures of PR{sub L76V} in complexes with DRV and SQV were determined at resolutions of 1.45-1.46 {angstrom}. Compared to the corresponding PR complexes, the mutated Val76 lacks hydrophobic interactions with Asp30, Lys45, Ile47, and Thr74 and exhibits closer interactions with Val32 and Val56. The bound DRV lacks one hydrogen bond with the main chain of Asp30 in PR{sub L76V} relative to PR, possibly accounting for the resistance to DRV. SQV shows slightly improved polar interactions with PR{sub L76V} compared to those with PR. Although the L76V mutation significantly slows the N-terminal autoprocessing of the precursor TFR-PR{sub L76V} to give rise to the mature PR{sub L76V}, the coselected M46I mutation counteracts the effect by enhancing this rate but renders the TFR-PRM46I/L76V precursor less responsive to inhibition by 6 {micro}M LPV while preserving inhibition by SQV and DRV. The correlation of lowered stability, higher K{sub d}, and impaired autoprocessing with reduced internal hydrophobic contacts suggests a novel molecular mechanism for drug resistance.

  5. HIV-1 Group O Genotypes and Phenotypes: Relationship to Fitness and Susceptibility to Antiretroviral Drugs.

    PubMed

    Tebit, Denis M; Patel, Hamish; Ratcliff, Annette; Alessandri, Elodie; Liu, Joseph; Carpenter, Crystal; Plantier, Jean-Christophe; Arts, Eric J

    2016-07-01

    Despite only 30,000 group O HIV-1 infections, a similar genetic diversity is observed among the O subgroups H (head) and T (tail) (previously described as subtypes A, B) as in the 9 group M subtypes (A-K). Group O isolates bearing a cysteine at reverse transcriptase (RT) position 181, predominantly the H strains are intrinsically resistant to non-nucleoside reverse transcriptase inhibitors (NNRTIs). However, their susceptibility to newer antiretroviral drugs such as etravirine, maraviroc, raltegravir (RAL), and elvitegravir (EVG) remains relatively unknown. We tested a large collection of HIV-1 group O strains for their susceptibility to four classes of antiretroviral drugs namely nucleoside RT, non-nucleoside RT, integrase, and entry inhibitors knowing in advance the intrinsic resistance to NNRTIs. Drug target regions were sequenced to determine various polymorphisms and were phylogenetically analyzed. Replication kinetics and fitness assays were performed in U87-CD4(+)CCR5 and CXCR4 cells and peripheral blood mononuclear cells. With all antiretroviral drugs, group O HIV-1 showed higher variability in IC50 values than group M HIV-1. The mean IC50 values for entry and nucleoside reverse transcriptase inhibitor (NRTI) were similar for group O and M HIV-1 isolates. Despite similar susceptibility to maraviroc, the various phenotypic algorithms failed to predict CXCR4 usage based on the V3 Env sequences of group O HIV-1 isolates. Decreased sensitivity of group O HIV-1 to integrase or NNRTIs had no relation to replicative fitness. Group O HIV-1 isolates were 10-fold less sensitive to EVG inhibition than group M HIV-1. These findings suggest that in regions where HIV-1 group O is endemic, first line treatment regimens combining two NRTIs with RAL may provide more sustained virologic responses than the standard regimens involving an NNRTI or protease inhibitors. PMID:26861573

  6. HIV-1 integrase inhibitor resistance and its clinical implications.

    PubMed

    Blanco, Jose-Luis; Varghese, Vici; Rhee, Soo-Yon; Gatell, Jose M; Shafer, Robert W

    2011-05-01

    With the approval in 2007 of the first integrase inhibitor (INI), raltegravir, clinicians became better able to suppress virus replication in patients infected with human immunodeficiency virus type 1 (HIV-1) who were harboring many of the most highly drug-resistant viruses. Raltegravir also provided clinicians with additional options for first-line therapy and for the simplification of regimens in patients with stable virological suppression. Two additional INIs in advanced clinical development-elvitegravir and S/GSK1349572-may prove equally versatile. However, the INIs have a relatively low genetic barrier to resistance in that 1 or 2 mutations are capable of causing marked reductions in susceptibility to raltegravir and elvitegravir, the most well-studied INIs. This perspective reviews the genetic mechanisms of INI resistance and their implications for initial INI therapy, the treatment of antiretroviral-experienced patients, and regimen simplification. PMID:21459813

  7. Computational Prediction of HIV-1 Resistance to Protease Inhibitors.

    PubMed

    Hosseini, Ali; Alibés, Andreu; Noguera-Julian, Marc; Gil, Victor; Paredes, Roger; Soliva, Robert; Orozco, Modesto; Guallar, Victor

    2016-05-23

    The development of mutations in HIV-1 protease (PR) hinders the activity of antiretroviral drugs, forcing changes in drug prescription. Most resistance assessments used to date rely on expert-based rules on predefined sets of stereotypical mutations; such an information-driven approach cannot capture new polymorphisms or be applied for new drugs. Computational modeling could provide a more general assessment of drug resistance and could be made available to clinicians through the Internet. We have created a protocol involving sequence comparison and all-atom protein-ligand induced fit simulations to predict resistance at the molecular level. We first compared our predictions with the experimentally determined IC50 values of darunavir, amprenavir, ritonavir, and indinavir from reference PR mutants displaying different resistance levels. We then performed analyses on a large set of variants harboring more than 10 mutations. Finally, several sequences from real patients were analyzed for amprenavir and darunavir. Our computational approach detected all of the genotype changes triggering high-level resistance, even those involving a large number of mutations. PMID:27082876

  8. The Genetic Basis of HIV-1 Resistance to Reverse Transcriptase and Protease Inhibitors

    PubMed Central

    Shafer, Robert W.; Kantor, Rami; Gonzales, Matthew J.

    2008-01-01

    HIV-1 drug resistance is caused by mutations in the reverse transcriptase (RT) and protease enzymes, the molecular targets of antiretroviral therapy. At the beginning of the year 2000, two expert panels recommended that HIV-1 RT and protease susceptibility testing be used to help select antiretroviral drugs for HIV-1-infected patients. Genotypic assays have been developed to detect HIV-1 mutations known to confer antiretroviral drug resistance. Genotypic assays using dideoxynucleoside sequencing provide extensive insight into the presence of drug-resistant variants in the population of viruses within an individual. However, the interpretation of these assays in clinical settings is formidable because of the large numbers of drug resistance mutations and because these mutations interact with one another and emerge in complex patterns. In addition, cross-resistance between antiretroviral drugs is greater than that anticipated from initial in vitro studies. This review summarises the published data linking HIV-1 RT and protease mutations to in vitro and clinical resistance to the currently available nucleoside RT inhibitors, non-nucleoside RT inhibitors, and protease inhibitors. PMID:19096725

  9. Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission

    PubMed Central

    Hauser, Andrea; Kuecherer, Claudia; Kunz, Andrea; Dabrowski, Piotr Wojtek; Radonić, Aleksandar; Nitsche, Andreas; Theuring, Stefanie; Bannert, Norbert; Sewangi, Julius; Mbezi, Paulina; Dugange, Festo; Harms, Gundel; Meixenberger, Karolin

    2015-01-01

    Background Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). Methods Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. Results Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2–3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. Conclusions Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in

  10. Molecular diversity of HIV-1 and surveillance of transmitted drug resistance variants among treatment Naïve patients, 5 years after active introduction of HAART in Kuala Lumpur, Malaysia.

    PubMed

    Ong, Lai Yee; Razak, Siti Nur Humaira; Lee, Yeat Mei; Sri La Sri Ponnampalavanar, Sasheela; Syed Omar, Sharifah Faridah; Azwa, Raja Iskandar; Tee, Kok Keng; Kamarulzaman, Adeeba

    2014-01-01

    Expansion of antiretroviral treatment programs have led to the growing concern for the development of antiretroviral drug resistance. The aims were to assess the prevalence of drug resistant HIV-1 variants and to identify circulating subtypes among HAART-naïve patients. Plasma specimens from N = 100 HIV+ HAART-naïve adult were collected between March 2008 and August 2010 and viral RNA were extracted for nested PCR and sequenced. PR-RT sequences were protein aligned and checked for transmitted drug resistance mutations. Phylogenetic reconstruction and recombination analysis were performed to determine the genotypes. Based on the WHO consensus guidelines, none of the recruited patients had any transmitted drug resistance mutations. When analyzed against the Stanford guidelines, 35% of patients had at least one reported mutation that may reduce drug susceptibility to PI (24%), NRTI (5%), and NNRTI (14%). The commonly detected mutation that may affect current first line therapy was V179D (3%), which may lead to reduced susceptibility to NNRTI. The predominant circulating HIV-1 genotypes were CRF01_AE (51%) and CRF33_01B (17%). The prevalence of unique recombinant forms (URF) was 7%; five distinct recombinant structures involving CRF01_AE and subtype B' were observed, among them a cluster of three isolates that could form a novel circulating recombinant form (CRF) candidate. Transmitted drug resistance prevalence among HAART-naïve patients was low in this cohort of patients in Kuala Lumpur despite introduction of HAART 5 years ago. Owing to the high genetic diversity, continued molecular surveillance can identify the persistent emergence of HIV-1 URF and novel CRF with significant epidemiological impact. PMID:24127302

  11. Trends in Transmission of Drug Resistance and Prevalence of Non-B Subtypes in Patients with Acute or Recent HIV-1 Infection in Barcelona in the Last 16 Years (1997-2012)

    PubMed Central

    Nicolas, David; Parera, Marta; López-Diéguez, María; Romero, Anabel; Agüero, Fernando; Marcos, María Ángeles; Manzardo, Christian; Zamora, Laura; Gómez-Carrillo, Manuel; Gatell, José María; Pumarola, Tomás; Miró, José María

    2015-01-01

    Objectives To evaluate the prevalence of transmitted drug resistance (TDR) and non-B subtypes in patients with acute/recent HIV-1 infection in Barcelona during the period 1997-2012. Methods Patients from the “Hospital Clínic Primary HIV-1 Infection Cohort” with a genotyping test performed within 180 days of infection were included. The 2009 WHO List of Mutations for Surveillance of Transmitted HIV-1 Drug Resistance was used for estimating the prevalence of TDR and phylogenetic analysis for subtype determination. Results 189 patients with acute/recent HIV-1 infection were analyzed in 4 time periods (1997-2000, n=28; 2001-4, n=42; 2005-8, n=55 and 2009-12, n=64). The proportion of patients with acute/recent HIV-1 infection with respect to the total of newly HIV-diagnosed patients in our center increased over the time and was 2.18%, 3.82%, 4.15% and 4.55% for the 4 periods, respectively (p=0.005). The global prevalence of TDR was 9%, or 17.9%, 9.5%, 3.6% and 9.4% by study period (p=0.2). The increase in the last period was driven by protease-inhibitor and nucleoside-reverse-transcriptase-inhibitor resistance mutations while non-nucleoside-reverse-transcriptase inhibitor TDR and TDR of more than one family decreased. The overall prevalence of non-B subtypes was 11.1%, or 0%, 4.8%, 9.1% and 20.3 by study period (p=0.01). B/F recombinants, B/G recombinants and subtype F emerged in the last period. We also noticed an increase in the number of immigrant patients (p=0.052). The proportion of men-who-have-sex-with-men (MSM) among patients with acute/recent HIV-1 infection increased over the time (p=0.04). Conclusions The overall prevalence of TDR in patients with acute/recent HIV-1 infection in Barcelona was 9%, and it has stayed relatively stable in recent years. Non-B subtypes and immigrants proportions progressively increased. PMID:26039689

  12. Substrate-induced stable enzyme-inhibitor complex formation allows tight binding of novel 2-aminopyrimidin-4(3H)-ones to drug-resistant HIV-1 reverse transcriptase mutants.

    PubMed

    Samuele, Alberta; Facchini, Marcella; Rotili, Dante; Mai, Antonello; Artico, Marino; Armand-Ugón, Mercedes; Esté, José A; Maga, Giovanni

    2008-09-01

    We recently reported the synthesis and biological evaluation of a novel series of 5-alkyl-2-(N,N-disubstituted)amino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones (F(2)-N,N-DABOs). These compounds are highly active against both wild-type HIV-1 and the K103N, Y181C, and Y188L mutant strains. Herein we present novel 6-(2-chloro-6-fluorophenylalkyl)-N,N-DABO (2-Cl-6-F-N,N-DABO) derivatives and investigate the molecular basis for their high-affinity binding to HIV-1 reverse transcriptase (RT). Our results show that the new compounds display higher association rates than the difluoro derivatives toward wild-type HIV-1 RT or drug-resistant RT mutant forms. We also show that they preferentially associate to either the free enzyme or the enzyme-nucleic acid binary complex, and that this binding is stabilized upon formation of the ternary complex between HIV-1 RT and both the nucleic acid and nucleotide substrates. Interestingly, one compound showed dissociation rates from the ternary complex with RT mutants K103N and Y181I 10-20-fold slower than from the corresponding complex with wild-type RT. PMID:18465760

  13. The Combination of the R263K and T66I Resistance Substitutions in HIV-1 Integrase Is Incompatible with High-Level Viral Replication and the Development of High-Level Drug Resistance

    PubMed Central

    Liang, Jiaming; Mesplède, Thibault; Oliveira, Maureen; Anstett, Kaitlin

    2015-01-01

    ABSTRACT The R263K substitution in integrase has been selected in tissue culture with dolutegravir (DTG) and has been reported for several treatment-experienced individuals receiving DTG as part of salvage therapy. The R263K substitution seems to be incompatible with the presence of common resistance mutations associated with raltegravir (RAL), a different integrase strand transfer inhibitor (INSTI). T66I is a substitution that is common in individuals who have developed resistance against a different INSTI termed elvitegravir (EVG), but it is not known whether these two mutations might be compatible in the context of resistance against DTG or what impact the combination of these substitutions might have on resistance against INSTIs. E138K is a common secondary substitution observed with various primary resistance substitutions in RAL- and EVG-treated individuals. Viral infectivity, replicative capacity, and resistance against INSTIs were measured in cell-based assays. Strand transfer and 3′ processing activities were measured biochemically. The combination of the R263K and T66I substitutions decreased HIV-1 infectivity, replicative capacity, and strand transfer activity. The addition of the E138K substitution partially compensated for these deficits and resulted in high levels of resistance against EVG but not against DTG or RAL. These findings suggest that the presence of the T66I substitution will not compromise the activity of DTG and may also help to prevent the additional generation of the R263K mutation. Our observations support the use of DTG in second-line therapy for individuals who experience treatment failure with EVG due to the T66I substitution. IMPORTANCE The integrase strand transfer inhibitors (INSTIs) elvitegravir and dolutegravir are newly developed inhibitors against human immunodeficiency virus type 1 (HIV-1). HIV drug-resistant mutations in integrase that can arise in individuals treated with elvitegravir commonly include the T66I

  14. Role and modulation of drug transporters in HIV-1 therapy.

    PubMed

    Alam, Camille; Whyte-Allman, Sana-Kay; Omeragic, Amila; Bendayan, Reina

    2016-08-01

    Current treatment of human immunodeficiency virus type-1 (HIV-1) infection involves a combination of antiretroviral drugs (ARVs) that target different stages of the HIV-1 life cycle. This strategy is commonly referred to as highly active antiretroviral therapy (HAART) or combined antiretroviral therapy (cART). Membrane-associated drug transporters expressed ubiquitously in mammalian systems play a crucial role in modulating ARV disposition during HIV-1 infection. Members of the ATP-binding cassette (ABC) and solute carrier (SLC) transporter superfamilies have been shown to interact with ARVs, including those that are used as part of first-line treatment regimens. As a result, the functional expression of drug transporters can influence the distribution of ARVs at specific sites of infection. In addition, pathological factors related to HIV-1 infection and/or ARV therapy itself can alter transporter expression and activity, thus further contributing to changes in ARV disposition and the effectiveness of HAART. This review summarizes current knowledge on the role of drug transporters in regulating ARV transport in the context of HIV-1 infection. PMID:27181050

  15. Design, Synthesis, and Evaluation of Thiophene[3,2-d]pyrimidine Derivatives as HIV-1 Non-nucleoside Reverse Transcriptase Inhibitors with Significantly Improved Drug Resistance Profiles.

    PubMed

    Kang, Dongwei; Fang, Zengjun; Li, Zhenyu; Huang, Boshi; Zhang, Heng; Lu, Xueyi; Xu, Haoran; Zhou, Zhongxia; Ding, Xiao; Daelemans, Dirk; De Clercq, Erik; Pannecouque, Christophe; Zhan, Peng; Liu, Xinyong

    2016-09-01

    We designed and synthesized a series of human immunodeficiency virus type 1 (HIV-1) non-nucleoside reverse transcriptase inhibitors (NNRTIs) with a piperidine-substituted thiophene[3,2-d]pyrimidine scaffold, employing a strategy of structure-based molecular hybridization and substituent decorating. Most of the synthesized compounds exhibited broad-spectrum activity with low (single-digit) nanomolar EC50 values toward a panel of wild-type (WT), single-mutant, and double-mutant HIV-1 strains. Compound 27 was the most potent; compared with ETV, its antiviral efficacy was 3-fold greater against WT, 5-7-fold greater against Y181C, Y188L, E138K, and F227L+V106A, and nearly equipotent against L100I and K103N, though somewhat weaker against K103N+Y181C. Importantly, 27 has lower cytotoxicity (CC50 > 227 μM) and a huge selectivity index (SI) value (ratio of CC50/EC50) of >159101. 27 also showed favorable, drug-like pharmacokinetic and safety properties in rats in vivo. Molecular docking studies and the structure-activity relationships provide important clues for further molecular elaboration. PMID:27541578

  16. Assessing the HIV-1 Epidemic in Brazilian Drug Users: A Molecular Epidemiology Approach

    PubMed Central

    Guimarães, Monick Lindenmeyer; Marques, Bianca Cristina Leires; Bertoni, Neilane; Teixeira, Sylvia Lopes Maia; Morgado, Mariza Gonçalves; Bastos, Francisco Inácio

    2015-01-01

    Person who inject illicit substances have an important role in HIV-1 blood and sexual transmission and together with person who uses heavy non-injecting drugs may have less than optimal adherence to anti-retroviral treatment and eventually could transmit resistant HIV variants. Unfortunately, molecular biology data on such key population remain fragmentary in most low and middle-income countries. The aim of the present study was to assess HIV infection rates, evaluate HIV-1 genetic diversity, drug resistance, and to identify HIV transmission clusters in heavy drug users (DUs). For this purpose, DUs were recruited in the context of a Respondent-Driven Sampling (RDS) study in different Brazilian cities during 2009. Overall, 2,812 individuals were tested for HIV, and 168 (6%) of them were positive, of which 19 (11.3%) were classified as recent seroconverters, corresponding to an estimated incidence rate of 1.58%/year (95% CI 0.92–2.43%). Neighbor joining phylogenetic trees from env and pol regions and bootscan analyses were employed to subtype the virus from132 HIV-1-infected individuals. HIV-1 subtype B was prevalent in most of the cities under analysis, followed by BF recombinants (9%-35%). HIV-1 subtype C was the most prevalent in Curitiba (46%) and Itajaí (86%) and was also detected in Brasília (9%) and Campo Grande (20%). Pure HIV-1F infections were detected in Rio de Janeiro (9%), Recife (6%), Salvador (6%) and Brasília (9%). Clusters of HIV transmission were assessed by Maximum likelihood analyses and were cross-compared with the RDS network structure. Drug resistance mutations were verified in 12.2% of DUs. Our findings reinforce the importance of the permanent HIV-1 surveillance in distinct Brazilian cities due to viral resistance and increasing subtype heterogeneity all over Brazil, with relevant implications in terms of treatment monitoring, prophylaxis and vaccine development. PMID:26536040

  17. Drug Susceptibility and Viral Fitness of HIV-1 with Integrase Strand Transfer Inhibitor Resistance Substitution Q148R or N155H in Combination with Nucleoside/Nucleotide Reverse Transcriptase Inhibitor Resistance Substitutions

    PubMed Central

    Miller, Michael D.; White, Kirsten L.

    2015-01-01

    In clinical trials of coformulated elvitegravir (EVG), cobicistat (COBI), emtricitabine (FTC), and tenofovir disoproxil fumarate (TDF), emergent drug resistance predominantly involved the FTC resistance substitution M184V/I in reverse transcriptase (RT), with or without the tenofovir (TFV) resistance substitution K65R, accompanied by a primary EVG resistance substitution (E92Q, N155H, or Q148R) in integrase (IN). We previously reported that the RT-K65R, RT-M184V, and IN-E92Q substitutions lacked cross-class phenotypic resistance and replicative fitness compensation. As a follow-up, the in vitro characteristics of mutant HIV-1 containing RT-K65R and/or RT-M184V with IN-Q148R or IN-N155H were also evaluated, alone and in combination, for potential interactions. Single mutants displayed reduced susceptibility to their corresponding inhibitor classes, with no cross-class resistance. Viruses with IN-Q148R or IN-N155H exhibited reduced susceptibility to EVG (137- and 40-fold, respectively) that was not affected by the addition of RT-M184V or RT-K65R/M184V. All viruses containing RT-M184V were resistant to FTC (>1,000-fold). Mutants with RT-K65R had reduced susceptibility to TFV (3.3- to 3.6-fold). Without drugs present, the viral fitness of RT and/or IN mutants was diminished relative to that of the wild type in the following genotypic order: wild type > RT-M184V ≥ IN-N155H ≈ IN-Q148R ≥ RT-M184V + IN-N155H ≥ RT-M184V + IN-Q148R ≥ RT-K65R/M184V + IN-Q148R ≈ RT-K65R/M184V + IN-N155H. In the presence of drug concentrations approaching physiologic levels, drug resistance counteracted replication defects, allowing single mutants to outcompete the wild type with one drug present and double mutants to outcompete single mutants with two drugs present. These results suggest that during antiretroviral treatment with multiple drugs, the development of viruses with combinations of resistance substitutions may be favored despite diminished viral fitness. PMID:26574015

  18. The higher barrier of darunavir and tipranavir resistance for HIV-1 protease

    SciTech Connect

    Wang, Yong; Liu, Zhigang; Brunzelle, Joseph S.; Kovari, Iulia A.; Dewdney, Tamaria G.; Reiter, Samuel J.; Kovari, Ladislau C.

    2011-11-17

    Darunavir and tipranavir are two inhibitors that are active against multi-drug resistant (MDR) HIV-1 protease variants. In this study, the invitro inhibitory efficacy was tested against a MDR HIV-1 protease variant, MDR 769 82T, containing the drug resistance mutations of 46L/54V/82T/84V/90M. Crystallographic and enzymatic studies were performed to examine the mechanism of resistance and the relative maintenance of potency. The key findings are as follows: (i) The MDR protease exhibits decreased susceptibility to all nine HIV-1 protease inhibitors approved by the US Food and Drug Administration (FDA), among which darunavir and tipranavir are the most potent; (ii) the threonine 82 mutation on the protease greatly enhances drug resistance by altering the hydrophobicity of the binding pocket; (iii) darunavir or tipranavir binding facilitates closure of the wide-open flaps of the MDR protease; and (iv) the remaining potency of tipranavir may be preserved by stabilizing the flaps in the inhibitor-protease complex while darunavir maintains its potency by preserving protein main chain hydrogen bonds with the flexible P2 group. These results could provide new insights into drug design strategies to overcome multi-drug resistance of HIV-1 protease variants.

  19. [Companion Diagnostics for Selecting Antiretroviral Drugs against HIV-1].

    PubMed

    Fukutake, Katsuyuki

    2015-11-01

    Currently, the treatment of human immunodeficiency virus involves combination therapy, as antiretroviral therapy(ART). The treatment has improved steadily since the advent of potent combination therapy in 1996. New drugs that offer new mechanisms of action, improvements in potency and activity even against multidrug-resistant viruses, dosing convenience, and tolerability have been approved. Among ART with useful drugs, there are two important examinations before starting the treatment using the two kinds of drug. CCR5 co-receptor antagonists, maraviroc, prevent HIV entry into target cells by binding to CCR5 receptors. Genotypic assays have been developed that can determine or predict the co-receptor tropism(i.e., CCR5, CXCR4, or both) of the patient's dominant virus population. The assay for HIV-1 co-receptor usage should be performed whenever the use of a CCR5 antagonist is being considered. One of the nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs), abacavir, is an important agent to develop recommended regimens for antiretroviral therapy. Serious and sometimes fatal hypersensitivity reactions have been associated with abacavir-containing products, ZIAGEN, Epzicom, and Triumeq. Patients who carry the HLA-B*5701 allele are at high-risk of a hypersensitivity reaction to abacavir. Prior to initiating therapy with abacavir, performing a screening test for the HLA-B*5701 allele is recommended. [Review]. PMID:26995879

  20. Dolutegravir-Selected HIV-1 Containing the N155H and R263K Resistance Substitutions Does Not Acquire Additional Compensatory Mutations under Drug Pressure That Lead to Higher-Level Resistance and Increased Replicative Capacity

    PubMed Central

    Anstett, Kaitlin; Fusco, Robert; Cutillas, Vincent; Mesplède, Thibault

    2015-01-01

    ABSTRACT We have previously shown that the addition of the raltegravir/elvitegavir (RAL/EVG) primary resistance mutation N155H to the R263K dolutegravir (DTG) resistance mutation partially compensated for the fitness cost imposed by R263K while also slightly increasing DTG resistance in vitro (K. Anstett, T. Mesplede, M. Oliveira, V. Cutillas, and M. A. Wainberg, J Virol 89:4681–4684, 2015, doi:10.1128/JVI.03485-14). Since many patients failing RAL/EVG are given DTG as part of rescue therapy, and given that the N155H substitution often is found in combination with other compensatory resistance mutations in such individuals, we investigated the effects of multiple such substitutions within integrase (IN) on each of integrase function, HIV-1 infectivity, and levels of drug resistance. To this end, each of the L74M, E92Q, T97A, E157Q, and G163R substitutions were introduced into NL4.3 subtype B HIV-1 vectors harboring N155H and R263K in tandem [termed NL4.3IN(N155H/R263K)]. Relevant recombinant integrase enzymes also were expressed, and purified and biochemical assays of strand transfer efficiency as well as viral infectivity and drug resistance studies were performed. We found that the addition of T97A, E157Q, or G163R somewhat improved the affinity of INN155H/R263K for its target DNA substrate, while the presence of L74M or E92Q had a negative effect on this process. However, viral infectivity was significantly decreased from that of NL4.3IN(N155H/R263K) after the addition of each tertiary mutation, and no increases in levels of DTG resistance were observed. This work shows that the compensatory mutations that evolve after N155H under continued DTG or RAL/EVG pressure in patients are unable to improve either enzyme efficiency or viral infectivity in an N155H/R263K background. IMPORTANCE In contrast to other drugs, dolutegravir has not selected for resistance in HIV-positive individuals when used in first-line therapy. We had previously shown that HIV containing

  1. Increasing HIV-1 Drug Resistance Between 2010 and 2012 in Adults Participating in Population-Based HIV Surveillance in Rural KwaZulu-Natal, South Africa.

    PubMed

    Manasa, Justen; Danaviah, Siva; Lessells, Richard; Elshareef, Muna; Tanser, Frank; Wilkinson, Eduan; Pillay, Sureshnee; Mthiyane, Hloniphile; Mwambi, Henry; Pillay, Deenan; de Oliveira, Tulio

    2016-08-01

    As more human immunodeficiency virus (HIV)-infected patients access combination antiretroviral therapy (cART), higher proportions of newly infected patients may be infected with drug-resistant viruses. Regular surveillance of transmitted drug resistance (TDR) is required in southern Africa where high rates of transmission persist despite rapid expansion of ART. Dried blood spot samples from cART-naive participants from two rounds of an annual population-based HIV surveillance program in rural KwaZulu-Natal were tested for HIV RNA, and samples with HIV RNA >10,000 copies/ml were genotyped for drug resistance. The 2009 surveillance of drug resistance mutation (SDRM) list was used for drug resistance interpretation. The data were added to previously published data from the same program, and the χ(2) test for trend was used to test for trend in estimated prevalence of any TDR. Seven hundred and one participants' data were analyzed: 67 (2010), 381 (2011), and 253 (2012). No TDR was detected in 2010. Years 2011 and 2012 had 18 participants with SDRMs 4.7% and 7.1%, respectively (p = .02, χ(2) test for trend). The nonnucleoside reverse transcriptase inhibitor mutation, K103N, was the most common mutation, occurring in 27 (3.8%) of the participants, while nucleoside reverse transcriptase inhibitor (NRTI) SDRMs were detected in 10 (1.4%) of the participants, of whom eight had only a single NRTI SDRM. The increase in levels of drug resistance observed in this population could be a signal of increasing transmission of drug-resistant HIV. Thus, continued surveillance is critical to inform public health policies around HIV treatment and prevention. PMID:27002368

  2. Increasing HIV-1 Drug Resistance Between 2010 and 2012 in Adults Participating in Population-Based HIV Surveillance in Rural KwaZulu-Natal, South Africa

    PubMed Central

    Danaviah, Siva; Lessells, Richard; Elshareef, Muna; Tanser, Frank; Wilkinson, Eduan; Pillay, Sureshnee; Mthiyane, Hloniphile; Mwambi, Henry; Pillay, Deenan; de Oliveira, Tulio

    2016-01-01

    Abstract As more human immunodeficiency virus (HIV)–infected patients access combination antiretroviral therapy (cART), higher proportions of newly infected patients may be infected with drug-resistant viruses. Regular surveillance of transmitted drug resistance (TDR) is required in southern Africa where high rates of transmission persist despite rapid expansion of ART. Dried blood spot samples from cART-naive participants from two rounds of an annual population-based HIV surveillance program in rural KwaZulu-Natal were tested for HIV RNA, and samples with HIV RNA >10,000 copies/ml were genotyped for drug resistance. The 2009 surveillance of drug resistance mutation (SDRM) list was used for drug resistance interpretation. The data were added to previously published data from the same program, and the χ2 test for trend was used to test for trend in estimated prevalence of any TDR. Seven hundred and one participants' data were analyzed: 67 (2010), 381 (2011), and 253 (2012). No TDR was detected in 2010. Years 2011 and 2012 had 18 participants with SDRMs 4.7% and 7.1%, respectively (p = .02, χ2 test for trend). The nonnucleoside reverse transcriptase inhibitor mutation, K103N, was the most common mutation, occurring in 27 (3.8%) of the participants, while nucleoside reverse transcriptase inhibitor (NRTI) SDRMs were detected in 10 (1.4%) of the participants, of whom eight had only a single NRTI SDRM. The increase in levels of drug resistance observed in this population could be a signal of increasing transmission of drug-resistant HIV. Thus, continued surveillance is critical to inform public health policies around HIV treatment and prevention. PMID:27002368

  3. Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System.

    PubMed

    Cheung, Ka-Wai; Peng, Qiaoli; He, Liufen; Cai, Kanru; Jiang, Qiang; Zhou, Boping; To, Sabrina Wai-Chi; Yam, Wing-Cheong; Liu, Li; Chen, Zhiwei; Wang, Hui

    2016-01-01

    The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China. PMID:27092551

  4. Rapid and Simultaneous Detection of Major Drug Resistance Mutations in Reverse Transcriptase Gene for HIV-1 CRF01_AE, CRF07_BC and Subtype B in China Using Sequenom MassARRAY® System

    PubMed Central

    He, Liufen; Cai, Kanru; Jiang, Qiang; Zhou, Boping; To, Sabrina Wai-Chi; Yam, Wing-Cheong; Liu, Li; Chen, Zhiwei; Wang, Hui

    2016-01-01

    The development of a rapid, high-throughput and cost-effective HIV-1 drug resistance (HIV-DR) testing system is a challenge for areas consisting different HIV-1 strains. In this study, we established a broadly reactive multiplex assay that could simultaneously detect major drug resistance mutations at 8 loci, which are associated with resistance to commonly used nucleoside reverse transcriptase inhibitors (NRTIs) and Non-nucleoside reverse transcriptase inhibitors (NNRTIs), in specimens of HIV-1 CRF01_AE, CRF07_BC and subtype B, the three major circulating strains in China, using the matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) provided by Sequenom MassARRAY® system. To establish the assay, pol gene fragments were prepared from the plasma viral RNA of 159 patients by nested PCR and the presence of wild type and mutant alleles at the 8 loci were analyzed by MALDI-TOF MS. In terms of loci, the detection rate of the alleles was greater than 97% for M41L, K65R, M184V and G190A, 91.2% for K101E/Q/P, 91.2% for T215F/Y, 89.9% for K103N/S and 80.5% for L210W. In terms of individuals, 80% of the alleles were detected in 95.4% CRF01_AE patients, 100% CRF07_BC patients and 83.3% subtype B patients. Importantly, the MALDI-TOF MS results were concordant to the drug resistance profiles of patients obtained from conventional sequencing analysis after excluded the failed detections. Using plasmid templates, the assay was estimated to be sensitive to detect drug resistant variants at level about 20% of the circulating viral population. The capability of this assay to detect mixed viral populations was further verified by two different patient specimens. In conclusion, this study evaluated the use of Sequenom MassARRAY® system for high-throughput detection of HIV-DR mutations towards the commonly used reverse transcriptase inhibitors in China. PMID:27092551

  5. A Pragmatic Approach to HIV-1 Drug Resistance Determination in Resource-Limited Settings by Use of a Novel Genotyping Assay Targeting the Reverse Transcriptase-Encoding Region Only

    PubMed Central

    Bronze, Michelle; Wallis, Carole L.; Stuyver, Lieven; Steegen, Kim; Balinda, Sheila; Kityo, Cissy; Stevens, Wendy; Rinke de Wit, Tobias F.; Schuurman, Rob

    2013-01-01

    In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples. PMID:23536405

  6. A pragmatic approach to HIV-1 drug resistance determination in resource-limited settings by use of a novel genotyping assay targeting the reverse transcriptase-encoding region only.

    PubMed

    Aitken, Susan C; Bronze, Michelle; Wallis, Carole L; Stuyver, Lieven; Steegen, Kim; Balinda, Sheila; Kityo, Cissy; Stevens, Wendy; Rinke de Wit, Tobias F; Schuurman, Rob

    2013-06-01

    In resource-limited settings (RLS), reverse transcriptase (RT) inhibitors form the backbone of first-line treatment regimens. We have developed a simplified HIV-1 drug resistance genotyping assay targeting the region of RT harboring all major RT inhibitor resistance mutation positions, thus providing all relevant susceptibility data for first-line failures, coupled with minimal cost and labor. The assay comprises a one-step RT-PCR amplification reaction, followed by sequencing using one forward and one reverse primer, generating double-stranded coverage of RT amino acids (aa) 41 to 238. The assay was optimized for all major HIV-1 group M subtypes in plasma and dried blood spot (DBS) samples using a panel of reference viruses for HIV-1 subtypes A to D, F to H, and circulating recombinant form 01_AE (CRF01_AE) and applied to 212 clinical plasma samples and 25 DBS samples from HIV-1-infected individuals from Africa and Europe. The assay was subsequently transferred to Uganda and applied locally on clinical plasma samples. All major HIV-1 subtypes could be detected with an analytical sensitivity of 5.00E+3 RNA copies/ml for plasma and DBS. Application of the assay on 212 clinical samples from African subjects comprising subtypes A to D, F to H (rare), CRF01_AE, and CRF02_AG at a viral load (VL) range of 6.71E+2 to 1.00E+7 (median, 1.48E+5) RNA copies/ml was 94.8% (n = 201) successful. Application on clinical samples in Uganda demonstrated a comparable success rate. Genotyping of clinical DBS samples, all subtype C with a VL range of 1.02E+3 to 4.49E+5 (median, 1.42E+4) RNA copies/ml, was 84.0% successful. The described assay greatly reduces hands-on time and the costs required for genotyping and is ideal for use in RLS, as demonstrated in a reference laboratory in Uganda and its successful application on DBS samples. PMID:23536405

  7. Drug-Eluting Fibers for HIV-1 Inhibition and Contraception

    PubMed Central

    Ball, Cameron; Krogstad, Emily; Chaowanachan, Thanyanan; Woodrow, Kim A.

    2012-01-01

    Multipurpose prevention technologies (MPTs) that simultaneously prevent sexually transmitted infections (STIs) and unintended pregnancy are a global health priority. Combining chemical and physical barriers offers the greatest potential to design effective MPTs, but integrating both functional modalities into a single device has been challenging. Here we show that drug-eluting fiber meshes designed for topical drug delivery can function as a combination chemical and physical barrier MPT. Using FDA-approved polymers, we fabricated nanofiber meshes with tunable fiber size and controlled degradation kinetics that facilitate simultaneous release of multiple agents against HIV-1, HSV-2, and sperm. We observed that drug-loaded meshes inhibited HIV-1 infection in vitro and physically obstructed sperm penetration. Furthermore, we report on a previously unknown activity of glycerol monolaurate (GML) to potently inhibit sperm motility and viability. The application of drug-eluting nanofibers for HIV-1 prevention and sperm inhibition may serve as an innovative platform technology for drug delivery to the lower female reproductive tract. PMID:23209601

  8. Drug-eluting fibers for HIV-1 inhibition and contraception.

    PubMed

    Ball, Cameron; Krogstad, Emily; Chaowanachan, Thanyanan; Woodrow, Kim A

    2012-01-01

    Multipurpose prevention technologies (MPTs) that simultaneously prevent sexually transmitted infections (STIs) and unintended pregnancy are a global health priority. Combining chemical and physical barriers offers the greatest potential to design effective MPTs, but integrating both functional modalities into a single device has been challenging. Here we show that drug-eluting fiber meshes designed for topical drug delivery can function as a combination chemical and physical barrier MPT. Using FDA-approved polymers, we fabricated nanofiber meshes with tunable fiber size and controlled degradation kinetics that facilitate simultaneous release of multiple agents against HIV-1, HSV-2, and sperm. We observed that drug-loaded meshes inhibited HIV-1 infection in vitro and physically obstructed sperm penetration. Furthermore, we report on a previously unknown activity of glycerol monolaurate (GML) to potently inhibit sperm motility and viability. The application of drug-eluting nanofibers for HIV-1 prevention and sperm inhibition may serve as an innovative platform technology for drug delivery to the lower female reproductive tract. PMID:23209601

  9. Comparative Fitness of Multi-Dideoxynucleoside-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) in an In Vitro Competitive HIV-1 Replication Assay

    PubMed Central

    Kosalaraksa, Pope; Kavlick, Mark F.; Maroun, Victor; Le, Richard; Mitsuya, Hiroaki

    1999-01-01

    We examined whether human immunodeficiency virus type 1 (HIV-1) fitness was altered upon the acquisition of a set or subset of five mutations (A62V, V75I, F77L, F116Y, and Q151M) in the pol gene, which confers resistance to multiple dideoxynucleosides (MDR), as well as the zidovudine resistance-associated mutation T215Y, using a competitive HIV-1 replication assay in a setting of an HXB2D genetic background. Target H9 cells were exposed to a 50:50 mixture of paired infectious molecular clones, and HIV-1 in the culture supernatant was transmitted to new cultures every 7 to 10 days. The polymerase-encoding region of the virus was sequenced at various time points, and the relative proportion of the two viral populations was determined. In the absence of drugs, the comparative order for replicative fitness was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-1151 > wild-type HIV-1 (HIV-1wt) > HIV-175/77/116/151 > HIV-1151/215 > HIV-1215. In the presence of zidovudine or didanosine, the order was HIV-162/75/77/116/151 > HIV-177/116/151 > HIV-175/77/116/151 > HIV-1151 > HIV-1215. HIV-1215S(TCC), a putative intermediate infectious clone for HIV-1215, replicated comparably to HIV-1wt, while two putative intermediates for HIV-1151 [HIV-1151L(CTG) and HIV-1151K(AAG)] replicated much less efficiently than HIV-1wt and HIV-1151, suggesting that for HIV-1151 to develop, two base substitutions are likely to occur concurrently or within a short interval. These data may illustrate the molecular basis by which HIV-1151 emerges much less frequently than HIV-1215. The present data also demonstrate that several MDR HIV-1 variants are more fit than HIV-1wt in the absence of drugs and that resistance-associated mutations and drug pressure are critical variates for HIV-1 fitness. PMID:10364282

  10. Drug-Induced Reactivation of Apoptosis Abrogates HIV-1 Infection

    PubMed Central

    Hanauske-Abel, Hartmut M.; Saxena, Deepti; Palumbo, Paul E.; Hanauske, Axel-Rainer; Luchessi, Augusto D.; Cambiaghi, Tavane D.; Hoque, Mainul; Spino, Michael; Gandolfi, Darlene D'Alliessi; Heller, Debra S.; Singh, Sukhwinder; Park, Myung Hee; Cracchiolo, Bernadette M.; Tricta, Fernando; Connelly, John; Popowicz, Anthony M.; Cone, Richard A.; Holland, Bart; Pe’ery, Tsafi; Mathews, Michael B.

    2013-01-01

    HIV-1 blocks apoptosis, programmed cell death, an innate defense of cells against viral invasion. However, apoptosis can be selectively reactivated in HIV-infected cells by chemical agents that interfere with HIV-1 gene expression. We studied two globally used medicines, the topical antifungal ciclopirox and the iron chelator deferiprone, for their effect on apoptosis in HIV-infected H9 cells and in peripheral blood mononuclear cells infected with clinical HIV-1 isolates. Both medicines activated apoptosis preferentially in HIV-infected cells, suggesting that the drugs mediate escape from the viral suppression of defensive apoptosis. In infected H9 cells, ciclopirox and deferiprone enhanced mitochondrial membrane depolarization, initiating the intrinsic pathway of apoptosis to execution, as evidenced by caspase-3 activation, poly(ADP-ribose) polymerase proteolysis, DNA degradation, and apoptotic cell morphology. In isolate-infected peripheral blood mononuclear cells, ciclopirox collapsed HIV-1 production to the limit of viral protein and RNA detection. Despite prolonged monotherapy, ciclopirox did not elicit breakthrough. No viral re-emergence was observed even 12 weeks after drug cessation, suggesting elimination of the proviral reservoir. Tests in mice predictive for cytotoxicity to human epithelia did not detect tissue damage or activation of apoptosis at a ciclopirox concentration that exceeded by orders of magnitude the concentration causing death of infected cells. We infer that ciclopirox and deferiprone act via therapeutic reclamation of apoptotic proficiency (TRAP) in HIV-infected cells and trigger their preferential elimination. Perturbations in viral protein expression suggest that the antiretroviral activity of both drugs stems from their ability to inhibit hydroxylation of cellular proteins essential for apoptosis and for viral infection, exemplified by eIF5A. Our findings identify ciclopirox and deferiprone as prototypes of selectively cytocidal

  11. Molecular Gymnastics: Mechanisms of HIV-1 Resistance to CCR5 Antagonists and Impact on Virus Phenotypes.

    PubMed

    Roche, Michael; Borm, Katharina; Flynn, Jacqueline K; Lewin, Sharon R; Churchill, Melissa J; Gorry, Paul R

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) enters host cells through the binding of its envelope glycoproteins (Env) to the host cell receptor CD4 and then subsequent binding to a chemokine coreceptor, either CCR5 or CXCR4. CCR5 antagonists are a relatively recent class addition to the armamentarium of anti-HIV-1 drugs. These compounds act by binding to a hydrophobic pocket formed by the transmembrane helices of CCR5 and altering the conformation of the extracellular domains, such that they are no longer recognized by Env. Maraviroc is the first drug within this class to be licenced for use in HIV-1 therapy regimens. HIV resistance to CCR5 antagonists occurs either through outgrowth of pre-existing CXCR4-using viruses, or through acquisition of the ability of CCR5-using HIV-1 to use the antagonist bound form of CCR5. In the latter scenario, the mechanism underlying resistance is through complex alterations in the way that resistant Envs engage CCR5. These significant changes are unlikely to occur without consequence to the viral entry phenotype and may also open up new avenues to target CCR5 antagonist resistant viruses. This review discusses the mechanism of action of CCR5 antagonists, how HIV resistance to CCR5 antagonists occurs, and the subsequent effects on Env function. PMID:26324043

  12. Potent inhibitors of HIV-1 integrase display a two-step, slow-binding inhibition mechanism which is absent in a drug-resistant T66I/M154I mutant.

    PubMed

    Garvey, Edward P; Schwartz, Benjamin; Gartland, Margaret J; Lang, Scott; Halsey, Wendy; Sathe, Ganesh; Carter, H Luke; Weaver, Kurt L

    2009-02-24

    Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs. PMID:19178153

  13. Low-frequency drug-resistant HIV-1 and risk of virological failure to first-line NNRTI-based ART: a multicohort European case–control study using centralized ultrasensitive 454 pyrosequencing

    PubMed Central

    Cozzi-Lepri, Alessandro; Noguera-Julian, Marc; Di Giallonardo, Francesca; Schuurman, Rob; Däumer, Martin; Aitken, Sue; Ceccherini-Silberstein, Francesca; D'Arminio Monforte, Antonella; Geretti, Anna Maria; Booth, Clare L.; Kaiser, Rolf; Michalik, Claudia; Jansen, Klaus; Masquelier, Bernard; Bellecave, Pantxika; Kouyos, Roger D.; Castro, Erika; Furrer, Hansjakob; Schultze, Anna; Günthard, Huldrych F.; Brun-Vezinet, Francoise; Paredes, Roger; Metzner, Karin J.; Paredes, Roger; Metzner, Karin J.; Cozzi-Lepri, Alessandro; Schuurman, Rob; Brun-Vezinet, Francoise; Günthard, Huldrych; Ceccherini-Silberstein, Francesca; Kaiser, Rolf; Geretti, Anna Maria; Brockmeyer, Norbert; Masquelier, Bernard; Dabis, F.; Bruyand, M.; Chêne, G.; Dabis, F.; Lawson-Ayayi, S.; Thiébaut, R.; Wittkop, L.; André, K.; Bonnal, F.; Bonnet, F.; Bernard, N.; Caunègre, L.; Cazanave, C.; Ceccaldi, J.; Chossat, I.; Courtaud, K.; Dauchy, F. A.; De Witte, S.; Dupon, M.; Dupont, A.; Duffau, P.; Dutronc, H.; Farbos, S.; Gaborieau, V.; Gemain, M. C.; Gerard, Y.; Greib, C.; Hessamfar, M.; Lacoste, D.; Lataste, P.; Lazaro, E.; Longy-Boursier, M.; Malvy, D.; Meraud, J. P.; Mercié, P.; Monlun, E.; Morlat, P.; Neau, D.; Ochoa, A.; Pellegrin, J. L.; Pistone, T.; Receveur, M. C.; Schmeltz, J. Roger; Tchamgoué, S.; Vandenhende, M. A.; Vareil, M.O.; Viallard, J. F.; Moreau, J. F.; Pellegrin, I.; Fleury, H.; Lafon, M. E.; Masquelier, B.; Reigadas, S.; Trimoulet, P.; Bouchet, S.; Breilh, D.; Molimard, M.; Titier, K.; Haramburu, F.; Miremont-Salamé., G.; Blaizeau, M. J.; Decoin, M.; Delaune, J.; Delveaux, S.; D'Ivernois, C.; Hanapier, C.; Leleux, O.; Lenaud, E.; Uwamaliya-Nziyumvira, B.; Sicard, X.; Geffard, S.; Le Marec, F.; Conte, V.; Frosch, A.; Leray, J.; Palmer, G.; Touchard, D.; Bonnet, F.; Breilh, D.; Chêne, G.; Dabis, F.; Dupon, M.; Fleury, H.; Malvy, D.; Mercié, P.; Morlat, P.; Neau, D.; Pellegrin, I.; Pellegrin, J. L.; Bouchet, S.; Gaborieau, V.; Lacoste, D.; Tchamgoué, S.; Thiébaut, R.; Losso, M.; Kundro, M.; Ramos Mejia, J. M.; Vetter, N.; Zangerle, R.; Karpov, I.; Vassilenko, A.; Mitsura, V. M.; Suetnov, O.; Clumeck, N.; De Wit, S.; Delforge, M.; Florence, E.; Vandekerckhove, L.; Hadziosmanovic, V.; Kostov, K.; Begovac, J.; Machala, L.; Jilich, D.; Sedlacek, D.; Nielsen, J.; Kronborg, G.; Benfield, T.; Larsen, M.; Gerstoft, J.; Katzenstein, T.; Hansen, A.-B. E.; Skinhøj, P.; Pedersen, C.; Ostergaard, L.; Dragsted, U. B.; Nielsen, L. N.; Zilmer, K.; Smidt, Jelena; Ristola, M.; Katlama, C.; Viard, J. P.; Girard, P. M.; Vanhems, P.; Pradier, C.; Dabis, F.; Neau, D.; Duvivier, C.; Rockstroh, J.; Schmidt, R.; van Lunzen, J.; Degen, O.; Stellbrink, H. J.; Bickel, M.; Bogner, J.; Fätkenheuer, G.; Kosmidis, J.; Gargalianos, P.; Xylomenos, G.; Perdios, J.; Sambatakou, H.; Banhegyi, D.; Gottfredsson, M.; Mulcahy, F.; Yust, I.; Turner, D.; Burke, M.; Pollack, S.; HassounRambam, G.; Elinav, H.; HaouziHadassah, M.; EspositoI, R.; Mazzotta, F.; Vullo, V.; Moroni, M.; Andreoni, M.; Angarano, G.; Antinori, A.; Castelli, F.; Cauda, R.; Di Perri, G.; Galli, M.; Iardino, R.; Ippolito, G.; Lazzarin, A.; Perno, C. F.; von Schloesser, F.; Viale, P.; Monforte, A. D'Arminio; Antinori, A.; Castagna, A.; Ceccherini-Silberstein, F.; Cozzi-Lepri, A.; Girardi, E.; Lo Caputo, S.; Mussini, C.; Puoti, M.; Andreoni, M.; Ammassari, A.; Antinori, A.; Balotta, C.; Bonfanti, P.; Bonora, S.; Borderi, M.; Capobianchi, M. R.; Castagna, A.; Ceccherini-Silberstein, F.; Cingolani, A.; Cinque, P.; Cozzi-Lepri, A.; De Luca, A.; Di Biagio, A.; Girardi, E.; Gianotti, N.; Gori, A.; Guaraldi, G.; Lapadula, G.; Lichtner, M.; Lo Caputo, S.; Madeddu, G.; Maggiolo, F.; Marchetti, G.; Marcotullio, S.; Monno, L.; Mussini, C.; Puoti, M.; Quiros Roldan, E.; Rusconi, S.; Cozzi-Lepri, A.; Cicconi, P.; Fanti, I.; Formenti, T.; Galli, L.; Lorenzini, P.; Carletti, F.; Carrara, S.; Castrogiovanni, A.; Di Caro, A.; Petrone, F.; Prota, G.; Quartu, S.; Giacometti, A.; Costantini, A.; Mazzoccato, S.; Angarano, G.; Monno, L.; Santoro, C.; Maggiolo, F.; Suardi, C.; Viale, P.; Vanino, E.; Verucchi, G.; Castelli, F.; Quiros Roldan, E.; Minardi, C.; Quirino, T.; Abeli, C.; Manconi, P. E.; Piano, P.; Vecchiet, J.; Falasca, K.; Sighinolfi, L.; Segala, D.; Mazzotta, F.; Lo Caputo, S.; Cassola, G.; Viscoli, C.; Alessandrini, A.; Piscopo, R.; Mazzarello, G.; Mastroianni, C.; Belvisi, V.; Bonfanti, P.; Caramma, I.; Chiodera, A.; Castelli, A. P.; Galli, M.; Lazzarin, A.; Rizzardini, G.; Puoti, M.; D'Arminio Monforte, A.; Ridolfo, A. L.; Piolini, R.; Castagna, A.; Salpietro, S.; Carenzi, L.; Moioli, M. C.; Tincati, C.; Marchetti, G.; Mussini, C.; Puzzolante, C.; Gori, A.

    2015-01-01

    Objectives It is still debated if pre-existing minority drug-resistant HIV-1 variants (MVs) affect the virological outcomes of first-line NNRTI-containing ART. Methods This Europe-wide case–control study included ART-naive subjects infected with drug-susceptible HIV-1 as revealed by population sequencing, who achieved virological suppression on first-line ART including one NNRTI. Cases experienced virological failure and controls were subjects from the same cohort whose viraemia remained suppressed at a matched time since initiation of ART. Blinded, centralized 454 pyrosequencing with parallel bioinformatic analysis in two laboratories was used to identify MVs in the 1%–25% frequency range. ORs of virological failure according to MV detection were estimated by logistic regression. Results Two hundred and sixty samples (76 cases and 184 controls), mostly subtype B (73.5%), were used for the analysis. Identical MVs were detected in the two laboratories. 31.6% of cases and 16.8% of controls harboured pre-existing MVs. Detection of at least one MV versus no MVs was associated with an increased risk of virological failure (OR = 2.75, 95% CI = 1.35–5.60, P = 0.005); similar associations were observed for at least one MV versus no NRTI MVs (OR = 2.27, 95% CI = 0.76–6.77, P = 0.140) and at least one MV versus no NNRTI MVs (OR = 2.41, 95% CI = 1.12–5.18, P = 0.024). A dose–effect relationship between virological failure and mutational load was found. Conclusions Pre-existing MVs more than double the risk of virological failure to first-line NNRTI-based ART. PMID:25336166

  14. Prevalent Polymorphisms in Wild-Type HIV-1 Integrase Are Unlikely To Engender Drug Resistance to Dolutegravir (S/GSK1349572)

    PubMed Central

    Hasan, Samiul; Madsen, Heather; Horton, Joseph; DeAnda, Felix; Martin-Carpenter, Louise; Sato, Akihiko; Cuffe, Robert; Chen, Shuguang; Underwood, Mark; Nichols, Garrett

    2013-01-01

    The majority of HIV-1 integrase amino acid sites are highly conserved, suggesting that most are necessary to carry out the critical structural and functional roles of integrase. We analyzed the 34 most variable sites in integrase (>10% variability) and showed that prevalent polymorphic amino acids at these positions did not affect susceptibility to the integrase inhibitor dolutegravir (S/GSK1349572), as demonstrated both in vitro (in site-directed mutagenesis studies) and in vivo (in a phase IIa study of dolutegravir monotherapy in HIV-infected individuals). Ongoing clinical trials will provide additional data on the virologic activity of dolutegravir across subject viruses with and without prevalent polymorphic substitutions. PMID:23295935

  15. Contribution of the 80s loop of HIV-1 protease to the multidrug-resistance mechanism: crystallographic study of MDR769 HIV-1 protease variants

    SciTech Connect

    Yedidi, Ravikiran S.; Proteasa, Georghe; Martinez, Jorge L.; Vickrey, John F.; Martin, Philip D.; Wawrzak, Zdzislaw; Liu, Zhigang; Kovari, Iulia A.; Kovari, Ladislau C.

    2011-09-06

    The flexible flaps and the 80s loops (Pro79-Ile84) of HIV-1 protease are crucial in inhibitor binding. Previously, it was reported that the crystal structure of multidrug-resistant 769 (MDR769) HIV-1 protease shows a wide-open conformation of the flaps owing to conformational rigidity acquired by the accumulation of mutations. In the current study, the effect of mutations on the conformation of the 80s loop of MDR769 HIV-1 protease variants is reported. Alternate conformations of Pro81 (proline switch) with a root-mean-square deviation of 3-4.8 {angstrom} in the C{alpha} atoms of the I10V mutant and a side chain with a 'flipped-out' conformation in the A82F mutant cause distortion in the S1/S1' binding pockets that affects inhibitor binding. The A82S and A82T mutants show local changes in the electrostatics of inhibitor binding owing to the mutation from nonpolar to polar residues. In summary, the crystallographic studies of four variants of MDR769 HIV-1 protease presented in this article provide new insights towards understanding the drug-resistance mechanism as well as a basis for design of future protease inhibitors with enhanced potency.

  16. HIV-1 Tat Protein Enhances Expression and Function of Breast Cancer Resistance Protein.

    PubMed

    Zhou, Yancong; Zhang, Kun; Yin, Xiaojie; Nie, Qichang; Ma, Yonggang

    2016-01-01

    ATP binding cassette (ABC) transporters can transfer a variety of antiviral agents from the cytoplasm to body fluid, which results in a reduced intracellular concentration of the drugs. Proteins of HIV-1, e.g., Tat and gp120, altered some types of ABC transporter expression in brain microvascular endothelial cells and astrocytes. However, the effect of Tat on ABC transporters in T lymphocytes is unclear. In this study the status of breast cancer resistance protein (BCRP) in Tat expressing cell lines was examined with real-time PCR and flow cytometry. It was found that HIV-1 Tat protein upregulated BCRP expression and enhanced efflux mediated by BCRP significantly, which could inhibit antiviral drugs from entering infected cells and interfere with the therapeutic effect of HAART. PMID:26367065

  17. Crystal Engineering of HIV-1 Reverse Transcriptase for structure-Based Drug Design

    SciTech Connect

    Bauman,J.; Das, K.; Ho, W.; Baweja, M.; Himmel, D.; Clark, A.; Oren, D.; Shatkin, A.; Arnold, E.

    2008-01-01

    HIV-1 reverse transcriptase (RT) is a primary target for anti-AIDS drugs. Structures of HIV-1 RT, usually determined at {approx}2.5-3.0 Angstroms resolution, are important for understanding enzyme function and mechanisms of drug resistance in addition to being helpful in the design of RT inhibitors. Despite hundreds of attempts, it was not possible to obtain the structure of a complex of HIV-1 RT with TMC278, a nonnucleoside RT inhibitor (NNRTI) in advanced clinical trials. A systematic and iterative protein crystal engineering approach was developed to optimize RT for obtaining crystals in complexes with TMC278 and other NNRTIs that diffract X-rays to 1.8 Angstroms resolution. Another form of engineered RT was optimized to produce a high-resolution apo-RT crystal form, reported here at 1.85 Angstroms resolution, with a distinct RT conformation. Engineered RTs were mutagenized using a new, flexible and cost effective method called methylated overlap-extension ligation independent cloning. Our analysis suggests that reducing the solvent content, increasing lattice contacts, and stabilizing the internal low-energy conformations of RT are critical for the growth of crystals that diffract to high resolution. The new RTs enable rapid crystallization and yield high-resolution structures that are useful in designing/developing new anti-AIDS drugs.

  18. Drug Resistance

    MedlinePlus

    HIV Treatment Drug Resistance (Last updated 3/1/2016; last reviewed 3/1/2016) Key Points As HIV multiplies in the ... the risk of drug resistance. What is HIV drug resistance? Once a person becomes infected with HIV, ...

  19. Importance of polar solvation and configurational entropy for design of antiretroviral drugs targeting HIV-1 protease.

    PubMed

    Kar, Parimal; Lipowsky, Reinhard; Knecht, Volker

    2013-05-16

    Both KNI-10033 and KNI-10075 are high affinity preclinical HIV-1 protease (PR) inhibitors with affinities in the picomolar range. In this work, the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method has been used to investigate the potency of these two HIV-1 PR inhibitors against the wild-type and mutated proteases assuming that potency correlates with the affinity of the drugs for the target protein. The decomposition of the binding free energy reveals the origin of binding affinities or mutation-induced affinity changes. Our calculations indicate that the mutation I50V causes drug resistance against both inhibitors. On the other hand, we predict that the mutant I84V causes drug resistance against KNI-10075 while KNI-10033 is more potent against the I84V mutant compared to wild-type protease. Drug resistance arises mainly from unfavorable shifts in van der Waals interactions and configurational entropy. The latter indicates that neglecting changes in configurational entropy in the computation of relative binding affinities as often done is not appropriate in general. For the bound complex PR(I50V)-KNI-10075, an increased polar solvation free energy also contributes to the drug resistance. The importance of polar solvation free energies is revealed when interactions governing the binding of KNI-10033 or KNI-10075 to the wild-type protease are compared to the inhibitors darunavir or GRL-06579A. Although the contributions from intermolecular electrostatic and van der Waals interactions as well as the nonpolar component of the solvation free energy are more favorable for PR-KNI-10033 or PR-KNI-10075 compared to PR-DRV or PR-GRL-06579A, both KNI-10033 and KNI-10075 show a similar affinity as darunavir and a lower binding affinity relative to GRL-06579A. This is because of the polar solvation free energy which is less unfavorable for darunavir or GRL-06579A relative to KNI-10033 or KNI-10075. The importance of the polar solvation as revealed here

  20. Design and synthesis of HIV-1 protease inhibitors for a long-acting injectable drug application.

    PubMed

    Kesteleyn, Bart; Amssoms, Katie; Schepens, Wim; Hache, Geerwin; Verschueren, Wim; Van De Vreken, Wim; Rombauts, Klara; Meurs, Greet; Sterkens, Patrick; Stoops, Bart; Baert, Lieven; Austin, Nigel; Wegner, Jörg; Masungi, Chantal; Dierynck, Inge; Lundgren, Stina; Jönsson, Daniel; Parkes, Kevin; Kalayanov, Genadiy; Wallberg, Hans; Rosenquist, Asa; Samuelsson, Bertil; Van Emelen, Kristof; Thuring, Jan Willem

    2013-01-01

    The design and synthesis of novel HIV-1 protease inhibitors (PIs) (1-22), which display high potency against HIV-1 wild-type and multi-PI-resistant HIV-mutant clinical isolates, is described. Lead optimization was initiated from compound 1, a Phe-Phe hydroxyethylene peptidomimetic PI, and was directed towards the discovery of new PIs suitable for a long-acting (LA) injectable drug application. Introducing a heterocyclic 6-methoxy-3-pyridinyl or a 6-(dimethylamino)-3-pyridinyl moiety (R(3)) at the para-position of the P1' benzyl fragment generated compounds with antiviral potency in the low single digit nanomolar range. Halogenation or alkylation of the metabolic hot spots on the various aromatic rings resulted in PIs with high stability against degradation in human liver microsomes and low plasma clearance in rats. Replacing the chromanolamine moiety (R(1)) in the P2 protease binding site by a cyclopentanolamine or a cyclohexanolamine derivative provided a series of high clearance PIs (16-22) with EC(50)s on wild-type HIV-1 in the range of 0.8-1.8 nM. PIs 18 and 22, formulated as nanosuspensions, showed gradual but sustained and complete release from the injection site over two months in rats, and were therefore identified as interesting candidates for a LA injectable drug application for treating HIV/AIDS. PMID:23177258

  1. Structural basis of HIV-1 resistance to AZT by excision

    SciTech Connect

    Tu, Xiongying; Das, Kalyan; Han, Qianwei; Bauman, Joseph D.; Clark, Jr., Arthur D.; Hou, Xiaorong; Frenkel, Yulia V.; Gaffney, Barbara L.; Jones, Roger A.; Boyer, Paul L.; Hughes, Stephen H.; Sarafianos, Stefan G.; Arnold, Eddy

    2011-11-23

    Human immunodeficiency virus (HIV-1) develops resistance to 3'-azido-2',3'-deoxythymidine (AZT, zidovudine) by acquiring mutations in reverse transcriptase that enhance the ATP-mediated excision of AZT monophosphate from the 3' end of the primer. The excision reaction occurs at the dNTP-binding site, uses ATP as a pyrophosphate donor, unblocks the primer terminus and allows reverse transcriptase to continue viral DNA synthesis. The excision product is AZT adenosine dinucleoside tetraphosphate (AZTppppA). We determined five crystal structures: wild-type reverse transcriptase-double-stranded DNA (RT-dsDNA)-AZTppppA; AZT-resistant (AZTr; M41L D67N K70R T215Y K219Q) RT-dsDNA-AZTppppA; AZTr RT-dsDNA terminated with AZT at dNTP- and primer-binding sites; and AZTr apo reverse transcriptase. The AMP part of AZTppppA bound differently to wild-type and AZTr reverse transcriptases, whereas the AZT triphosphate part bound the two enzymes similarly. Thus, the resistance mutations create a high-affinity ATP-binding site. The structure of the site provides an opportunity to design inhibitors of AZT-monophosphate excision.

  2. Selected Drugs with Reported Secondary Cell-Differentiating Capacity Prime Latent HIV-1 Infection for Reactivation

    PubMed Central

    Shishido, Takao; Wolschendorf, Frank; Duverger, Alexandra; Wagner, Frederic; Kappes, John; Jones, Jennifer

    2012-01-01

    Reactivation of latent HIV-1 infection is considered our best therapeutic means to eliminate the latent HIV-1 reservoir. Past therapeutic attempts to systemically trigger HIV-1 reactivation using single drugs were unsuccessful. We thus sought to identify drug combinations consisting of one component that would lower the HIV-1 reactivation threshold and a synergistic activator. With aclacinomycin and dactinomycin, we initially identified two FDA-approved drugs that primed latent HIV-1 infection in T cell lines and in primary T cells for reactivation and facilitated complete reactivation at the population level. This effect was correlated not with the reported primary drug effects but with the cell-differentiating capacity of the drugs. We thus tested other cell-differentiating drugs/compounds such as cytarabine and aphidicolin and found that they also primed latent HIV-1 infection for reactivation. This finding extends the therapeutic promise of N′-N′-hexamethylene-bisacetamide (HMBA), another cell-differentiating agent that has been reported to trigger HIV-1 reactivation, into the group of FDA-approved drugs. To this end, it is also noteworthy that suberoylanilide hydroxamic acid (SAHA), a polar compound that was initially developed as a second-generation cell-differentiating agent using HMBA as a structural template and which is now marketed as the histone deacetylase (HDAC) inhibitor vorinostat, also has been reported to trigger HIV-1 reactivation. Our findings suggest that drugs with primary or secondary cell-differentiating capacity should be revisited as HIV-1-reactivating agents as some could potentially be repositioned as candidate drugs to be included in an induction therapy to trigger HIV-1 reactivation. PMID:22696646

  3. Selected drugs with reported secondary cell-differentiating capacity prime latent HIV-1 infection for reactivation.

    PubMed

    Shishido, Takao; Wolschendorf, Frank; Duverger, Alexandra; Wagner, Frederic; Kappes, John; Jones, Jennifer; Kutsch, Olaf

    2012-09-01

    Reactivation of latent HIV-1 infection is considered our best therapeutic means to eliminate the latent HIV-1 reservoir. Past therapeutic attempts to systemically trigger HIV-1 reactivation using single drugs were unsuccessful. We thus sought to identify drug combinations consisting of one component that would lower the HIV-1 reactivation threshold and a synergistic activator. With aclacinomycin and dactinomycin, we initially identified two FDA-approved drugs that primed latent HIV-1 infection in T cell lines and in primary T cells for reactivation and facilitated complete reactivation at the population level. This effect was correlated not with the reported primary drug effects but with the cell-differentiating capacity of the drugs. We thus tested other cell-differentiating drugs/compounds such as cytarabine and aphidicolin and found that they also primed latent HIV-1 infection for reactivation. This finding extends the therapeutic promise of N'-N'-hexamethylene-bisacetamide (HMBA), another cell-differentiating agent that has been reported to trigger HIV-1 reactivation, into the group of FDA-approved drugs. To this end, it is also noteworthy that suberoylanilide hydroxamic acid (SAHA), a polar compound that was initially developed as a second-generation cell-differentiating agent using HMBA as a structural template and which is now marketed as the histone deacetylase (HDAC) inhibitor vorinostat, also has been reported to trigger HIV-1 reactivation. Our findings suggest that drugs with primary or secondary cell-differentiating capacity should be revisited as HIV-1-reactivating agents as some could potentially be repositioned as candidate drugs to be included in an induction therapy to trigger HIV-1 reactivation. PMID:22696646

  4. HIV-1 Alters Intestinal Expression of Drug Transporters and Metabolic Enzymes: Implications for Antiretroviral Drug Disposition.

    PubMed

    Kis, Olena; Sankaran-Walters, Sumathi; Hoque, M Tozammel; Walmsley, Sharon L; Dandekar, Satya; Bendayan, Reina

    2016-05-01

    This study investigated the effects of HIV-1 infection and antiretroviral therapy (ART) on the expression of intestinal drug efflux transporters, i.e., P-glycoprotein (Pgp), multidrug resistance-associated proteins (MRPs), and breast cancer resistance protein (BCRP), and metabolic enzymes, such as cytochrome P450s (CYPs), in the human upper intestinal tract. Intestinal biopsy specimens were obtained from HIV-negative healthy volunteers, ART-naive HIV-positive (HIV(+)) subjects, and HIV(+) subjects receiving ART (10 in each group). Intestinal tissue expression of drug transporters and metabolic enzymes was examined by microarray, real-time quantitative reverse transcription-PCR (qPCR), and immunohistochemistry analyses. Microarray analysis demonstrated significantly lower expression of CYP3A4 and ABCC2/MRP2 in the HIV(+) ART-naive group than in uninfected subjects. qPCR analysis confirmed significantly lower expression of ABCC2/MRP2 in ART-naive subjects than in the control group, while CYP3A4 and ABCG2/BCRP showed a trend toward decreased expression. Protein expression of MRP2 and BCRP was also significantly lower in the HIV(+) naive group than in the control group and was partially restored to baseline levels in HIV(+) subjects receiving ART. In contrast, gene and protein expression of ABCB1/Pgp was significantly increased in HIV(+) subjects on ART relative to HIV(+) ART-naive subjects. These data demonstrate that the expression of drug-metabolizing enzymes and efflux transporters is significantly altered in therapy-naive HIV(+) subjects and in those receiving ART. Since CYP3A4, Pgp, MRPs, and BCRP metabolize or transport many antiretroviral drugs, their altered expression with HIV infection may negatively impact drug pharmacokinetics in HIV(+) subjects. This has clinical implications when using data from healthy volunteers to guide ART. PMID:26902756

  5. Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase

    PubMed Central

    Kuroda, Daniel G.; Bauman, Joseph D.; Challa, J. Reddy; Patel, Disha; Troxler, Thomas; Das, Kalyan; Arnold, Eddy; Hochstrasser, Robin M.

    2013-01-01

    The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase and retain potency against drug-resistance mutations. Our discovery that water molecules play an essential role in the drug binding is reported. Femtosecond experiments and theory expose molecular level dynamics of rilpivirine bound to HIV-1 reverse transcriptase. The two nitrile substituents (-CN), one on each arm of the drug, have vibrational spectra consistent with their protein environments being similar in crystals and in solutions. Two-dimensional vibrational-echo spectroscopy reveals a dry environment for one nitrile while unexpectedly the other is hydrogen-bonded to a mobile water molecule, not identified in earlier X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure indeed reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond, despite the enlargement and structural variation of the binding pocket, may help retain the potency of rilpivirine against the pocket mutations. PMID:23422558

  6. Drug-induced lipotoxicity: lipodystrophy associated with HIV-1 infection and antiretroviral treatment.

    PubMed

    Villarroya, Francesc; Domingo, Pere; Giralt, Marta

    2010-03-01

    A subset of HIV-1-infected patients undergoing antiretroviral treatment develops a lipodystrophy syndrome. It is characterized by loss of peripheral subcutaneous adipose tissue (face, limbs, buttocks), visceral fat accumulation, and, in some cases, lipomatosis, especially in the dorsocervical area. In addition, these patients show metabolic alterations reminiscent of the metabolic syndrome, particularly dyslipidemia and insulin resistance. These alterations lead to enhanced cardiovascular risk in patients and favor the development of diabetes. Although a complex combination of HIV-1 infection and drug treatment-related events triggers the syndrome, lipotoxicity appears to contribute to the development of the syndrome. Active lipolysis in subcutaneous fat, combined with impaired fat storage capacity in the subcutaneous depot, drive ectopic deposition of lipids, either in the visceral depot or in nonadipose sites. Both hepatic steatosis and increased lipid content in skeletal muscle take place and surely contribute to systemic metabolic alterations, especially insulin resistance. Pancreatic function may also be affected by the exposure to high levels of fatty acids; together with direct effects of antiretroviral drugs, this may contribute to impaired insulin release and a prodiabetic state in the patients. Addressing lipotoxicity as a pathogenic actor in the lipodystrophy syndrome should be considered in strategies for treating and/or preventing the morphological alterations and systemic metabolic disturbances associated with lipodystrophy. PMID:19800025

  7. Snapshot of the equilibrium dynamics of a drug bound to HIV-1 reverse transcriptase

    NASA Astrophysics Data System (ADS)

    Kuroda, Daniel G.; Bauman, Joseph D.; Challa, J. Reddy; Patel, Disha; Troxler, Thomas; Das, Kalyan; Arnold, Eddy; Hochstrasser, Robin M.

    2013-03-01

    The anti-AIDS drug rilpivirine undergoes conformational changes to bind HIV-1 reverse transcriptase (RT), which is an essential enzyme for the replication of HIV. These changes allow it to retain potency against mutations that otherwise would render the enzyme resistant. Here we report that water molecules play an essential role in this binding process. Femtosecond experiments and theory expose the molecular level dynamics of rilpivirine bound to HIV-1 RT. Two nitrile substituents, one on each arm of the drug, are used as vibrational probes of the structural dynamics within the binding pocket. Two-dimensional vibrational echo spectroscopy reveals that one nitrile group is unexpectedly hydrogen-bonded to a mobile water molecule, not identified in previous X-ray structures. Ultrafast nitrile-water dynamics are confirmed by simulations. A higher (1.51 Å) resolution X-ray structure also reveals a water-drug interaction network. Maintenance of a crucial anchoring hydrogen bond may help retain the potency of rilpivirine against pocket mutations despite the structural variations they cause.

  8. MULTIPLE WAYS OF TARGETING APOBEC3/VIF INTERACTIONS FOR ANTI-HIV-1 DRUG DEVELOPMENT

    PubMed Central

    Smith, Jessica L.; Bu, Wei; Burdick, Ryan C.; Pathak, Vinay K.

    2009-01-01

    Human immunodeficiency virus type 1 (HIV-1) infections and the resulting acquired immunodeficiency syndrome (AIDS) pandemic remain a global challenge in the absence of a protective vaccine and because of rapid selection of drug-resistant viral variants in response to all currently available antiviral therapies. Development of new and highly active antiviral agents would greatly facilitate effective clinical management of HIV-1 infections and delay the onset of AIDS. Recent advances in our understanding of intracellular immunity conferred by host cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F), and the mechanism by which the virally encoded Virion Infectivity Factor (Vif) protein induces their proteasomal degradation, provide fresh opportunities for the development of novel antiviral treatments. Interestingly, the interactions between Vif-A3G and Vif-A3F that overcome this host defense mechanism are structurally distinct, and provide two potential targets for antiviral drug development. This review provides an overview of the current knowledge of APOBEC3/Vif interactions and recent efforts to target these interactions for antiviral drug development. PMID:19837465

  9. Interaction between Tat and Drugs of Abuse during HIV-1 Infection and Central Nervous System Disease

    PubMed Central

    Maubert, Monique E.; Pirrone, Vanessa; Rivera, Nina T.; Wigdahl, Brian; Nonnemacher, Michael R.

    2016-01-01

    In many individuals, drug abuse is intimately linked with HIV-1 infection. In addition to being associated with one-third of all HIV-1 infections in the United States, drug abuse also plays a role in disease progression and severity in HIV-1-infected patients, including adverse effects on the central nervous system (CNS). Specific systems within the brain are known to be damaged in HIV-1-infected individuals and this damage is similar to that observed in drug abuse. Even in the era of anti-retroviral therapy (ART), CNS pathogenesis occurs with HIV-1 infection, with a broad range of cognitive impairment observed, collectively referred to as HIV-1-associated neurocognitive disorders (HAND). A number of HIV-1 proteins (Tat, gp120, Nef, Vpr) have been implicated in the etiology of pathogenesis and disease as a result of the biologic activity of the extracellular form of each of the proteins in a number of tissues, including the CNS, even in ART-suppressed patients. In this review, we have made Tat the center of attention for a number of reasons. First, it has been shown to be synthesized and secreted by HIV-1-infected cells in the CNS, despite the most effective suppression therapies available to date. Second, Tat has been shown to alter the functions of several host factors, disrupting the molecular and biochemical balance of numerous pathways contributing to cellular toxicity, dysfunction, and death. In addition, the advantages and disadvantages of ART suppression with regard to controlling the genesis and progression of neurocognitive impairment are currently under debate in the field and are yet to be fully determined. In this review, we discuss the individual and concerted contributions of HIV-1 Tat, drug abuse, and ART with respect to damage in the CNS, and how these factors contribute to the development of HAND in HIV-1-infected patients. PMID:26793168

  10. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9.

    PubMed

    Yoder, Kristine E; Bundschuh, Ralf

    2016-01-01

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance. PMID:27404981

  11. Host Double Strand Break Repair Generates HIV-1 Strains Resistant to CRISPR/Cas9

    PubMed Central

    Yoder, Kristine E.; Bundschuh, Ralf

    2016-01-01

    CRISPR/Cas9 genome editing has been proposed as a therapeutic treatment for HIV-1 infection. CRISPR/Cas9 induced double strand breaks (DSBs) targeted to the integrated viral genome have been shown to decrease production of progeny virus. Unfortunately HIV-1 evolves rapidly and may readily produce CRISPR/Cas9 resistant strains. Here we used next-generation sequencing to characterize HIV-1 strains that developed resistance to six different CRISPR/Cas9 guide RNAs (gRNAs). Reverse transcriptase (RT) derived base substitution mutations were commonly found at sites encoding unpaired bases of RNA stem-loop structures. In addition to RT mutations, insertion and/or deletion (indel) mutations were common. Indels localized to the CRISPR/Cas9 cleavage site were major contributors to CRISPR gRNA resistance. While most indels at non-coding regions were a single base pair, 3 base pair indels were observed when a coding region of HIV-1 was targeted. The DSB repair event may preserve the HIV-1 reading frame, while destroying CRISPR gRNA homology. HIV-1 may be successfully edited by CRISPR/Cas9, but the virus remains competent for replication and resistant to further CRISPR/Cas9 targeting at that site. These observations strongly suggest that host DSB repair at CRISPR/Cas9 cleavage sites is a novel and important pathway that may contribute to HIV-1 therapeutic resistance. PMID:27404981

  12. Antiretroviral Genotypic Resistance Mutations in HIV-1 Infected Korean Patients with Virologic Failure

    PubMed Central

    Chin, Bum Sik; Choi, Ju-Yeon; Choi, Jin Young; Kim, Gab Jung; Kee, Mee-Kyung; Kim, June Myung

    2009-01-01

    Resistance assays are useful in guiding decisions for patients experiencing virologic failure (VF) during highly-active antiretroviral therapy (HAART). We investigated antiretroviral resistance mutations in 41 Korean human immunodeficiency virus type 1 (HIV-1) infected patients with VF and observed immunologic/virologic response 6 months after HAART regimen change. Mean HAART duration prior to resistance assay was 45.3±27.5 months and commonly prescribed HAART regimens were zidovudine/lamivudine/nelfinavir (22.0%) and zidovudine/lamivudine/efavirenz (19.5%). Forty patients (97.6%) revealed intermediate to high-level resistance to equal or more than 2 antiretroviral drugs among prescribed HAART regimen. M184V/I mutation was observed in 36 patients (87.7%) followed by T215Y/F (41.5%) and M46I/L (34%). Six months after resistance assay and HAART regimen change, median CD4+ T cell count increased from 168 cells/µL (interquartile range [IQR], 62-253) to 276 cells/µL (IQR, 153-381) and log viral load decreased from 4.65 copies/mL (IQR, 4.18-5.00) to 1.91 copies/mL (IQR, 1.10-3.60) (P<0.001 for both values). The number of patients who accomplished viral load <400 copies/mL was 26 (63.4%) at 6 months follow-up. In conclusion, many Korean HIV-1 infected patients with VF are harboring strains with multiple resistance mutations and immunologic/virologic parameters are improved significantly after genotypic resistance assay and HAART regimen change. PMID:19949656

  13. HIV-1 Resistance to the Capsid-Targeting Inhibitor PF74 Results in Altered Dependence on Host Factors Required for Virus Nuclear Entry

    PubMed Central

    Zhou, Jing; Price, Amanda J.; Halambage, Upul D.; James, Leo C.

    2015-01-01

    ABSTRACT During HIV-1 infection of cells, the viral capsid plays critical roles in reverse transcription and nuclear entry of the virus. The capsid-targeting small molecule PF74 inhibits HIV-1 at early stages of infection. HIV-1 resistance to PF74 is complex, requiring multiple amino acid substitutions in the viral CA protein. Here we report the identification and analysis of a novel PF74-resistant mutant encoding amino acid changes in both domains of CA, three of which are near the pocket where PF74 binds. Interestingly, the mutant virus retained partial PF74 binding, and its replication was stimulated by the compound. The mutant capsid structure was not significantly perturbed by binding of PF74; rather, the mutations inhibited capsid interactions with CPSF6 and Nup153 and altered HIV-1 dependence on these host factors and on TNPO3. Moreover, the replication of the mutant virus was markedly impaired in activated primary CD4+ T cells and macrophages. Our results suggest that HIV-1 escapes a capsid-targeting small molecule inhibitor by altering the virus's dependence on host factors normally required for entry into the nucleus. They further imply that clinical resistance to inhibitors targeting the PF74 binding pocket is likely to be strongly limited by functional constraints on HIV-1 evolution. IMPORTANCE The HIV-1 capsid plays critical roles in early steps of infection and is an attractive target for therapy. Here we show that selection for resistance to a capsid-targeting small molecule inhibitor can result in viral dependence on the compound. The mutant virus was debilitated in primary T cells and macrophages—cellular targets of infection in vivo. The mutations also altered the virus's dependence on cellular factors that are normally required for HIV-1 entry into the nucleus. This work provides new information regarding mechanisms of HIV-1 resistance that should be useful in efforts to develop clinically useful drugs targeting the HIV-1 capsid. PMID:26109731

  14. Cocaine modulates HIV-1 integration in primary CD4+ T cells: implications in HIV-1 pathogenesis in drug-abusing patients

    PubMed Central

    Addai, Amma B.; Pandhare, Jui; Paromov, Victor; Mantri, Chinmay K.; Pratap, Siddharth; Dash, Chandravanu

    2015-01-01

    Epidemiologic studies suggest that cocaine abuse worsens HIV-1 disease progression. Increased viral load has been suggested to play a key role for the accelerated HIV disease among cocaine-abusing patients. The goal of this study was to investigate whether cocaine enhances proviral DNA integration as a mechanism to increase viral load. We infected CD4+ T cells that are the primary targets of HIV-1 in vivo and treated the cells with physiologically relevant concentrations of cocaine (1 µM–100 µM). Proviral DNA integration in the host genome was measured by nested qPCR. Our results illustrated that cocaine from 1 µM through 50 µM increased HIV-1 integration in CD4+ T cells in a dose-dependent manner. As integration can be modulated by several early postentry steps of HIV-1 infection, we examined the direct effects of cocaine on viral integration by in vitro integration assays by use of HIV-1 PICs. Our data illustrated that cocaine directly increases viral DNA integration. Furthermore, our MS analysis showed that cocaine is able to enter CD4+ T cells and localize to the nucleus-. In summary, our data provide strong evidence that cocaine can increase HIV-1 integration in CD4+ T cells. Therefore, we hypothesize that increased HIV-1 integration is a novel mechanism by which cocaine enhances viral load and worsens disease progression in drug-abusing HIV-1 patients. PMID:25691383

  15. In vitro model of mycobacteria and HIV-1 co-infection for drug discovery.

    PubMed

    Vijayakumar, Sudhamathi; Finney John, Sarah; Nusbaum, Rebecca J; Ferguson, Monique R; Cirillo, Jeffrey D; Olaleye, Omonike; Endsley, Janice J

    2013-12-01

    Tuberculosis (TB) has become a global health threat in the wake of the Human Immunodeficiency Virus (HIV) pandemic and is the leading cause of death in people with HIV/AIDS. Treatment of patients with Mycobacterium tuberculosis (Mtb)/HIV co-infection is complicated by drug interactions and toxicity that present huge challenges for clinical intervention. Discovery efforts to identify novel compounds with increased effectiveness and decreased drug-drug interactions against Mtb, HIV-1, or both, would be greatly aided by the use of a co-infection model for screening drug libraries. Currently, inhibitors of Mtb are screened independently in mycobacterial cell cultures or target based biochemical screens and less often in macrophages or peripheral blood leukocytes. Similarly, HIV-1 drugs are screened in vitro independently from anti-mycobacterial compounds. Here, we describe an in vitro model where primary human peripheral blood mononuclear cells or monocyte-derived macrophages are infected with Mycobacterium bovis BCG and HIV-1, and used to evaluate drug toxicity and activity in a co-infection setting. Our results with standard compounds (e.g. Azidothymidine, Rifampicin) demonstrate the utility of this in vitro model to evaluate drug effectiveness relevant to cellular toxicity, HIV-1 replication, and intracellular mycobacterial growth, through the use of ELISA, bacterial enumeration, and multi-variate flow cytometry. This model and associated assays have great value in accelerating the discovery of compounds for use in Mtb/HIV-1 co-infected patients. PMID:24388652

  16. Vpu-Mediated Counteraction of Tetherin Is a Major Determinant of HIV-1 Interferon Resistance

    PubMed Central

    Kmiec, Dorota; Iyer, Shilpa S.; Stürzel, Christina M.; Sauter, Daniel; Hahn, Beatrice H.

    2016-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) groups M, N, O, and P are the result of independent zoonotic transmissions of simian immunodeficiency viruses (SIVs) infecting great apes in Africa. Among these, only Vpu proteins of pandemic HIV-1 group M strains evolved potent activity against the restriction factor tetherin, which inhibits virus release from infected cells. Thus, effective Vpu-mediated tetherin antagonism may have been a prerequisite for the global spread of HIV-1. To determine whether this particular function enhances primary HIV-1 replication and interferon resistance, we introduced mutations into the vpu genes of HIV-1 group M and N strains to specifically disrupt their ability to antagonize tetherin, but not other Vpu functions, such as degradation of CD4, down-modulation of CD1d and NTB-A, and suppression of NF-κB activity. Lack of particular human-specific adaptations reduced the ability of HIV-1 group M Vpu proteins to enhance virus production and release from primary CD4+ T cells at high levels of type I interferon (IFN) from about 5-fold to 2-fold. Interestingly, transmitted founder HIV-1 strains exhibited higher virion release capacity than chronic control HIV-1 strains irrespective of Vpu function, and group M viruses produced higher levels of cell-free virions than an N group HIV-1 strain. Thus, efficient virus release from infected cells seems to play an important role in the spread of HIV-1 in the human population and requires a fully functional Vpu protein that counteracts human tetherin. PMID:27531907

  17. Structural Basis of Why Nelfinavir-Resistant D30N Mutant of HIV-1 Protease Remains Susceptible to Saquinavir.

    PubMed

    Prashar, Vishal; Bihani, Subhash C; Ferrer, Jean-Luc; Hosur, Madhusoodan V

    2015-09-01

    Although anti-HIV-1 protease drugs nelfinavir (NFV) and saquinavir (SQV) share common functional groups, D30N is a major resistance mutation against NFV but remains susceptible to SQV. We have determined the crystal structure of D30N mutant-tethered HIV-1 protease in complex with SQV to 1.79 Å resolution. Structural analysis showed that SQV forms two direct hydrogen bonds with the main chain atoms of the residues Asp29 and Asp30 that are not observed in the D30N-NFV complex. Apart from maintaining these two main chain hydrogen bonds, the P2-asparagine of SQV forms an additional hydrogen bond to the mutated side chain of the residue 30. These could be the reasons why D30N is not a drug resistance mutation against SQV. This structure supports the previous studies showing that the interactions between a potential inhibitor and backbone atoms of the enzyme are important to maintain potency against drug-resistant HIV-1 protease. PMID:25487655

  18. Compensatory Substitutions in the HIV-1 Capsid Reduce the Fitness Cost Associated with Resistance to a Capsid-Targeting Small-Molecule Inhibitor

    PubMed Central

    Shi, Jiong; Zhou, Jing; Halambage, Upul D.; Shah, Vaibhav B.; Burse, Mallori J.; Wu, Hua; Blair, Wade S.; Butler, Scott L.

    2014-01-01

    ABSTRACT The HIV-1 capsid plays multiple roles in infection and is an emerging therapeutic target. The small-molecule HIV-1 inhibitor PF-3450074 (PF74) blocks HIV-1 at an early postentry stage by binding the viral capsid and interfering with its function. Selection for resistance resulted in accumulation of five amino acid changes in the viral CA protein, which collectively reduced binding of the compound to HIV-1 particles. In the present study, we dissected the individual and combinatorial contributions of each of the five substitutions Q67H, K70R, H87P, T107N, and L111I to PF74 resistance, PF74 binding, and HIV-1 infectivity. Q67H, K70R, and T107N each conferred low-level resistance to PF74 and collectively conferred strong resistance. The substitutions K70R and L111I impaired HIV-1 infectivity, which was partially restored by the other substitutions at positions 67 and 107. PF74 binding to HIV-1 particles was reduced by the Q67H, K70R, and T107N substitutions, consistent with the location of these positions in the inhibitor-binding pocket. Replication of the 5Mut virus was markedly impaired in cultured macrophages, reminiscent of the previously reported N74D CA mutant. 5Mut substitutions also reduced the binding of the host protein CPSF6 to assembled CA complexes in vitro and permitted infection of cells expressing the inhibitory protein CPSF6-358. Our results demonstrate that strong resistance to PF74 requires accumulation of multiple substitutions in CA to inhibit PF74 binding and compensate for fitness impairments associated with some of the sequence changes. IMPORTANCE The HIV-1 capsid is an emerging drug target, and several small-molecule compounds have been reported to inhibit HIV-1 infection by targeting the capsid. Here we show that resistance to the capsid-targeting inhibitor PF74 requires multiple amino acid substitutions in the binding pocket of the CA protein. Three changes in CA were necessary to inhibit binding of PF74 while maintaining viral

  19. Structure of the HIV-1 reverse transcriptase Q151M mutant: insights into the inhibitor resistance of HIV-1 reverse transcriptase and the structure of the nucleotide-binding pocket of Hepatitis B virus polymerase

    SciTech Connect

    Nakamura, Akiyoshi; Tamura, Noriko; Yasutake, Yoshiaki

    2015-10-23

    The structure of the HIV-1 reverse transcriptase Q151M mutant was determined at a resolution of 2.6 Å in space group P321. Hepatitis B virus polymerase (HBV Pol) is an important target for anti-HBV drug development; however, its low solubility and stability in vitro has hindered detailed structural studies. Certain nucleotide reverse transcriptase (RT) inhibitors (NRTIs) such as tenofovir and lamivudine can inhibit both HBV Pol and Human immunodeficiency virus 1 (HIV-1) RT, leading to speculation on structural and mechanistic analogies between the deoxynucleotide triphosphate (dNTP)-binding sites of these enzymes. The Q151M mutation in HIV-1 RT, located at the dNTP-binding site, confers resistance to various NRTIs, while maintaining sensitivity to tenofovir and lamivudine. The residue corresponding to Gln151 is strictly conserved as a methionine in HBV Pol. Therefore, the structure of the dNTP-binding pocket of the HIV-1 RT Q151M mutant may reflect that of HBV Pol. Here, the crystal structure of HIV-1 RT Q151M, determined at 2.6 Å resolution, in a new crystal form with space group P321 is presented. Although the structure of HIV-1 RT Q151M superimposes well onto that of HIV-1 RT in a closed conformation, a slight movement of the β-strands (β2–β3) that partially create the dNTP-binding pocket was observed. This movement might be caused by the introduction of the bulky thioether group of Met151. The structure also highlighted the possibility that the hydrogen-bonding network among amino acids and NRTIs is rearranged by the Q151M mutation, leading to a difference in the affinity of NRTIs for HIV-1 RT and HBV Pol.

  20. Two subtypes of HIV-1 among injection-drug users in southern China.

    PubMed

    Yu, X F; Chen, J; Shao, Y; Beyrer, C; Lai, S

    1998-04-25

    The rate of HIV-1 infection has increased steadily in China by about 80% annually and by the end of September 1997, 8277 HIV-1 cases had been reported, of whom more than 75% were IV drug users (IVDUs). UNAIDS, however, has estimated that up to 200,000 people could actually be infected with HIV-1 in China. Guangxi Province borders Yunnan province in the west and Vietnam to the south, and is a major transit area for heroin trafficking from the opium-growing region of Laos and Myanmar. Phylogenetic analyses of HIV-1 env sequences (C2-V3) obtained from 14 IVDUs found that 9 subjects from Pingxiang City were infected with subtype E and 5 from Baise City with subtype C. The 9 subtype E and 5 subtype C HIV-1 sequences were clustered together within each group, with significant bootstrap values of 100% and 95%, respectively. The subtype E sequences were more closely related to HIV-1 subtype E from Thailand than to those from Africa, and the subtype C sequences were clustered more closely to those from India than to those from Africa. Study results suggest 2 epidemiologically unrelated epidemics and 2 different sources; subtype C probably transmitted from Yunnan to Baise City through drug trafficking and IVDU interaction, and subtype E coming into China from Vietnam. PMID:9643749

  1. High-Resolution Structures of HIV-1 Reverse Transcriptase/TMC278 Complexes: Strategic Flexibility Explains Potency Against Resistance Mutations

    SciTech Connect

    Das,K.; Bauman, J.; Clark, Jr., A.; Frenkel, Y.; Lewi, P.; Shatkin, A.; Hughes, S.; Arnold, E.

    2008-01-01

    TMC278 is a diarylpyrimidine (DAPY) nonnucleoside reverse transcriptase inhibitor (NNRTI) that is highly effective in treating wild-type and drug-resistant HIV-1 infections in clinical trials at relatively low doses ({approx}25-75 mg/day). We have determined the structure of wild-type HIV-1 RT complexed with TMC278 at 1.8 Angstroms resolution, using an RT crystal form engineered by systematic RT mutagenesis. This high-resolution structure reveals that the cyanovinyl group of TMC278 is positioned in a hydrophobic tunnel connecting the NNRTI-binding pocket to the nucleic acid-binding cleft. The crystal structures of TMC278 in complexes with the double mutant K103N/Y181C (2.1 Angstroms ) and L100I/K103N HIV-1 RTs (2.9 Angstroms ) demonstrated that TMC278 adapts to bind mutant RTs. In the K103N/Y181C RT/TMC278 structure, loss of the aromatic ring interaction caused by the Y181C mutation is counterbalanced by interactions between the cyanovinyl group of TMC278 and the aromatic side chain of Y183, which is facilitated by an {approx}1.5 Angstroms shift of the conserved Y183MDD motif. In the L100I/K103N RT/TMC278 structure, the binding mode of TMC278 is significantly altered so that the drug conforms to changes in the binding pocket primarily caused by the L100I mutation. The flexible binding pocket acts as a molecular 'shrink wrap' that makes a shape complementary to the optimized TMC278 in wild-type and drug-resistant forms of HIV-1 RT. The crystal structures provide a better understanding of how the flexibility of an inhibitor can compensate for drug-resistance mutations.

  2. Computational Analysis of HIV-1 Resistance Based on Gene Expression Profiles and the Virus-Host Interaction Network

    PubMed Central

    Huang, Tao; Xu, Zhongping; Chen, Lei; Cai, Yu-Dong; Kong, Xiangyin

    2011-01-01

    A very small proportion of people remain negative for HIV infection after repeated HIV-1 viral exposure, which is called HIV-1 resistance. Understanding the mechanism of HIV-1 resistance is important for the development of HIV-1 vaccines and Acquired Immune Deficiency Syndrome (AIDS) therapies. In this study, we analyzed the gene expression profiles of CD4+ T cells from HIV-1-resistant individuals and HIV-susceptible individuals. One hundred eighty-five discriminative HIV-1 resistance genes were identified using the Minimum Redundancy-Maximum Relevance (mRMR) and Incremental Feature Selection (IFS) methods. The virus protein target enrichment analysis of the 185 HIV-1 resistance genes suggested that the HIV-1 protein nef might play an important role in HIV-1 infection. Moreover, we identified 29 infection information exchanger genes from the 185 HIV-1 resistance genes based on a virus-host interaction network analysis. The infection information exchanger genes are located on the shortest paths between virus-targeted proteins and are important for the coordination of virus infection. These proteins may be useful targets for AIDS prevention or therapy, as intervention in these pathways could disrupt communication with virus-targeted proteins and HIV-1 infection. PMID:21394196

  3. Ex vivo analysis identifies effective HIV-1 latency-reversing drug combinations.

    PubMed

    Laird, Gregory M; Bullen, C Korin; Rosenbloom, Daniel I S; Martin, Alyssa R; Hill, Alison L; Durand, Christine M; Siliciano, Janet D; Siliciano, Robert F

    2015-05-01

    Reversal of HIV-1 latency by small molecules is a potential cure strategy. This approach will likely require effective drug combinations to achieve high levels of latency reversal. Using resting CD4+ T cells (rCD4s) from infected individuals, we developed an experimental and theoretical framework to identify effective latency-reversing agent (LRA) combinations. Utilizing ex vivo assays for intracellular HIV-1 mRNA and virion production, we compared 2-drug combinations of leading candidate LRAs and identified multiple combinations that effectively reverse latency. We showed that protein kinase C agonists in combination with bromodomain inhibitor JQ1 or histone deacetylase inhibitors robustly induce HIV-1 transcription and virus production when directly compared with maximum reactivation by T cell activation. Using the Bliss independence model to quantitate combined drug effects, we demonstrated that these combinations synergize to induce HIV-1 transcription. This robust latency reversal occurred without release of proinflammatory cytokines by rCD4s. To extend the clinical utility of our findings, we applied a mathematical model that estimates in vivo changes in plasma HIV-1 RNA from ex vivo measurements of virus production. Our study reconciles diverse findings from previous studies, establishes a quantitative experimental approach to evaluate combinatorial LRA efficacy, and presents a model to predict in vivo responses to LRAs. PMID:25822022

  4. C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir

    PubMed Central

    Aoki, Manabu; Hayashi, Hironori; Yedidi, Ravikiran S.; Martyr, Cuthbert D.; Takamatsu, Yuki; Aoki-Ogata, Hiromi; Nakamura, Teruya; Nakata, Hirotomo; Das, Debananda; Yamagata, Yuriko; Ghosh, Arun K.

    2015-01-01

    vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRVR. Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRVR. The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRVR. PMID:26581995

  5. HIV-1 protease mutations and protease inhibitor cross-resistance.

    PubMed

    Rhee, Soo-Yon; Taylor, Jonathan; Fessel, W Jeffrey; Kaufman, David; Towner, William; Troia, Paolo; Ruane, Peter; Hellinger, James; Shirvani, Vivian; Zolopa, Andrew; Shafer, Robert W

    2010-10-01

    The effects of many protease inhibitor (PI)-selected mutations on the susceptibility to individual PIs are unknown. We analyzed in vitro susceptibility test results on 2,725 HIV-1 protease isolates. More than 2,400 isolates had been tested for susceptibility to fosamprenavir, indinavir, nelfinavir, and saquinavir; 2,130 isolates had been tested for susceptibility to lopinavir; 1,644 isolates had been tested for susceptibility to atazanavir; 1,265 isolates had been tested for susceptibility to tipranavir; and 642 isolates had been tested for susceptibility to darunavir. We applied least-angle regression (LARS) to the 200 most common mutations in the data set and identified a set of 46 mutations associated with decreased PI susceptibility of which 40 were not polymorphic in the eight most common HIV-1 group M subtypes. We then used least-squares regression to ascertain the relative contribution of each of these 46 mutations. The median number of mutations associated with decreased susceptibility to each PI was 28 (range, 19 to 32), and the median number of mutations associated with increased susceptibility to each PI was 2.5 (range, 1 to 8). Of the mutations with the greatest effect on PI susceptibility, I84AV was associated with decreased susceptibility to eight PIs; V32I, G48V, I54ALMSTV, V82F, and L90M were associated with decreased susceptibility to six to seven PIs; I47A, G48M, I50V, L76V, V82ST, and N88S were associated with decreased susceptibility to four to five PIs; and D30N, I50L, and V82AL were associated with decreased susceptibility to fewer than four PIs. This study underscores the greater impact of nonpolymorphic mutations compared with polymorphic mutations on decreased PI susceptibility and provides a comprehensive quantitative assessment of the effects of individual mutations on susceptibility to the eight clinically available PIs. PMID:20660676

  6. Phenotypic assays and sequencing are less sensitive than point mutation assays for detection of resistance in mixed HIV-1 genotypic populations.

    PubMed

    Van Laethem, K; Van Vaerenbergh, K; Schmit, J C; Sprecher, S; Hermans, P; De Vroey, V; Schuurman, R; Harrer, T; Witvrouw, M; Van Wijngaerden, E; Stuyver, L; Van Ranst, M; Desmyter, J; De Clercq, E; Vandamme, A M

    1999-10-01

    The sensitivity and discriminatory power of the 151 and 215 amplification refractory mutation system (ARMS) were evaluated, and their performance for the detection of drug resistance in mixed genotypic populations of the reverse transcription (RT) gene of HIV-1 were compared with T7 sequencing, cycle sequencing, the line probe assay (LiPA) HIV-1 RT test, and the recombinant virus assay (RVA). ARMS and the LiPA HIV-1 RT test were shown to be able to detect minor variants that in particular cases comprised only 1%. T7 sequencing on an ALF semiautomated sequencer could correctly score mixtures only when variants were present at 50%. Cycle sequencing on an ABI PRISM 310 improved the sensitivity for mixtures to about 25%. Using RVA, it was shown that at least 50% of the virus population needed to carry the resistance mutation at codon 184 to afford phenotypic resistance against lamivudine. The two point mutation assays therefore proved to be more sensitive methods than sequencing and RVA to reliably determine a gradual shift in HIV-1 drug resistance mutations in follow-up of patients infected with HIV-1. In 4 of 5 treated patients who were followed by ARMS, a gradual shift in resistant genotypic populations was observed during a period of 6 to 19 months. For 1 patient, a shift from wild to mutant type at position 151 occurred within 2 months, without mixed genotypic intermediate type's being detected. PMID:10843523

  7. CRF07_BC Strain Dominates the HIV-1 Epidemic in Injection Drug Users in Liangshan Prefecture of Sichuan, China.

    PubMed

    Li, Lin; Wei, Daying; Hsu, Wan-Ling; Li, Tianyi; Gui, Tao; Wood, Charles; Liu, Yongjian; Li, Hanping; Bao, Zuoyi; Liu, Siyang; Wang, Xiaolin; Li, Jingyun

    2015-05-01

    The Liangshan prefecture in Sichuan province is an area in China severely affected by the HIV epidemic, with intravenous drug use (IDU) as the main risk factor. No reports on HIV subtypes prevalent in IDUs in Liangshan prefecture could be found. In this study, we have characterized the genotypes of HIV-1 in the IDU population in Liangshan prefecture and further determined the phylogenetic relationship of the CRF07_BC strains to HIV-1 sequences from the other regions of China, including Xinjiang and Yunnan provinces, to explore the pattern and possible diffusion pathway of HIV-1 in these regions. HIV-1-seropositive drug-naive IDUs identified in Liangshan prefecture, Sichuan province were enrolled in 2009. Full-length gag and pol genes were amplified by reverse transcription and nested PCR and then sequenced. All of the sequences were subtyped. Phylogenetic trees were constructed using the neighbor-joining and maximum likelihood methods. Divergence times were estimated using a Bayesian molecular clock approach. CRF07_BC was found to be the predominant strain in IDUs in Liangshan prefecture (95.5%). The CRF07_BC strains from Liangshan prefecture were found to be intermixed with those from Yunnan province in phylogenetic trees. The CRF07_BC sequences from Xinjiang province can be grouped into several clusters, suggesting that the expansion of the CRF07_BC epidemic in Xinjiang province was the result of a local epidemic driven by multiple independent introductions in the late 1990s. Only low-level drug-resistant viruses were found in the IDU population. CRF07_BC strains from Liangshan prefecture were more similar to those from Yunnan province than those from Xinjiang province. This finding will contribute to our understanding of the distribution, the evolution, and the potential source of CRF07_BC founder strains, and will also provide useful information for the development of strategies to prevent transmission. PMID:25560398

  8. CRF07_BC Strain Dominates the HIV-1 Epidemic in Injection Drug Users in Liangshan Prefecture of Sichuan, China

    PubMed Central

    Li, Lin; Wei, Daying; Hsu, Wan-Ling; Li, Tianyi; Gui, Tao; Wood, Charles; Liu, Yongjian; Li, Hanping; Bao, Zuoyi; Liu, Siyang; Wang, Xiaolin

    2015-01-01

    Abstract The Liangshan prefecture in Sichuan province is an area in China severely affected by the HIV epidemic, with intravenous drug use (IDU) as the main risk factor. No reports on HIV subtypes prevalent in IDUs in Liangshan prefecture could be found. In this study, we have characterized the genotypes of HIV-1 in the IDU population in Liangshan prefecture and further determined the phylogenetic relationship of the CRF07_BC strains to HIV-1 sequences from the other regions of China, including Xinjiang and Yunnan provinces, to explore the pattern and possible diffusion pathway of HIV-1 in these regions. HIV-1-seropositive drug-naive IDUs identified in Liangshan prefecture, Sichuan province were enrolled in 2009. Full-length gag and pol genes were amplified by reverse transcription and nested PCR and then sequenced. All of the sequences were subtyped. Phylogenetic trees were constructed using the neighbor-joining and maximum likelihood methods. Divergence times were estimated using a Bayesian molecular clock approach. CRF07_BC was found to be the predominant strain in IDUs in Liangshan prefecture (95.5%). The CRF07_BC strains from Liangshan prefecture were found to be intermixed with those from Yunnan province in phylogenetic trees. The CRF07_BC sequences from Xinjiang province can be grouped into several clusters, suggesting that the expansion of the CRF07_BC epidemic in Xinjiang province was the result of a local epidemic driven by multiple independent introductions in the late 1990s. Only low-level drug-resistant viruses were found in the IDU population. CRF07_BC strains from Liangshan prefecture were more similar to those from Yunnan province than those from Xinjiang province. This finding will contribute to our understanding of the distribution, the evolution, and the potential source of CRF07_BC founder strains, and will also provide useful information for the development of strategies to prevent transmission. PMID:25560398

  9. HIV-1 drug discovery: targeting folded RNA structures with branched peptides.

    PubMed

    Wynn, Jessica E; Santos, Webster L

    2015-06-01

    Human immunodeficiency virus type 1 (HIV-1) is an RNA virus that is prone to high rates of mutation. While the disease is managed with current antiretroviral therapies, drugs with a new mode of action are needed. A strategy towards this goal is aimed at targeting the native three-dimensional fold of conserved RNA structures. This perspective highlights medium-sized peptides and peptidomimetics used to target two conserved RNA structures of HIV-1. In particular, branched peptides have the capacity to bind in a multivalent fashion, utilizing a large surface area to achieve the necessary affinity and selectivity toward the target RNA. PMID:25958855

  10. HIV-1 Drug Discovery: Targeting Folded RNA Structures With Branched Peptides

    PubMed Central

    Wynn, Jessica E.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) is an RNA virus that is prone to high rates of mutation. While the disease is managed with current antiretroviral therapies, drugs with a new mode of action are needed. A strategy towards this goal is aimed at targeting the native three-dimensional fold of conserved RNA structures. This perspective highlights medium-sized peptides and peptidomimetics used to target two conserved RNA structures of HIV-1. In particular, branched peptides have the capacity to bind in a multivalent fashion, utilizing a large surface area to achieve the necessary affinity and selectivity toward the target RNA. PMID:25958855

  11. Sub-Epidemics Explain Localized High Prevalence of Reduced Susceptibility to Rilpivirine in Treatment-Naive HIV-1-Infected Patients: Subtype and Geographic Compartmentalization of Baseline Resistance Mutations

    PubMed Central

    Van Laethem, Kristel; Gomes, Perpetua; Baele, Guy; Pineda-Peña, Andrea-Clemencia; Vandamme, Anne-Mieke; Camacho, Ricardo J.; Abecasis, Ana B.

    2016-01-01

    Abstract Objective: The latest nonnucleoside reverse transcriptase inhibitor (NNRTI) rilpivirine (RPV) is indicated for human immunodeficiency virus type-1 (HIV-1) patients initiating antiretroviral treatment, but the extent of genotypic RPV resistance in treatment-naive patients outside clinical trials is poorly defined. Study Design: This retrospective observational study of clinical data from Belgium and Portugal evaluates genotypic information from HIV-1 drug-naive patients obtained for the purpose of drug resistance testing. Rilpivirine resistance-associated mutations (RPV-RAMs) were defined based on clinical trials, phenotypic studies, and expert-based resistance algorithms. Viral susceptibility to RPV alone and to the single-tablet regimen was estimated using expert-based resistance algorithms. Results: In 4,631 HIV-1 treatment-naive patients infected with diverse HIV-1 subtypes, major RPV-RAMs were detected in 4.6%, while complete viral susceptibility to RPV was estimated in 95% of patients. Subtype C- and F1-infected patients displayed the highest levels of reduced viral susceptibility at baseline, respectively 13.2% and 9.3%, mainly due to subtype- and geographic-dependent occurrence of RPV-RAMs E138A and A98G as natural polymorphisms. Strikingly, a founder effect in Portugal resulted in a 138A prevalence of 13.2% in local subtype C-infected treatment-naive patients. The presence of transmitted drug resistance did not impact our estimates. Conclusion: RPV is the first HIV-1 inhibitor for which, in the absence of transmitted drug resistance, intermediate or high-level genotypic resistance can be detected in treatment-naive patients. The extent of RPV susceptibility in treatment-naive patients differs depending on the HIV-1 subtype and dynamics of local compartmentalized epidemics. The highest prevalence of reduced susceptibility was found to be 15.7% in Portuguese subtype C-infected treatment-naive patients. In this context, even in the absence of

  12. Resistance and replicative capacity of HIV-1 strains selected in vivo by long-term enfuvirtide treatment.

    PubMed

    Menzo, S; Castagna, A; Monachetti, A; Hasson, H; Danise, A; Carini, E; Bagnarelli, P; Lazzarin, A; Clementi, M

    2004-04-01

    Enfuvirtide is the prototype member of a new class of anti HIV-1 agents, the fusion inhibitors (FI). In recent clinical trials, the compound has shown its efficacy in combination with other antiretroviral agents in vivo. However mutant strains resistant to the action of the drug arise quite rapidly in vitro and in vivo. To analyze the process of selection and evolution of HIV-1 strains resistant to enfuvirtide in vivo and to evaluate the impact of resistance on viral fitness, 12 HIV-1 infected subjects treated with T20 (enfuvirtide) for at least one year were included in the study. Gp41-coding sequences were amplified from plasma samples of these subjects at baseline and at different time points during treatment. Seven of the 12 subjects showed selection of gp41 mutations under the selective pressure of enfuvirtide. In particular, these mutations clustered in two distinct regions: (i) a mutational hot-spot localized, as previously described, in the first residues of the N-HR domain, with position number 38 as the most heavily mutated, but including also a G36V, a N42D/T, a N43D, a L44M and a L45M; (ii) other mutations were localized further downstream, within N-HR/C-HR junction and in the C-HR. A recombinant assay specifically designed for the determination of HIV-1 phenotype to FI was developed and validated. Using this assay, we observed that all of the 7 mutated clones displayed substantially reduced susceptibility to T20, IC50 ranging from 0.6 to12.8 microg/ml (>100 fold change). The residues whose mutation was associated with a potent reduction in susceptibility were V38, N42, and N43, other positions such as G36, N44 and L45 playing a minor role. None of the mutant HIV isolates showed cross-resistance to T-1249. By the same method, the HIV-1 replicative capacity of the recombinant clones was tested in the absence of drugs, and for each subject, pre-therapy clones were compared to post-therapy ones. In 3/7 subjects a significant decrease in replicative

  13. Positional adaptability in the design of mutation-resistant nonnucleoside HIV-1 reverse transcriptase inhibitors: a supramolecular perspective.

    PubMed

    Bruccoleri, Aldo

    2013-01-01

    Drug resistance is a key cause of failed treatment of HIV infection. The efficacy of nonnucleoside reverse transcriptase-inhibiting (NNRTI) drugs is impaired by the rapid emergence of drug-resistant mutations. The literature supports the idea that purposefully designed flexible NNRTIs at an active site may help overcome drug resistance. It is proposed here that the usual "lock and key" model, with respect to NNRTI drug design, be expanded to consider creating "master keys" that would automatically adjust conformations to fit all of the "locks" mutations may make. The present work introduces the novel perspective of designing and creating supramolecular assemblies as potential NNRTIs (instead of the relatively more rigid single-molecule inhibitors). Specifically, flexible self-assembling quinhydrone supramolecular dimers formed from quinonoid monomers (designed to be highly flexible NNRTIs themselves) will be offered as a working example of this new perspective in NNRTI drug design. Quinonoid compounds have demonstrated binding interactions at various sites of the HIV-1 RT enzyme, including the elusive ribonuclease H area. Quinhydrone self-organized dimers have at some point in their molecular architecture a noncovalently interacting donor-acceptor ring pair complex. This complex is at the heart of the increased torsional, rotational, and translational motion this species will experience at a particular active site. Flexible supramolecular assemblies, together with their flexible monomer components, may offer a critical advantage in retaining potency against a wide range of drug-resistant HIV-1 RTs. This new supramolecular perspective may also have broader implications in the general field of antimicrobial drug design. PMID:22938539

  14. Different Effects of Nonnucleoside Reverse Transcriptase Inhibitor Resistance Mutations on Cytotoxic T Lymphocyte Recognition between HIV-1 Subtype B and Subtype A/E Infections

    PubMed Central

    Kuse, Nozomi; Rahman, Mohammad Arif; Murakoshi, Hayato; Tran, Giang Van; Chikata, Takayuki; Koyanagi, Madoka; Nguyen, Kinh Van; Gatanaga, Hiroyuki; Oka, Shinichi

    2015-01-01

    ABSTRACT The effect of antiretroviral drug resistance mutations on cytotoxic T lymphocyte (CTL) recognition has been analyzed in HIV-1 subtype B infections, but it remains unclear in infections by other HIV-1 subtypes that are epidemic in countries where antiretroviral drugs are not effectively used. We investigated the effect of nonnucleoside reverse transcriptase (RT) inhibitor (NNRTI)-resistance mutations (Y181C, Y181I, and Y181V) on epitope recognition by CTLs specific for 3 different HIV-1 epitopes (HLA-A*02:01-restricted IV10, HLA-B*35:01-restricted NY9, and HLA-C*12:02-restricted KY9) in subtype B and subtype A/E infections and the accumulation of these mutations in treatment-naive Japanese and Vietnamese. These NNRTI-resistance mutations critically affected NY9-specific and KY9-specific T cell responses in the subtype B infections, whereas they showed a different effect on IV10-specific T cell responses among the subtype B-infected individuals. These mutations affected IV10-specific T cell responses but weakly affected NY9-specific T cell responses in the subtype A/E infections. The substitution at position 3 of NY9 epitope which was found in the subtype A/E virus differently influenced the peptide binding to HLA-B*35:01, suggesting that the differences in peptide binding may result in the differences in T cell recognition between the subtype B virus and A/E virus infections. The Y181C mutation was found to be accumulating in treatment-naive Vietnamese infected with the subtype A/E virus. The present study demonstrated different effects of NNRTI-resistance RT181 mutations on CTL responses between the 2 subtype infections. The Y181C mutation may influence HIV-1 control by the CTLs in Vietnam, since this mutation has been accumulating in treatment-naive Vietnamese. IMPORTANCE Antiretroviral therapy leads to the emergence of drug-resistant HIV-1, resulting in virological and clinical failures. Though HIV-1-specific CTLs play a critical role in HIV-1 infection

  15. Drug Resistance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Drug resistance refers to both intrinsic and acquired abilities of cells or organisms to become insensitive or refractory to chemotherapeutic intervention. The advent of antibiotics is considered one of the most important medicinal developments in human history, which has led to significantly reduce...

  16. Antiretroviral genotypic resistance in plasma RNA and whole blood DNA in HIV-1 infected patients failing HAART.

    PubMed

    Saracino, Annalisa; Gianotti, Nicola; Marangi, Marianna; Cibelli, Donatella C; Galli, Andrea; Punzi, Grazia; Monno, Laura; Lazzarin, Adriano; Angarano, Gioacchino

    2008-10-01

    The extent to which HIV-1 proviral DNA mutations cause clinically relevant antiretroviral resistance is still controversial. Paired plasma HIV-1 RNA and whole blood DNA were compared in patients failing HAART to investigate if the additional knowledge of archived mutations could improve the selection of potentially active drugs. Seventy-three HIV-1-infected patients with first/second HAART failure were studied before starting a new regimen based on RNA genotyping. Follow-up data after a 12-week therapy were available. DNA genotyping was retrospectively performed on stored whole blood samples and mutational profiles were compared to those from RNA. The mean number of IAS pol mutations was significantly higher in RNA (4.45 +/- 2.76) than in DNA (2.88 +/- 2.47) (P < 0.001). DNA genotyping provided a 6% increase in detection of resistance-associated mutations. Among 64/73 patients showing discordant DNA/RNA profiles, 54 (84%) also differed for predicted active drugs. 16/73 (22%) patients had >or=1 mutation revealed by DNA genotyping alone, probably affecting therapy success in 2/16. However, neither RNA/DNA discordance nor detection of isolated DNA mutations were statistically associated with outcome. In conclusion, plasma RNA remains the elective choice for HIV genotyping in patients with therapy failure, even if the detection of proviral resistance-associated mutations, not simultaneously found in RNA, is a frequent event. Therefore, in some cases DNA plus RNA genotyping might assist in choosing more accurately subsequent antiretroviral regimens. PMID:18712823

  17. Consensus HIV-1 subtype A integrase and its raltegravir-resistant variants: design and characterization of the enzymatic properties.

    PubMed

    Shadrina, Olga; Krotova, Olga; Agapkina, Julia; Knyazhanskaya, Ekaterina; Korolev, Sergey; Starodubova, Elizaveta; Viklund, Alecia; Lukashov, Vladimir; Magnani, Mauro; Medstrand, Patrik; Karpov, Vadim; Gottikh, Marina; Isaguliants, Maria

    2014-07-01

    Model studies of the subtype B and non-subtype B integrases are still required to compare their susceptibility to antiretroviral drugs, evaluate the significance of resistance mutations and identify the impact of natural polymorphisms on the level of enzymatic reactivity. We have therefore designed the consensus integrase of the HIV-1 subtype A strain circulating in the former Soviet Union territory (FSU-A) and two of its variants with mutations of resistance to the strand transfer inhibitor raltegravir. Their genes were synthesized, and expressed in E coli; corresponding His-tagged proteins were purified using the affinity chromatography. The enzymatic properties of the consensus integrases and their sensitivity to raltegravir were examined in a series of standard in vitro reactions and compared to the properties of the integrase of HIV-1 subtype B strain HXB2. The consensus enzyme demonstrated similar DNA-binding properties, but was significantly more active than HXB-2 integrase in the reactions of DNA cleavage and integration. All integrases were equally susceptible to inhibition by raltegravir and elvitegravir, indicating that the sporadic polymorphisms inherent to the HXB-2 enzyme have little effect on its susceptibility to drugs. Insensitivity of the mutated enzymes to the inhibitors of strand transfer occurred at a cost of a 30-90% loss of the efficacies of both 3'-processing and strand transfer. This is the first study to describe the enzymatic properties of the consensus integrase of HIV-1 clade A and the effects of the resistance mutations when the complex actions of sporadic sequence polymorphisms are excluded. PMID:24594066

  18. Analysis of the Zidovudine Resistance Mutations T215Y, M41L, and L210W in HIV-1 Reverse Transcriptase

    PubMed Central

    Boyer, Paul L.; Das, Kalyan; Arnold, Eddy

    2015-01-01

    Although anti-human immunodeficiency virus type 1 (HIV-1) therapies have become more sophisticated and more effective, drug resistance continues to be a major problem. Zidovudine (azidothymidine; AZT) was the first nucleoside reverse transcriptase (RT) inhibitor (NRTI) approved for the treatment of HIV-1 infections and is still being used, particularly in the developing world. This drug targets the conversion of single-stranded RNA to double-stranded DNA by HIV-1 RT. However, resistance to the drug quickly appeared both in viruses replicating in cells in culture and in patients undergoing AZT monotherapy. The primary resistance pathway selects for mutations of T215 that change the threonine to either a tyrosine or a phenylalanine (T215Y/F); this resistance pathway involves an ATP-dependent excision mechanism. The pseudo-sugar ring of AZT lacks a 3′ OH; RT incorporates AZT monophosphate (AZTMP), which blocks the end of the viral DNA primer. AZT-resistant forms of HIV-1 RT use ATP in an excision reaction to unblock the 3′ end of the primer strand, allowing its extension by RT. The T215Y AZT resistance mutation is often accompanied by two other mutations, M41L and L210W. In this study, the roles of these mutations, in combination with T215Y, were examined to determine whether they affect polymerization and excision by HIV-1 RT. The M41L mutation appears to help restore the DNA polymerization activity of RT containing the T215Y mutation and also enhances AZTMP excision. The L210W mutation plays a similar role, but it enhances excision by RTs that carry the T215Y mutation when ATP is present at a low concentration. PMID:26324274

  19. Probing Multidrug-Resistance and Protein-Ligand Interactions with Oxatricyclic Designed Ligands in HIV-1 Protease Inhibitors

    SciTech Connect

    Ghosh, Arun K.; Xu, Chun-Xiao; Rao, Kalapala V.; Baldridge, Abigail; Agniswamy, Johnson; Wang, Yuan-Fang; Weber, Irene T.; Aoki, Manabu; Miguel, Salcedo Pedro; Amano, Masayuki; Mitsuya, Hiroaki

    2010-10-29

    We report the design, synthesis, biological evaluation, and X-ray crystallographic analysis of a new class of HIV-1 protease inhibitors. Compound 4 proved to be an extremely potent inhibitor toward various multidrug-resistant HIV-1 variants, representing a near 10-fold improvement over darunavir (DRV). Compound 4 also blocked protease dimerization with at least 10-fold greater potency than DRV.

  20. Unique Flap Conformation in an HIV-1 Protease with High-Level Darunavir Resistance.

    PubMed

    Nakashima, Masaaki; Ode, Hirotaka; Suzuki, Koji; Fujino, Masayuki; Maejima, Masami; Kimura, Yuki; Masaoka, Takashi; Hattori, Junko; Matsuda, Masakazu; Hachiya, Atsuko; Yokomaku, Yoshiyuki; Suzuki, Atsuo; Watanabe, Nobuhisa; Sugiura, Wataru; Iwatani, Yasumasa

    2016-01-01

    Darunavir (DRV) is one of the most powerful protease inhibitors (PIs) for treating human immunodeficiency virus type-1 (HIV-1) infection and presents a high genetic barrier to the generation of resistant viruses. However, DRV-resistant HIV-1 infrequently emerges from viruses exhibiting resistance to other protease inhibitors. To address this resistance, researchers have gathered genetic information on DRV resistance. In contrast, few structural insights into the mechanism underlying DRV resistance are available. To elucidate this mechanism, we determined the crystal structure of the ligand-free state of a protease with high-level DRV resistance and six DRV resistance-associated mutations (including I47V and I50V), which we generated by in vitro selection. This crystal structure showed a unique curling conformation at the flap regions that was not found in the previously reported ligand-free protease structures. Molecular dynamics simulations indicated that the curled flap conformation altered the flap dynamics. These results suggest that the preference for a unique flap conformation influences DRV binding. These results provide new structural insights into elucidating the molecular mechanism of DRV resistance and aid to develop PIs effective against DRV-resistant viruses. PMID:26870021

  1. Unique Flap Conformation in an HIV-1 Protease with High-Level Darunavir Resistance

    PubMed Central

    Nakashima, Masaaki; Ode, Hirotaka; Suzuki, Koji; Fujino, Masayuki; Maejima, Masami; Kimura, Yuki; Masaoka, Takashi; Hattori, Junko; Matsuda, Masakazu; Hachiya, Atsuko; Yokomaku, Yoshiyuki; Suzuki, Atsuo; Watanabe, Nobuhisa; Sugiura, Wataru; Iwatani, Yasumasa

    2016-01-01

    Darunavir (DRV) is one of the most powerful protease inhibitors (PIs) for treating human immunodeficiency virus type-1 (HIV-1) infection and presents a high genetic barrier to the generation of resistant viruses. However, DRV-resistant HIV-1 infrequently emerges from viruses exhibiting resistance to other protease inhibitors. To address this resistance, researchers have gathered genetic information on DRV resistance. In contrast, few structural insights into the mechanism underlying DRV resistance are available. To elucidate this mechanism, we determined the crystal structure of the ligand-free state of a protease with high-level DRV resistance and six DRV resistance-associated mutations (including I47V and I50V), which we generated by in vitro selection. This crystal structure showed a unique curling conformation at the flap regions that was not found in the previously reported ligand-free protease structures. Molecular dynamics simulations indicated that the curled flap conformation altered the flap dynamics. These results suggest that the preference for a unique flap conformation influences DRV binding. These results provide new structural insights into elucidating the molecular mechanism of DRV resistance and aid to develop PIs effective against DRV-resistant viruses. PMID:26870021

  2. Full quantum mechanical study of binding of HIV-1 protease drugs

    NASA Astrophysics Data System (ADS)

    Zhang, Da W.; Zhang, John Z. H.

    Fully quantum mechanical studies of detailed binding interactions between HIV-1 protease and six FDA (Food and Drug Administration)-approved drugs (saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir) are carried out using a recently developed MFCC (molecular fractionation with conjugate caps) method. The MFCC calculation produces a quantum mechanical interaction spectrum for any protease drug binding complex. Detailed quantitative analysis on binding of lopinavir to specific residues of the protease is given from the current study. The present calculation shows that the dominant binding of lopinavir to the protease is through the formation of a strong hydrogen bond between the central hydroxyl group of the drug to the aspartate oxygen of Asp25 in one of the two chains of the protease (A chain). This is closely followed by hydrogen binding of the drug to Asp29 in the B chain and somewhat weak hydrogen bonding to Asp30, Gly27, Gly48, and Ile50 in both chains. By partitioning all six drugs into four building blocks besides the central component containing the hydroxyl group, MFCC calculation finds that block III has essentially no binding interaction with the protease and the major binding interactions of these drugs are from blocks II and IV, in addition to the dominant central hydroxyl group. This detailed quantitative information on drug binding to the protease is very useful in rational design of new and improved inhibitors of HIV-1 protease and its mutants.

  3. HIV-1 gp120 and Drugs of Abuse: Interactions in the Central Nervous System

    PubMed Central

    Silverstein, Peter S.; Shah, Ankit; Weemhoff, James; Kumar, Santosh; Singh, D.P.; Kumar, Anil

    2014-01-01

    HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs. PMID:22591361

  4. HIV-1 gp120 and drugs of abuse: interactions in the central nervous system.

    PubMed

    Silverstein, Peter S; Shah, Ankit; Weemhoff, James; Kumar, Santosh; Singh, D P; Kumar, Anil

    2012-07-01

    HIV-1 infection is a global public health problem with more than 34 million people living with HIV infection. Although great strides have been made in treating this epidemic with therapeutic agents, the increase in patient life span has been coincident with an increase in the prevalence of HIV-associated neurocognitive disorders (HAND). HAND is thought to result from the neurotoxic effects of viral proteins that are shed from HIV-infected microglial cells. One of the primary neurotoxins responsible for this effect is the HIV-1 glycoprotein gp120. Exposure of neurons to gp120 has been demonstrated to cause apoptosis in neurons, as well as numerous indirect effects such as an increase in inflammatory cytokines, an increase in oxidative stress, and an increase in permeability of the blood-brain barrier. In many patients, the use of drugs of abuse (DOA) exacerbates the neurotoxic effects of gp120. Cocaine, methamphetamine and morphine are three DOAs that are commonly used by those infected with HIV-1. All three of these DOAs have been demonstrated to increase oxidative stress in the CNS as well as to increase permeability of the blood-brain barrier. Numerous model systems have demonstrated that these DOAs have the capability of exacerbating the neurotoxic effects of gp120. This review will summarize the neurotoxic effects of gp120, the deleterious effects of cocaine, methamphetamine and morphine on the CNS, and the combined effects of gp120 in the context of these drugs. PMID:22591361

  5. Genotypic Testing for Human Immunodeficiency Virus Type 1 Drug Resistance

    PubMed Central

    Shafer, Robert W.

    2002-01-01

    There are 16 approved human immunodeficiency virus type 1 (HIV-1) drugs belonging to three mechanistic classes: protease inhibitors, nucleoside and nucleotide reverse transcriptase (RT) inhibitors, and nonnucleoside RT inhibitors. HIV-1 resistance to these drugs is caused by mutations in the protease and RT enzymes, the molecular targets of these drugs. Drug resistance mutations arise most often in treated individuals, resulting from selective drug pressure in the presence of incompletely suppressed virus replication. HIV-1 isolates with drug resistance mutations, however, may also be transmitted to newly infected individuals. Three expert panels have recommended that HIV-1 protease and RT susceptibility testing should be used to help select HIV drug therapy. Although genotypic testing is more complex than typical antimicrobial susceptibility tests, there is a rich literature supporting the prognostic value of HIV-1 protease and RT mutations. This review describes the genetic mechanisms of HIV-1 drug resistance and summarizes published data linking individual RT and protease mutations to in vitro and in vivo resistance to the currently available HIV drugs. PMID:11932232

  6. Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease.

    PubMed

    Louis, John M; Roche, Julien

    2016-07-01

    Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism. PMID:27170547

  7. Modulation of HIV-1 Gag NC/p1 cleavage efficiency affects protease inhibitor resistance and viral replicative capacity

    PubMed Central

    2012-01-01

    Background Mutations in the substrate of HIV-1 protease, especially changes in the NC/p1 cleavage site, can directly contribute to protease inhibitor (PI) resistance and also compensate for defects in viral replicative capacity (RC) due to a drug resistant protease. These NC/p1 changes are known to enhance processing of the Gag protein. To investigate the capacity of HIV-1 to modulate Gag cleavage and its consequences for PI resistance and RC, we performed a detailed enzymatic and virological analysis using a set of PI resistant NC/p1 variants (HXB2431V, HXB2436E+437T, HXB2437T and HXB2437V). Results Here, we demonstrate that single NC/p1 mutants, which displayed only a slight increase in PI resistance did not show an obvious change in RC. In contrast, the double NC/p1 mutant, which displayed a clear increase in processing efficiency and PI resistance, demonstrated a clear reduction in RC. Cleavage analysis showed that a tridecameric NC/p1 peptide representing the double NC/p1 mutant was cleaved in two specific ways instead of one. The observed decrease in RC for the double NC/p1 mutant (HXB2436E+437T) could (partially) be restored by either reversion of the 436E change or by acquisition of additional changes in the NC/p1 cleavage site at codon 435 or 438 as was revealed during in vitro evolution experiments. These changes not only restored RC but also reduced PI resistance levels. Furthermore these changes normalized Gag processing efficiency and obstructed the novel secondary cleavage site observed for the double NC/p1 mutant. Conclusions The results of this study clearly demonstrate that HIV-1 can modulate Gag processing and thereby PI resistance. Distinct increases in Gag cleavage and PI resistance result in a reduced RC that can only be restored by amino acid changes in NC/p1 which reduce Gag processing to an optimal rate. PMID:22462820

  8. Clinical Management of HIV Drug Resistance

    PubMed Central

    Cortez, Karoll J.; Maldarelli, Frank

    2011-01-01

    Combination antiretroviral therapy for HIV-1 infection has resulted in profound reductions in viremia and is associated with marked improvements in morbidity and mortality. Therapy is not curative, however, and prolonged therapy is complicated by drug toxicity and the emergence of drug resistance. Management of clinical drug resistance requires in depth evaluation, and includes extensive history, physical examination and laboratory studies. Appropriate use of resistance testing provides valuable information useful in constructing regimens for treatment-experienced individuals with viremia during therapy. This review outlines the emergence of drug resistance in vivo, and describes clinical evaluation and therapeutic options of the individual with rebound viremia during therapy. PMID:21994737

  9. Vitamin-D pathway genes and HIV-1 disease progression in injection drug users.

    PubMed

    Laplana, Marina; Sánchez-de-la-Torre, Manuel; Puig, Teresa; Caruz, Antonio; Fibla, Joan

    2014-07-15

    Vitamin-D has pleiotropic effects on calcium and bone metabolism, cellular growth control, cell differentiation and modulation of both innate and acquired immune response. Previous studies revealed the association of vitamin-D receptor gene (VDR) polymorphism with infection diseases including HIV-1 infection. To assess for association between polymorphisms of vitamin-D pathway genes CYP27B1, vitamin-D binding protein (VDBP) and VDR with HIV-1 infection, disease progression to acquired immunodeficiency syndrome (AIDS) was analysed according to CDC93 criteria in a cohort of 185 HIV-1 seroprevalent patients belonging to the injection drug users. Genotype data was obtained from rs10877012, rs3782130 and rs4646536 markers at CYP27B1 locus; rs7041 and rs4588 at VDBP locus; and rs11568820, rs4516035, rs2228570, rs1544410 and rs17878969 at VDR locus. Distribution of genotypes between patients grouped by outcome was compared by contingency table analysis. Marker-marker interaction was assessed by a MDR analysis. Assuming an additive model for VDR markers, a Kaplan-Meier survival analysis was employed to evaluate association with disease progression. Among vitamin-D pathway genes, VDR locus reveals specific 5'UTR and 3'UTR diplotype combinations associated with both, slower and faster progression to AIDS. Marker-marker interaction analysis indicates a strong interaction between VDR markers and a redundant effect for CYP27B1 markers. According to our results, VDR locus association follows an additive model in which increased genetic risk score for the VDR is directly correlated with AIDS progression rates. Our data supports a role of vitamin-D pathway gene variability on HIV-1 disease progression. PMID:24768180

  10. HIV-1 Protease with 20 Mutations Exhibits Extreme Resistance to Clinical Inhibitors through Coordinated Structural Rearrangements

    SciTech Connect

    Agniswamy, Johnson; Shen, Chen-Hsiang; Aniana, Annie; Sayer, Jane M.; Louis, John M.; Weber, Irene T.

    2012-06-28

    The escape mutant of HIV-1 protease (PR) containing 20 mutations (PR20) undergoes efficient polyprotein processing even in the presence of clinical protease inhibitors (PIs). PR20 shows >3 orders of magnitude decreased affinity for PIs darunavir (DRV) and saquinavir (SQV) relative to PR. Crystal structures of PR20 crystallized with yttrium, substrate analogue p2-NC, DRV, and SQV reveal three distinct conformations of the flexible flaps and diminished interactions with inhibitors through the combination of multiple mutations. PR20 with yttrium at the active site exhibits widely separated flaps lacking the usual intersubunit contacts seen in other inhibitor-free dimers. Mutations of residues 35-37 in the hinge loop eliminate interactions and perturb the flap conformation. Crystals of PR20/p2-NC contain one uninhibited dimer with one very open flap and one closed flap and a second inhibitor-bound dimer in the closed form showing six fewer hydrogen bonds with the substrate analogue relative to wild-type PR. PR20 complexes with PIs exhibit expanded S2/S2' pockets and fewer PI interactions arising from coordinated effects of mutations throughout the structure, in agreement with the strikingly reduced affinity. In particular, insertion of the large aromatic side chains of L10F and L33F alters intersubunit interactions and widens the PI binding site through a network of hydrophobic contacts. The two very open conformations of PR20 as well as the expanded binding site of the inhibitor-bound closed form suggest possible approaches for modifying inhibitors to target extreme drug-resistant HIV.

  11. The Effect of Clade-Specific Sequence Polymorphisms on HIV-1 Protease Activity and Inhibitor Resistance Pathways

    SciTech Connect

    Bandaranayake, Rajintha M.; Kolli, Madhavi; King, Nancy M.; Nalivaika, Ellen A.; Heroux, Annie; Kakizawa, Junko; Sugiura, Wataru; Schiffer, Celia A.

    2010-09-08

    The majority of HIV-1 infections around the world result from non-B clade HIV-1 strains. The CRF01{_}AE (AE) strain is seen principally in Southeast Asia. AE protease differs by {approx}10% in amino acid sequence from clade B protease and carries several naturally occurring polymorphisms that are associated with drug resistance in clade B. AE protease has been observed to develop resistance through a nonactive-site N88S mutation in response to nelfinavir (NFV) therapy, whereas clade B protease develops both the active-site mutation D30N and the nonactive-site mutation N88D. Structural and biochemical studies were carried out with wild-type and NFV-resistant clade B and AE protease variants. The relationship between clade-specific sequence variations and pathways to inhibitor resistance was also assessed. AE protease has a lower catalytic turnover rate than clade B protease, and it also has weaker affinity for both NFV and darunavir (DRV). This weaker affinity may lead to the nonactive-site N88S variant in AE, which exhibits significantly decreased affinity for both NFV and DRV. The D30N/N88D mutations in clade B resulted in a significant loss of affinity for NFV and, to a lesser extent, for DRV. A comparison of crystal structures of AE protease shows significant structural rearrangement in the flap hinge region compared with those of clade B protease and suggests insights into the alternative pathways to NFV resistance. In combination, our studies show that sequence polymorphisms within clades can alter protease activity and inhibitor binding and are capable of altering the pathway to inhibitor resistance.

  12. Antimicrobial (Drug) Resistance

    MedlinePlus

    ... Antimicrobial (Drug) Resistance Antibiotic-Resistant Mycobacterium tuberculosis (TB) Methicillin-Resistant Staphylococcus aureus (MRSA) Vancomycin-Resistant Enterococci (VRE) Multidrug-Resistant Neisseria ...

  13. Stage-dependent inhibition of HIV-1 replication by antiretroviral drugs in cell culture.

    PubMed

    Donahue, Daniel A; Sloan, Richard D; Kuhl, Björn D; Bar-Magen, Tamara; Schader, Susan M; Wainberg, Mark A

    2010-03-01

    Recent clinical trials have shown that the use of the HIV-1 integrase (IN) inhibitor raltegravir (RAL) results in drops in the viral load that are more rapid than those achieved by use of the reverse transcriptase (RT) inhibitor efavirenz. Previously, mathematical modeling of viral load decay that takes into account the stage of viral replication targeted by a drug has yielded data that closely approximate the clinical trial results. This model predicts greater inhibition of viral replication by drugs that act later in the viral replication cycle. In the present study, we have added drugs that target entry, reverse transcription, integration, or proteolytic processing to acutely infected cells and have shown modest viral inhibition by entry inhibitors, intermediate levels of inhibition by RT and IN inhibitors, and high levels of inhibition by protease inhibitors relative to the levels of growth for the no-drug controls. When dual or triple combinations of these drugs were added to acutely infected cells, we found that the levels of inhibition achieved by any given combination were comparable to those achieved by the latest-acting drug in the combination. In single-round infections in which the kinetics of reverse transcription and integration had been determined by quantitative PCR, addition of IN inhibitors at various times postinfection resulted in levels of inhibition equal to or greater than those achieved by addition of RT inhibitors. Collectively, our data provide in vitro evidence of the stage-dependent inhibition of HIV-1 by clinically relevant drugs. We discuss how stage-dependent inhibition helps to explain the unique viral load decay dynamics observed clinically with RAL. PMID:20038621

  14. RNAi-Mediated CCR5 Knockdown Provides HIV-1 Resistance to Memory T Cells in Humanized BLT Mice

    PubMed Central

    Shimizu, Saki; Ringpis, Gene-Errol; Marsden, Matthew D; Cortado, Ruth V; Wilhalme, Holly M; Elashoff, David; Zack, Jerome A; Chen, Irvin S Y; An, Dong Sung

    2015-01-01

    Transplantation of hematopoietic stem/progenitor cells (HSPC) modified with a lentiviral vector bearing a potent nontoxic short hairpin RNA (sh1005) directed to the HIV coreceptor CCR5 is capable of continuously producing CCR5 downregulated CD4+ T lymphocytes. Here, we characterized HIV-1 resistance of the sh1005-modified CD4+ T lymphocytes in vivo in humanized bone marrow/liver/thymus (hu BLT) mice. The sh1005-modified CD4+ T lymphocytes were positively selected in CCR5-tropic HIV-1–challenged mice. The sh1005-modified memory CD4+ T lymphocytes (the primary target of CCR5-tropic HIV-1) expressing sh1005 were maintained in lymphoid tissues in CCR5-tropic HIV-1–challenged mice. Frequencies of HIV-1 p24 expressing cells were significantly reduced in the sh1005-modified splenocytes by ex vivo cell stimulation confirming that CCR5 downregulated sh1005 modified cells are protected from viral infection. These results demonstrate that stable CCR5 downregulation through genetic modification of human HSPC by lentivirally delivered sh1005 is highly effective in providing HIV-1 resistance. Our results provide in vivo evidence in a relevant small animal model that sh1005 is a potent early-step anti-HIV reagent that has potential as a novel anti-HIV-1 HSPC gene therapeutic reagent for human applications. PMID:25689223

  15. Enabled clinical use of an HIV-1 attachment inhibitor through drug delivery.

    PubMed

    Timmins, Peter; Brown, Jonathan; Meanwell, Nicholas A; Hanna, George J; Zhu, Li; Kadow, John F

    2014-09-01

    The clinical advancement of HIV-1 attachment inhibitors was hindered initially by poor bioavailability. Attempts to identify improved candidates revealed that solubility and dissolution-rate-limited absorption are barriers to achieving adequate antiviral plasma levels. This was mitigated by forming nanosized drugs or by creating stabilised amorphous drug-polymer composites. In further improving drug potency and mitigating solubility-limited bioavailability, a candidate based on a phosphate ester prodrug was identified that, although having excellent bioavailability, exhibited unacceptable pharmacokinetics. Based on in silico modelling and a site of absorption study it was confirmed that creating an extended release formulation could provide the desired pharmacokinetic profile. The optimised formulation showed good antiviral activity when dosed employing a once or twice a day regimen. PMID:24727410

  16. Antiviral molecules correlate with vitamin D pathway genes and are associated with natural resistance to HIV-1 infection.

    PubMed

    Aguilar-Jimenez, Wbeimar; Zapata, Wildeman; Rugeles, María T

    2016-01-01

    The relationship between the immunomodulatory effects of Vitamin D (VitD) and the expression of anti-HIV-1 molecules has not been explored in HIV-1-exposed seronegative individuals (HESNs). Higher mRNA levels of cathelicidin and HAD-4 in oral-mucosa and peripheral-blood, along with higher CYP24A1 mRNA in vaginal-mucosa and lower TLR2 mRNA in endocervical-mucosa were found in HESNs compared to non-exposed controls. Furthermore, the mRNA of anti-HIV molecules Elafin, TRIM5, Cathelicidin, HAD-4 and RNase7, previously associated with natural resistance to HIV-1 infection, positively correlated with the mRNA expression of VDR in HESNs, suggesting the potential participation of VitD in natural resistance to HIV-1. PMID:27083474

  17. Natural polymorphism S119R of HIV-1 integrase enhances primary INSTI resistance.

    PubMed

    Hachiya, Atsuko; Ode, Hirotaka; Matsuda, Masakazu; Kito, Yumiko; Shigemi, Urara; Matsuoka, Kazuhiro; Imamura, Junji; Yokomaku, Yoshiyuki; Iwatani, Yasumasa; Sugiura, Wataru

    2015-07-01

    Integrase strand transfer inhibitors (INSTIs), which block proviral DNA integration into the host chromosome, are clinically effective against HIV-1 isolates exhibiting resistance to other classes of antiretroviral agents. Although naturally occurring amino acid variation has been less frequently observed in the integrase region, the functional constraints of this variation on primary INSTI resistance-associated mutations are not fully understood. In the present study, we focused on the S119G/R/P/T (S119X) polymorphisms, which are frequently observed in HIV-1 sequences derived from clinical specimens (naïve, n=458, 26%). The frequency of the S119X polymorphism together with Q148H/R (n=8, 63%) or N155H (n=12, 83%) was relatively high compared with that of naïve group. Our in vitro assays revealed that S119G/P/T alone exerted no effect on the susceptibility to INSTIs, whereas S119R enhanced the level of INSTI resistance induced by well-known INSTI resistance-associated mutations (Y143C, Q148H or N155H). Notably, the S119R polymorphism contributed to a significant (5.9-fold) increase in dolutegravir resistance caused by G140S/Q148H. Analysis of two cases of virological failure during raltegravir-based therapy showed that the accumulation and the rapid evolution of primary INSTI resistance-associated mutations coincided with the S119R mutation. These data highlight the role of the S119X polymorphism in INSTI resistance, and this polymorphism might be linked to the potential treatment outcome with INSTI-based therapy. PMID:25956162

  18. Relative Resistance of HLA-B to Downregulation by Naturally Occurring HIV-1 Nef Sequences

    PubMed Central

    Mahiti, Macdonald; Toyoda, Mako; Jia, Xiaofei; Kuang, Xiaomei T.; Mwimanzi, Francis; Mwimanzi, Philip; Walker, Bruce D.; Xiong, Yong; Brumme, Zabrina L.; Brockman, Mark A.

    2016-01-01

    ABSTRACT HIV-1 Nef binds to the cytoplasmic region of HLA-A and HLA-B and downregulates these molecules from the surface of virus-infected cells, thus evading immune detection by CD8+ T cells. Polymorphic residues within the HLA cytoplasmic region may affect Nef’s downregulation activity. However, the effects of HLA polymorphisms on recognition by primary Nef isolates remain elusive, as do the specific Nef regions responsible for downregulation of HLA-A versus HLA-B. Here, we examined 46 Nef clones isolated from chronically HIV-1 subtype B-infected subjects for their ability to downregulate various HLA-A, HLA-B, and HLA-C molecules on the surface of virus-infected cells. Overall, HLA-B exhibited greater resistance to Nef-mediated downregulation than HLA-A, regardless of the cell type examined. As expected, no Nef clone downregulated HLA-C. Importantly, the differential abilities of patient-derived Nef clones to downregulate HLA-A and HLA-B correlated inversely with the sensitivities of HIV-infected target cells to recognition by effector cells expressing an HIV-1 Gag-specific T cell receptor. Nef codon function analysis implicated amino acid variation at position 202 (Nef-202) in differentially affecting the ability to downregulate HLA-A and HLA-B, an observation that was subsequently confirmed by experiments using Nef mutants constructed by site-directed mutagenesis. The in silico and mutagenesis analyses further suggested that Nef-202 may interact with the C-terminal Cys-Lys-Val residues of HLA-A, which are absent in HLA-B. Taken together, the results show that natural polymorphisms within Nef modulate its interaction with natural polymorphisms in the HLA cytoplasmic tails, thereby affecting the efficiency of HLA downregulation and consequent recognition by HIV-specific T cells. These results thus extend our understanding of this complex pathway of retroviral immune evasion. PMID:26787826

  19. High Prevalence of HIV-1 Intersubtype B′/C Recombinants among Injecting Drug Users in Dehong, China

    PubMed Central

    Han, Xiaoxu; An, Minghui; Zhao, Bin; Duan, Song; Yang, Shaomin; Xu, Junjie; Zhang, Min; McGoogan, Jennifer M.; Takebe, Yutaka; Shang, Hong

    2013-01-01

    Objective To examine the distribution of HIV-1 genotypes among injecting drug users (IDUs) from Dehong, Yunnan province. Materials and Methods Blood samples from a total of 95 HIV-positive IDUs were retrospectively analyzed. Samples were collected between 2005 and 2009 from four cities in Dehong prefecture, western Yunnan province, the geographical origin of the HIV epidemic in China. HIV-1 gag, partial pol, vpr-env fragment, half-genome, or near-full-length sequences were analyzed to determine the HIV-1 genotypes of each subject. Results were compared with findings from past studies of IDUs in Dehong and in neighboring Myanmar. Results We observed a high prevalence of B′/C recombinants (82.4%) among IDUs in Dehong, the structural profiles of which do not match those previously reported in Dehong or in Myanmar. Furthermore, statistically significant differences in geographical and temporal distributions of HIV-1 genotypes were characterized by a predominance of HIV-1 B′/C recombinant forms among older subjects(p = 0.034), subjects from Longchuan district (p = 0.022), and subjects diagnosed between 2000 and 2004 (p = 0.004). Conclusions The increasing prevalence of multiple, new B′/C recombinant forms suggest that HIV-1 intersubtype recombination is substantial and ongoing in western Yunnan. This reflects the high-risk behavior of IDUs in this region and argues the need for stronger monitoring and prevention measures in Dehong and other high-prevalence areas around China. PMID:23741489

  20. Co-infections and transmission networks of HCV, HIV-1 and HPgV among people who inject drugs

    PubMed Central

    Tien Ng, Kim; Takebe, Yutaka; Bee Chook, Jack; Zhen Chow, Wei; Gan Chan, Kok; Abed Al-Darraji, Haider Abdulrazzaq; Kamarulzaman, Adeeba; Keng Tee, Kok

    2015-01-01

    Co-infections with human immunodeficiency virus type 1 (HIV-1) and human pegivirus (HPgV) are common in hepatitis C virus (HCV)-infected individuals. However, analysis on the evolutionary dynamics and transmission network profiles of these viruses among individuals with multiple infections remains limited. A total of 228 injecting drug users (IDUs), either HCV- and/or HIV-1-infected, were recruited in Kuala Lumpur, Malaysia. HCV, HIV-1 and HPgV genes were sequenced, with epidemic growth rates assessed by the Bayesian coalescent method. Based on the sequence data, mono-, dual- and triple-infection were detected in 38.8%, 40.6% and 20.6% of the subjects, respectively. Fifteen transmission networks involving HCV (subtype 1a, 1b, 3a and 3b), HIV-1 (CRF33_01B) and HPgV (genotype 2) were identified and characterized. Genealogical estimates indicated that the predominant HCV, HIV-1 and HPgV genotypes were introduced into the IDUs population through multiple sub-epidemics that emerged as early as 1950s (HCV), 1980s (HIV-1) and 1990s (HPgV). By determining the difference in divergence times between viral lineages (ΔtMRCA), we also showed that the frequency of viral co-transmission is low among these IDUs. Despite increased access to therapy and other harm reduction interventions, the continuous emergence and coexistence of new transmission networks suggest persistent multiple viral transmissions among IDUs. PMID:26459957

  1. Emerging pathogens: Dynamics, mutation and drug resistance

    SciTech Connect

    Perelson, A.S.; Goldstein, B.; Korber, B.T.

    1997-10-01

    This is the final report of a one-year, Laboratory Directed Research and Development (LDRD) project at Los Alamos National Laboratory (LANL). The objectives of this project were to develop models of the spread of pathogens, such as HIV-1 and influenza, in humans, and then to use the models to address the possibility of designing appropriate drug therapies that may limit the ability of the pathogen to escape treatment by mutating into a drug resistant form. We have developed a model of drug-resistance to amantidine and rimantadine, the two major antiviral drugs used to treat influenza, and have used the model to suggest treatment strategies during an epidemic.

  2. [Research development of HIV drug resistance].

    PubMed

    Zou, Wen; Liu, Ying; Wang, Jian; Gao, Guo-Jian; Dong, Ji-Peng; Xian, Qing-Fei

    2013-08-01

    Highly active antiretroviral combination therapy significantly reduced the mortality, but in the high-speed copying, high genetic variation and drug selection pressure under the effect of the increasingly serious problem of drug resistance greatly weakened the role of HAART inhibit viral replication and reduce antiviral treatment. This paper reports the latest trends in HIV drug-resistance in order to develop anti-HIV drugs in clinical programs, research and development of new guidance anti-HIV-1 strategy to bring guidance. PMID:24228557

  3. Sphingopeptides: dihydrosphingosine-based fusion inhibitors against wild-type and enfuvirtide-resistant HIV-1.

    PubMed

    Ashkenazi, Avraham; Viard, Mathias; Unger, Linor; Blumenthal, Robert; Shai, Yechiel

    2012-11-01

    Understanding the structural organization of lipids in the cell and viral membranes is essential for elucidating mechanisms of viral fusion that lead to entry of enveloped viruses into their host cells. The HIV lipidome shows a remarkable enrichment in dihydrosphingomyelin, an unusual sphingolipid formed by a dihydrosphingosine backbone. Here we investigated the ability of dihydrosphingosine to incorporate into the site of membrane fusion mediated by the HIV envelope (Env) protein. Dihydrosphingosine as well as cholesterol, fatty acid, and tocopherol was conjugated to highly conserved, short HIV-1 Env-derived peptides with no antiviral activity otherwise. We showed that dihydrosphingosine exclusively endowed nanomolar antiviral activity to the peptides (IC(50) as low as 120 nM) in HIV-1 infection on TZM-bl cells and on Jurkat T cells, as well as in the cell-cell fusion assay. These sphingopeptides were active against enfuvirtide-resistant and wild-type CXCR4 and CCR5 tropic HIV strains. The anti-HIV activity was determined by both the peptides and their dihydrosphingosine conjugate. Moreover, their mode of action involved accumulation in the cells and viruses and binding to membranes enriched in sphingomyelin and cholesterol. The data suggest that sphingopeptides are recruited to the HIV membrane fusion site and provide a general concept in developing inhibitors of sphingolipid-mediated biological systems. PMID:22872679

  4. A Randomized Trial of Therapeutic Drug Monitoring of Protease Inhibitors in Antiretroviral-Experienced, HIV-1-Infected Patients

    PubMed Central

    Demeter, Lisa M.; Jiang, Hongyu; Mukherjee, A. Lisa; Morse, Gene D.; DiFrancesco, Robin; Dykes, Carrie; Sista, Prakash; Bacheler, Lee; Klingman, Karin; Rinehart, Alex; Albrecht, Mary

    2009-01-01

    Objective Whether therapeutic drug monitoring of protease inhibitors (PIs) improves outcomes in HIV-infected patients is controversial. We evaluated this strategy in a randomized, open-label clinical trial, using a normalized inhibitory quotient (NIQ), which incorporates drug exposure and viral drug resistance. NIQs≤1 may predict poor outcome and identify patients who could benefit from dose escalation. Design/Methods Eligible patients had a viral load ≥1,000 copies/mL on a failing regimen, and began a new PI-containing regimen at entry. All FDA-approved PIs available during study recruitment (June 2002-May 2006) were allowed. One-hundred-eighty-three participants with NIQ≤1, based on their week 2 PI trough concentration and pre-entry drug resistance test, were randomized at week 4 to standard of care (SOC) or PI dose escalation (TDM). The primary endpoint was change in log10 plasma HIV-1 RNA concentration from randomization to 20 weeks later. Results Ninety-one subjects were randomized to SOC, 92 to TDM. NIQs increased more in the TDM arm compared to SOC (+69% versus +25%, p=0.01). Despite this, TDM and SOC arms showed no difference in outcome (+0.09 versus +0.02 log10, p=0.17). In retrospective subgroup analyses, patients with less HIV resistance to their PIs benefited from TDM (p=0.002), as did black and Hispanic patients (p=0.035 and 0.05, respectively). Differences between black and white patients persisted when accounting for PI susceptibility. Conclusions There was no overall benefit of TDM. In post-hoc, subgroup analyses, TDM appeared beneficial in black and Hispanic patients, and in patients whose virus retained some susceptibility to the PIs in their regimen. PMID:19114860

  5. Human X-DING-CD4 mediates resistance to HIV-1 infection through novel paracrine-like signaling

    PubMed Central

    Sachdeva, Rakhee; Li, Yuchang; Shilpi, Rasheda Y.; Simm, Malgorzata

    2015-01-01

    X-DING-CD4 is a novel phosphatase mediating antiviral responses to HIV-1 infection. This protein is constitutively expressed and secreted by HIV-1 resistant CD4+ T cells and its mRNA transcription is up-regulated in PBMCs from HIV-1 elite controllers. The secreted/soluble X-DING-CD4 protein form is of particular importance because it blocks virus transcription when added to HIV-1 susceptible cells. Our research aimed to determine the contribution of this factor to induction of antiviral response in target cells. We found that soluble X-DING-CD4 enters cells by endocytosis and influx of this protein induced transcription of IFN-α and endogenous X-DING-CD4 mRNA in transformed CD4+ T cells and primary macrophages. Treatment of HIV-1 susceptible cells with exogenous X-DING-CD4 caused depletion of phosphorylated p50 and p65 NF-κB subunits and a significant reduction in p50/p65 NF-κB binding to the HIV-1 LTR. Together, these findings indicate a novel antiviral mechanism mediated by the influx of soluble X-DING-CD4, its signaling to promote self-amplification and functional duality as an endogenous innate immunity effector and exogenous factor regulating gene expression in bystander cells. PMID:25581464

  6. Drug-Based Lead Discovery: The Novel Ablative Antiretroviral Profile of Deferiprone in HIV-1-Infected Cells and in HIV-Infected Treatment-Naive Subjects of a Double-Blind, Placebo-Controlled, Randomized Exploratory Trial

    PubMed Central

    Saxena, Deepti; Spino, Michael; Tricta, Fernando; Connelly, John; Cracchiolo, Bernadette M.; Hanauske, Axel-Rainer; D’Alliessi Gandolfi, Darlene; Mathews, Michael B.; Karn, Jonathan; Holland, Bart; Park, Myung Hee; Pe’ery, Tsafi; Palumbo, Paul E.; Hanauske-Abel, Hartmut M.

    2016-01-01

    Antiretrovirals suppress HIV-1 production yet spare the sites of HIV-1 production, the HIV-1 DNA-harboring cells that evade immune detection and enable viral resistance on-drug and viral rebound off-drug. Therapeutic ablation of pathogenic cells markedly improves the outcome of many diseases. We extend this strategy to HIV-1 infection. Using drug-based lead discovery, we report the concentration threshold-dependent antiretroviral action of the medicinal chelator deferiprone and validate preclinical findings by a proof-of-concept double-blind trial. In isolate-infected primary cultures, supra-threshold concentrations during deferiprone monotherapy caused decline of HIV-1 RNA and HIV-1 DNA; did not allow viral breakthrough for up to 35 days on-drug, indicating resiliency against viral resistance; and prevented, for at least 87 days off-drug, viral rebound. Displaying a steep dose-effect curve, deferiprone produced infection-independent deficiency of hydroxylated hypusyl-eIF5A. However, unhydroxylated deoxyhypusyl-eIF5A accumulated particularly in HIV-infected cells; they preferentially underwent apoptotic DNA fragmentation. Since the threshold, ascertained at about 150 μM, is achievable in deferiprone-treated patients, we proceeded from cell culture directly to an exploratory trial. HIV-1 RNA was measured after 7 days on-drug and after 28 and 56 days off-drug. Subjects who attained supra-threshold concentrations in serum and completed the protocol of 17 oral doses, experienced a zidovudine-like decline of HIV-1 RNA on-drug that was maintained off-drug without statistically significant rebound for 8 weeks, over 670 times the drug’s half-life and thus clearance from circulation. The uniform deferiprone threshold is in agreement with mapping of, and crystallographic 3D-data on, the active site of deoxyhypusyl hydroxylase (DOHH), the eIF5A-hydroxylating enzyme. We propose that deficiency of hypusine-containing eIF5A impedes the translation of mRNAs encoding proline

  7. HIV-1 resistance to neutralizing antibodies: Determination of antibody concentrations leading to escape mutant evolution.

    PubMed

    Magnus, Carsten; Reh, Lucia; Trkola, Alexandra

    2016-06-15

    Broadly neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) are considered vital components of novel therapeutics and blueprints for vaccine research. Yet escape to even the most potent of these antibodies is imminent in natural infection. Measures to define antibody efficacy and prevent mutant selection are thus urgently needed. Here, we derive a mathematical framework to predict the concentration ranges for which antibody escape variants can outcompete their viral ancestors, referred to as mutant selection window (MSW). When determining the MSW, we focus on the differential efficacy of neutralizing antibodies against HIV-1 in two canonical infection routes, free-virus infection and cell-cell transmission. The latter has proven highly effective in vitro suggesting its importance for both in vivo spread as well as for escaping targeted intervention strategies. We observed a range of MSW patterns that highlight the potential of mutants to arise in both transmission pathways and over wide concentration ranges. Most importantly, we found that only when the arising mutant has both, residual sensitivity to the neutralizing antibody and reduced infectivity compared to the parental virus, antibody dosing outside of the MSW to restrict mutant selection is possible. Emergence of mutants that provide complete escape and have no considerable fitness loss cannot be prevented by adjusting antibody doses. The latter may in part explain the ubiquitous resistance to neutralizing antibodies observed in natural infection and antibody treatment. Based on our findings, combinations of antibodies targeting different epitopes should be favored for antibody-based interventions as this may render complete resistance less likely to occur and also increase chances that multiple escapes result in severe fitness loss of the virus making longer-term antibody treatment more feasible. PMID:26494166

  8. Action of anti-HIV drugs and resistance: reverse transcriptase inhibitors and protease inhibitors.

    PubMed

    Imamichi, Tomozumi

    2004-01-01

    Currently, 20 drugs have been approved for Human Immunodeficiency Virus type-1 (HIV-1) clinical therapy. These drugs inhibit HIV-1 reverse transcriptase, protease, or virus entry. Introduction of a combination therapy with reverse transcriptase inhibitors and protease inhibitors has resulted in a drastic decrease in HIV-1 related mortality. Although the combination therapy can suppress viral replication below detection levels in current available assays, low levels of on-going viral replication still persist in some patients. Long-term administration of the combination therapy may increase selective pressure against viruses, and subsequently induce emergence of multiple drug-resistant HIV-1 variants. Attempts have been made to design novel antiretroviral drugs that would be able to suppress replication of the resistant variants. At present, several investigational drugs are being tested in clinical trials. These drugs target not only the resistant variants, but also improvement in oral bioavilability or other viral proteins such as HIV-1 integrase, ribonuclease H, and HIV-1 entry (CD4 attachment inhibitors, chemokine receptors antagonists, and fusion inhibitors). Understanding mechanism(s) of action of the drugs and mechanisms of drug resistance is necessary for successful designs in the next generation of anti-HIV-1 drugs. In this review, the mechanisms of action of reverse transcriptase- and protease-inhibitors, and the mechanism of resistance to these inhibitors, are described. PMID:15579086

  9. Simian-tropic HIV as a model to study drug resistance against integrase inhibitors.

    PubMed

    Wares, Melissa; Hassounah, Said; Mesplède, Thibault; Sandstrom, Paul A; Wainberg, Mark A

    2015-04-01

    Drug resistance represents a key aspect of human immunodeficiency virus (HIV) treatment failure. It is important to develop nonhuman primate models for studying issues of drug resistance and the persistence and transmission of drug-resistant viruses. However, relatively little work has been conducted using either simian immunodeficiency virus (SIV) or SIV/HIV recombinant viruses for studying resistance against integrase strand transfer inhibitors (INSTIs). Here, we used a T-cell-tropic SIV/HIV recombinant virus in which the capsid and vif regions of HIV-1 were replaced with their SIV counterparts (simian-tropic HIV-1 [stHIV-1](SCA,SVIF)) to study the impact of a number of drug resistance substitutions in the integrase coding region at positions E92Q, G118R, E138K, Y143R, S153Y, N155H, and R263K on drug resistance, viral infectivity, and viral replication capacity. Our results show that each of these substitutions exerted effects that were similar to their effects in HIV-1. Substitutions associated with primary resistance against dolutegravir were more detrimental to stHIV-1(SCA,SVIF) infectiousness than were resistance substitutions associated with raltegravir and elvitegravir, consistent with data that have been reported for HIV-1. These findings support the role of stHIV-1(SCA,SVIF) as a useful model with which to evaluate the role of INSTI resistance substitutions on viral persistence, transmissibility, and pathogenesis in a nonhuman primate model. PMID:25583721

  10. HIV-1 Envelope Resistance to Proteasomal Cleavage: Implications for Vaccine Induced Immune Responses

    PubMed Central

    Steers, Nicholas J.; Ratto-Kim, Silvia; de Souza, Mark S.; Currier, Jeffrey R.; Kim, Jerome H.; Michael, Nelson L.; Alving, Carl R.; Rao, Mangala

    2012-01-01

    Background Antigen processing involves many proteolytic enzymes such as proteasomes and cathepsins. The processed antigen is then presented on the cell surface bound to either MHC class I or class II molecules and induces/interacts with antigen-specific CD8+ and CD4+ T-cells, respectively. Preliminary immunological data from the RV144 phase III trial indicated that the immune responses were biased towards the Env antigen with a dominant CD4+ T-cell response. Methods In this study, we examined the susceptibility of HIV-1 Env-A244 gp120 protein, one of the protein boost subunits of the RV144 Phase III vaccine trial, to proteasomes and cathepsins and identified the generated peptide epitope repertoire by mass spectrometry. The peptide fragments were tested for cytokine production in CD4+ T-cell lines derived from RV144 volunteers. Results Env-A244 was resistant to proteasomes, thus diminishing the possibility of the generation of class I epitopes by the classical MHC class I pathway. However, Env-A244 was efficiently cleaved by cathepsins generating peptide arrays identified by mass spectrometry that contained both MHC class I and class II epitopes as reported in the Los Alamos database. Each of the cathepsins generated distinct degradation patterns containing regions of light and dense epitope clusters. The sequence DKKQKVHALF that is part of the V2 loop of gp120 produced by cathepsins induced a polyfunctional cytokine response including the generation of IFN-γ from CD4+ T-cell lines-derived from RV144 vaccinees. This sequence is significant since antibodies to the V1/V2-loop region correlated inversely with HIV-1 infection in the RV144 trial. Conclusions Based on our results, the susceptibility of Env-A244 to cathepsins and not to proteasomes suggests a possible mechanism for the generation of Env-specific CD4+T cell and antibody responses in the RV144 vaccinees. PMID:22880042

  11. Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure.

    PubMed

    Kyeyune, Fred; Gibson, Richard M; Nankya, Immaculate; Venner, Colin; Metha, Samar; Akao, Juliet; Ndashimye, Emmanuel; Kityo, Cissy M; Salata, Robert A; Mugyenyi, Peter; Arts, Eric J; Quiñones-Mateu, Miguel E

    2016-06-01

    Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more

  12. Genome editing of CXCR4 by CRISPR/cas9 confers cells resistant to HIV-1 infection.

    PubMed

    Hou, Panpan; Chen, Shuliang; Wang, Shilei; Yu, Xiao; Chen, Yu; Jiang, Meng; Zhuang, Ke; Ho, Wenzhe; Hou, Wei; Huang, Jian; Guo, Deyin

    2015-01-01

    Genome editing via CRISPR/Cas9 has become an efficient and reliable way to make precise, targeted changes to the genome of living cells. CXCR4 is a co-receptor for the human immunodeficiency virus type 1 (HIV-1) infection and has been considered as an important therapeutic target for AIDS. CXCR4 mediates viral entry into human CD4(+) cells by binding to envelope protein, gp120. Here, we show that human CXCR4 gene is efficiently disrupted by CRISPR/Cas9-mediated genome editing, leading to HIV-1 resistance of human primary CD4(+) T cells. We also show that the Cas9-mediated ablation of CXCR4 demonstrated high specificity and negligible off-target effects without affecting cell division and propagation. The precise and efficient genome editing of CXCR4 will provide a new strategy for therapeutic application against HIV-1 infection. PMID:26481100

  13. Endotoxin-Mediated Downregulation of Hepatic Drug Transporters in HIV-1 Transgenic Rats.

    PubMed

    Ghoneim, Ragia H; Piquette-Miller, Micheline

    2016-05-01

    Altered expression of drug transporters and metabolic enzymes is known to occur in infection-induced inflammation. We hypothesize that in human immunodeficiency virus (HIV)-infected individuals, further alteration could occur as a result of augmented inflammation. The HIV-1 transgenic (Tg) rat is used to simulate HIV pathologies associated with the presence of HIV viral proteins. Therefore, the objective of this study was to examine the effect of endotoxin administration on the gene expression of drug transporters in the liver of HIV-Tg rats. Male and female HIV-Tg and wild-type (WT) littermates were injected with 5 mg/kg endotoxin or saline (n= 7-9/group). Eighteen hours later, rats were euthanized and tissues were collected. Quantitative real-time polymerase chain reaction and Western blot analysis were used to measure hepatic gene and protein expression, respectively, and enzyme-linked immunosorbent assay was used to measure serum cytokine levels. Although an augmented inflammatory response was seen in HIV-Tg rats, similar endotoxin- mediated downregulation of Abcb1a, Abcc2, Abcg2, Abcb11, Slco1a1, Slco1a2, Slco1b2, Slc10a1, Slc22a1, Cyp3a2, and Cyp3a9 gene expression was seen in the HIV-Tg and WT groups. A significantly greater endotoxin- mediated downregulation of Ent1/Slc29a1 was seen in female HIV-Tg rats. Basal expression of inflammatory mediators was not altered in the HIV-Tg rat; likewise, the basal expression of most transporters was not significantly different between HIV-Tg and WT rats. Our findings suggest that hepatobiliary clearances of endogenous and exogenous substrates are altered in the HIV-Tg rat after endotoxin exposure. This is of particular importance because HIV-infected individuals frequently present with bacterial or viral infections, which are a potential source for drug-disease interactions. PMID:26977098

  14. Targeting TNF-Alpha in HIV-1 Infection.

    PubMed

    Kumar, Amit; Coquard, Laurie; Herbein, Georges

    2016-01-01

    Highly active antiretroviral therapy (HAART) has dramatically extended the lifespan and quality of life of individuals infected with human immunodeficiency virus type 1 (HIV-1). HAART comprises of a cocktail of various pharmacological inhibitors which interfere with almost every stages of HIV-1 life cycle. However, constant application of drugs often results in the evolution of hostpathogen relationship resulting in the emergence of drug resistant viral strains. Drug resistant HIV-1 is a potent threat for the humankind. Therefore, there is a constant need to search for novel therapeutic molecules. HIV-1 infection results in the depletion of CD4+/CD8+T cells and alters the cytokine network in the infected individuals. Tumor necrosis factor alpha (TNF-alpha), a proinflammatory cytokine, plays a critical role in HIV-1 pathogenesis. HIV-1 utilizes the TNF-alpha signaling pathway for expanding its reservoir. Several HIV-1 proteins mimic and regulate the TNF-alpha signaling pathway. TNF-alpha inhibitors have been used in several inflammatory pathologies with success to some extent. In the present mini review we will discuss the role of TNF-alpha in HIV-1 pathogenesis. Furthermore we will evaluate the TNF-alpha inhibitors as an additional therapeutic option for HIV-1 infection. PMID:26073859

  15. Human immunodeficiency virus type 1 (HIV-1) strains selected for resistance against the HIV-1-specific [2',5'-bis-O-(tert-butyldimethylsilyl)-3'-spiro- 5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D-pentofurano syl (TSAO) nucleoside analogues retain sensitivity to HIV-1-specific nonnucleoside inhibitors.

    PubMed Central

    Balzarini, J; Karlsson, A; Vandamme, A M; Pérez-Pérez, M J; Zhang, H; Vrang, L; Oberg, B; Bäckbro, K; Unge, T; San-Félix, A

    1993-01-01

    We recently reported that a newly discovered class of nucleoside analogues--[2',5'-bis-O-(tert-butyldimethylsilyl)- 3'-spiro-5''-(4''-amino-1'',2''-oxathiole-2'',2''-dioxide)]-beta-D - pentofuranosyl derivatives of pyrimidines and purines (designated TSAO)--are highly specific inhibitors of human immunodeficiency virus type 1 (HIV-1) and targeted at the nonsubstrate binding site of HIV-1 reverse transcriptase (RT). We now find that HIV-1 strains selected for resistance against three different TSAO nucleoside derivatives retain sensitivity to the other HIV-1-specific nonnucleoside derivatives (tetrahydroimidazo[4,5,1-jk][1,4]benzodiazepin-2(1H)-one and -thione (TIBO), 1-[(2-hydroxyethoxy)methyl]-6-phenylthiothymine, nevirapine, and pyridinone L697,661, as well as to the nucleoside analogues 3'-azido-3'-deoxythymidine, ddI, ddC, and 9-(2-phosphonylmethoxyethyl)adenine. Pol gene nucleotide sequence analysis of the TSAO-resistant and -sensitive HIV-1 strains revealed a single amino acid substitution at position 138 (Glu-->Lys) in the RT of all TSAO-resistant HIV-1 strains. HIV-1 RT in which the Glu-138-->Lys substitution was introduced by site-directed mutagenesis and expressed in Escherichia coli could not be purified because of rapid degradation. However, HIV-1 RT containing the Glu-138-->Arg substitution was stable. It lost its sensitivity to the TSAO nucleosides but not to the other HIV-1-specific RT inhibitors (i.e., TIBO and pyridinone). Our findings point to a specific interaction of the 4''-amino group on the 3'-spiro-substituted ribose moiety of the TSAO nucleosides with the carboxylic acid group of glutamic acid at position 138 of HIV-1 RT. PMID:7688467

  16. Drug 9AA reactivates p21/Waf1 and Inhibits HIV-1 progeny formation

    PubMed Central

    Wu, Weilin; Kehn-Hall, Kylene; Pedati, Caitlin; Zweier, Lynnsey; Castro, Iris; Klase, Zachary; Dowd, Cynthia S; Dubrovsky, Larisa; Bukrinsky, Michael; Kashanchi, Fatah

    2008-01-01

    It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated. PMID:18348731

  17. Complexity and Dynamics of HIV-1 Chemokine Receptor Usage in a Multidrug-Resistant Adolescent

    PubMed Central

    Mainetti, Lara; Pignataro, Angela Rosa; Bigoloni, Alba; Tolazzi, Monica; Galli, Andrea; Nozza, Silvia; Castagna, Antonella; Sampaolo, Michela; Boeri, Enzo; Scarlatti, Gabriella

    2014-01-01

    Abstract Maraviroc (MVC) is licensed in clinical practice for patients with R5 virus and virological failure; however, in anecdotal reports, dual/mixed viruses were also inhibited. We retrospectively evaluated the evolution of HIV-1 coreceptor tropism in plasma and peripheral blood mononuclear cells (PBMCs) of an infected adolescent with a CCR5/CXCR4 Trofile profile who experienced an important but temporary immunological and virological response during a 16-month period of MVC-based therapy. Coreceptor usage of biological viral clones isolated from PBMCs was investigated in U87.CD4 cells expressing wild-type or chimeric CCR5 and CXCR4. Plasma and PBMC-derived viral clones were sequenced to predict coreceptor tropism using the geno2pheno algorithm from the V3 envelope sequence and pol gene-resistant mutations. From start to 8.5 months of MVC treatment only R5X4 viral clones were observed, whereas at 16 months the phenotype enlarged to also include R5 and X4 clones. Chimeric receptor usage suggested the preferential usage of the CXCR4 coreceptor by the R5X4 biological clones. According to phenotypic data, R5 viruses were susceptible, whereas R5X4 and X4 viruses were resistant to RANTES and MVC in vitro. Clones at 16 months, but not at baseline, showed an amino acidic resistance pattern in protease and reverse transcription genes, which, however, did not drive their tropisms. The geno2pheno algorithm predicted at baseline R5 viruses in plasma, and from 5.5 months throughout follow-up only CXCR4-using viruses. An extended methodological approach is needed to unravel the complexity of the phenotype and variation of viruses resident in the different compartments of an infected individual. The accurate evaluation of the proportion of residual R5 viruses may guide therapeutic intervention in highly experienced patients with limited therapeutic options. PMID:25275490

  18. HIV-1 suppression and durable control by combining single broadly neutralizing antibodies and antiretroviral drugs in humanized mice

    PubMed Central

    Horwitz, Joshua A.; Halper-Stromberg, Ariel; Mouquet, Hugo; Gitlin, Alexander D.; Tretiakova, Anna; Eisenreich, Thomas R.; Malbec, Marine; Gravemann, Sophia; Billerbeck, Eva; Dorner, Marcus; Büning, Hildegard; Schwartz, Olivier; Knops, Elena; Kaiser, Rolf; Seaman, Michael S.; Wilson, James M.; Rice, Charles M.; Ploss, Alexander; Bjorkman, Pamela J.; Klein, Florian; Nussenzweig, Michel C.

    2013-01-01

    Effective control of HIV-1 infection in humans is achieved using combinations of antiretroviral therapy (ART) drugs. In humanized mice (hu-mice), control of viremia can be achieved using either ART or by immunotherapy using combinations of broadly neutralizing antibodies (bNAbs). Here we show that treatment of HIV-1–infected hu-mice with a combination of three highly potent bNAbs not only resulted in complete viremic control but also led to a reduction in cell-associated HIV-1 DNA. Moreover, lowering the initial viral load by coadministration of ART and immunotherapy enabled prolonged viremic control by a single bNAb after ART was withdrawn. Similarly, a single injection of adeno-associated virus directing expression of one bNAb produced durable viremic control after ART was terminated. We conclude that immunotherapy reduces plasma viral load and cell-associated HIV-1 DNA and that decreasing the initial viral load enables single bNAbs to control viremia in hu-mice. PMID:24043801

  19. A novel small-molecule inhibitor of HIV-1 entry

    PubMed Central

    Heredia, Alonso; Latinovic, Olga S; Barbault, Florent; de Leeuw, Erik PH

    2015-01-01

    Background Antiretroviral therapy has transformed HIV-1 infection into a managed condition with near-normal life expectancy. However, a significant number of patients remain with limited therapeutic options due to HIV-1 resistance, side effects, or drug costs. Further, it is likely that current drugs will not retain efficacy, due to risks of side effects and transmitted resistance. Results We describe compound 5660386 (3-ethyl-2-[3-(1,3,3-trimethyl-1,3-dihydro-2H-indol-2-ylidene)-1-propen-1-yl]-1,3-benzothiazol-3-ium) as a novel inhibitor of HIV-1 entry. Compound 5660386 inhibits HIV-1 entry in cell lines and primary cells, binds to HIV-1 envelope protein, and inhibits the interaction of GP120 to CD4. Further, compound 5660386 showed a unique and broad-range activity against primary HIV-1 isolates from different subtypes and geographical areas. Conclusion Development of small-molecule entry inhibitors of HIV-1 such as 5660386 may lead to novel classes of anti-HIV-1 therapeutics. These inhibitors may be particularly effective against viruses resistant to current antiretroviral drugs and could have potential applications in both treatment and prevention. PMID:26491257

  20. Incompatible Natures of the HIV-1 Envelope in Resistance to the CCR5 Antagonist Cenicriviroc and to Neutralizing Antibodies

    PubMed Central

    Enomoto, Ikumi; Baba, Masanori

    2015-01-01

    Cenicriviroc is a CCR5 antagonist which prevents human immunodeficiency virus type 1 (HIV-1) from cellular entry. The CCR5-binding regions of the HIV-1 envelope glycoprotein are important targets for neutralizing antibodies (NAbs), and mutations conferring cenicriviroc resistance may therefore affect sensitivity to NAbs. Here, we used the in vitro induction of HIV-1 variants resistant to cenicriviroc or NAbs to examine the relationship between resistance to cenicriviroc and resistance to NAbs. The cenicriviroc-resistant variant KK652-67 (strain KK passaged 67 times in the presence of increasing concentrations of cenicriviroc) was sensitive to neutralization by NAbs against the V3 loop, the CD4-induced (CD4i) region, and the CD4-binding site (CD4bs), whereas the wild-type (WT) parental HIV-1 strain KKWT from which cenicriviroc-resistant strain KK652-67 was obtained was resistant to these NAbs. The V3 region of KK652-67 was important for cenicriviroc resistance and critical to the high sensitivity of the V3, CD4i, and CD4bs epitopes to NAbs. Moreover, induction of variants resistant to anti-V3 NAb 0.5γ and anti-CD4i NAb 4E9C from cenicriviroc-resistant strain KK652-67 resulted in reversion to the cenicriviroc-sensitive phenotype comparable to that of the parental strain, KKWT. Resistance to 0.5γ and 4E9C was caused by the novel substitutions R315K, G324R, and E381K in the V3 and C3 regions near the substitutions conferring cenicriviroc resistance. Importantly, these amino acid changes in the CCR5-binding region were also responsible for reversion to the cenicriviroc-sensitive phenotype. These results suggest the presence of key amino acid residues where resistance to cenicriviroc is incompatible with resistance to NAbs. This implies that cenicriviroc and neutralizing antibodies may restrict the emergence of variants resistant to each other. PMID:26525792

  1. Resolution of Specific Nucleotide Mismatches by Wild-Type and AZT-Resistant Reverse Transcriptases during HIV-1 Replication.

    PubMed

    Kharytonchyk, Siarhei; King, Steven R; Ndongmo, Clement B; Stilger, Krista L; An, Wenfeng; Telesnitsky, Alice

    2016-06-01

    A key contributor to HIV-1 genetic variation is reverse transcriptase errors. Some mutations result because reverse transcriptase (RT) lacks 3' to 5' proofreading exonuclease and can extend mismatches. However, RT also excises terminal nucleotides to a limited extent, and this activity contributes to AZT resistance. Because HIV-1 mismatch resolution has been studied in vitro but only indirectly during replication, we developed a novel system to study mismatched base pair resolution during HIV-1 replication in cultured cells using vectors that force template switching at defined locations. These vectors generated mismatched reverse transcription intermediates, with proviral products diagnostic of mismatch resolution mechanisms. Outcomes for wild-type (WT) RT and an AZT-resistant (AZT(R)) RT containing a thymidine analog mutation set-D67N, K70R, D215F, and K219Q-were compared. AZT(R) RT did not excise terminal nucleotides more frequently than WT, and for the majority of tested mismatches, both WT and AZT(R) RTs extended mismatches in more than 90% of proviruses. However, striking enzyme-specific differences were observed for one mispair, with WT RT preferentially resolving dC-rC pairs either by excising the mismatched base or switching templates prematurely, while AZT(R) RT primarily misaligned the primer strand, causing deletions via dislocation mutagenesis. Overall, the results confirmed HIV-1 RT's high capacity for mismatch extension during virus replication and revealed dramatic differences in aberrant intermediate resolution repertoires between WT and AZT(R) RTs on one mismatched replication intermediate. Correlating mismatch extension frequencies observed here with reported viral mutation rates suggests a complex interplay of nucleotide discrimination and mismatch extension drives HIV-1 mutagenesis. PMID:27075671

  2. Dolutegravir in Antiretroviral-Experienced Patients With Raltegravir- and/or Elvitegravir-Resistant HIV-1: 24-Week Results of the Phase III VIKING-3 Study

    PubMed Central

    Castagna, Antonella; Maggiolo, Franco; Penco, Giovanni; Wright, David; Mills, Anthony; Grossberg, Robert; Molina, Jean-Michel; Chas, Julie; Durant, Jacques; Moreno, Santiago; Doroana, Manuela; Ait-Khaled, Mounir; Huang, Jenny; Min, Sherene; Song, Ivy; Vavro, Cindy; Nichols, Garrett; Yeo, Jane M.; Aberg, J.; Akil, B.; Arribas, J. R.; Baril, J.-G.; Blanco Arévalo, J. L.; Blanco Quintana, F.; Blick, G.; Boix Martínez, V.; Bouchaud, O.; Branco, T.; Bredeek, U. F.; Castro Iglesias, M.; Clumeck, N.; Conway, B.; DeJesus, E.; Delassus, J.-L.; De Truchis, P.; Di Perri, G.; Di Pietro, M.; Duggan, J.; Duvivier, C.; Elion, R.; Eron, J.; Fish, D.; Gathe, J.; Haubrich, R.; Henderson, H.; Hicks, C.; Hocqueloux, L.; Hodder, S.; Hsiao, C.-B.; Katlama, C.; Kozal, M.; Kumar, P.; Lalla-Reddy, S.; Lazzarin, A.; Leoncini, F.; Llibre, J. M.; Mansinho, K.; Morlat, P.; Mounzer, K.; Murphy, M.; Newman, C.; Nguyen, T.; Nseir, B.; Philibert, P.; Pialoux, G.; Poizot-Martin, I.; Ramgopal, M.; Richmond, G.; Salmon Ceron, D.; Sax, P.; Scarsella, A.; Sension, M.; Shalit, P.; Sighinolfi, L.; Sloan, L.; Small, C.; Stein, D.; Tashima, K.; Tebas, P.; Torti, C.; Tribble, M.; Troisvallets, D.; Tsoukas, C.; Viciana Fernández, P.; Ward, D.; Wheeler, D.; Wilkin, T.; Yeni, G.-P.; Louise Martin-Carpenter, J.; Uhlenbrauck, Gina

    2014-01-01

    Background. The pilot phase IIb VIKING study suggested that dolutegravir (DTG), a human immunodeficiency virus (HIV) integrase inhibitor (INI), would be efficacious in INI-resistant patients at the 50 mg twice daily (BID) dose. Methods. VIKING-3 is a single-arm, open-label phase III study in which therapy-experienced adults with INI-resistant virus received DTG 50 mg BID while continuing their failing regimen (without raltegravir or elvitegravir) through day 7, after which the regimen was optimized with ≥1 fully active drug and DTG continued. The primary efficacy endpoints were the mean change from baseline in plasma HIV-1 RNA at day 8 and the proportion of subjects with HIV-1 RNA <50 c/mL at week 24. Results. Mean change in HIV-1 RNA at day 8 was −1.43 log10 c/mL, and 69% of subjects achieved <50 c/mL at week 24. Multivariate analyses demonstrated a strong association between baseline DTG susceptibility and response. Response was most reduced in subjects with Q148 + ≥2 resistance-associated mutations. DTG 50 mg BID had a low (3%) discontinuation rate due to adverse events, similar to INI-naive subjects receiving DTG 50 mg once daily. Conclusions. DTG 50 mg BID–based therapy was effective in this highly treatment-experienced population with INI-resistant virus. Clinical Trials Registration. www.clinicaltrials.gov (NCT01328041) and http://www.gsk-clinicalstudywww.gsk-clinicalstudyregister.com (112574). PMID:24446523

  3. The Antiviral Activity of Approved and Novel Drugs against HIV-1 Mutations Evaluated under the Consideration of Dose-Response Curve Slope

    PubMed Central

    Chang, Shuai; Zhuang, Daomin; Guo, Wei; Li, Lin; Zhang, Wenfu; Liu, Siyang; Li, Hanping; Liu, Yongjian; Bao, Zuoyi; Han, Jingwan

    2016-01-01

    Objectives This study was designed to identify common HIV-1 mutation complexes affecting the slope of inhibition curve, and to propose a new parameter incorporating both the IC50 and the slope to evaluate phenotypic resistance. Methods Utilizing site-directed mutagenesis, we constructed 22 HIV-1 common mutation complexes. IC50 and slope of 10 representative approved drugs and a novel agent against these mutations were measured to determine the resistance phenotypes. The values of new parameter incorporating both the IC50 and the slope of the inhibition curve were calculated, and the correlations between parameters were assessed. Results Depending on the class of drug, there were intrinsic differences in how the resistance mutations affected the drug parameters. All of the mutations resulted in large increases in the IC50s of nucleoside reverse transcriptase inhibitors. The effects of the mutations on the slope were the most apparent when examining their effects on the inhibition of non-nucleoside reverse transcriptase inhibitors and protease inhibitors. For example, some mutations, such as V82A, had no effect on IC50, but reduced the slope. We proposed a new concept, termed IIPatoxic, on the basis of IC50, slope and the maximum limiting concentrations of the drug. The IIPatoxic values of 10 approved drugs and 1 novel agent were calculated, and were closely related to the IIPmax values (r > 0.95, p < 0.001). Conclusions This study confirms that resistance mutations cannot be accurately assessed by IC50 alone, because it tends to underestimate the degree of resistance. The slope parameter is of very importance in the measurement of drug resistance and the effect can be applied to more complex patterns of resistance. This is the most apparent when testing the effects of the mutations on protease inhibitors activity. We also propose a new index, IIPatoxic, which incorporates both the IC50 and the slope. This new index could complement current IIP indices, thereby

  4. Analysis of nonnucleoside drug-resistant variants of human immunodeficiency virus type 1 reverse transcriptase.

    PubMed Central

    Boyer, P L; Currens, M J; McMahon, J B; Boyd, M R; Hughes, S H

    1993-01-01

    A number of chemically distinct nonnucleoside inhibitors of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) have been reported. Several lines of evidence, including the isolation of RT mutants that show cross resistance, suggest that, despite their structural diversity, many of these inhibitors bind to a common site on HIV-1 RT. We have recently reported that, on the basis of analyses of HIV-1/HIV-2 chimeras, the natural product calanolide A may interact with a different site or sites in HIV-1 RT. We have used BspMI cassette mutagenesis to prepare a collection of HIV-1 RT mutants that show resistance to the known members of the general class of nonnucleoside inhibitors. This collection of mutants can be used to determine whether a new drug will show cross resistance with known inhibitors and to define amino acid positions critical for the action of the drugs. The mutants were used to analyze calanolide A, 1H,3H-thiazolo[3,4-a]benzimidazole(4i), and the acyclic nucleoside analog 1-[(2-hydroxyethoxy)methyl]-6-(phenylthio)thymine. These analyses suggest that all three drugs interact with HIV-1 RT within the previously defined common binding site for nonnucleoside inhibitors. However, the drugs respond differently to the panel of drug-resistant HIV-1 RTs, indicating that while the binding sites of the drugs overlap they are not identical. PMID:7680393

  5. Increased density of neurons containing NADPH diaphorase and nitric oxide synthase in the cerebral cortex of patients with HIV-1 infection and drug abuse.

    PubMed

    Kuljis, Rodrigo O; Shapshak, Paul; Alcabes, Philip; Rodríguez de la Vega, Pura; Fujimura, Robert; Petito, Carol K

    2002-01-01

    To determine whether nitrogen monoxide (nitric oxide; NO) synthase (NOS) and NADPH diaphorase (NDP) co-containing cerebrocortical neurons (NOSN) neurons are affected in patients infected with human immunodeficiency virus type 1 (HIV-1) with and without associated intake of drugs of abuse, we examined the temporal neocortex of 24 individuals: 12 HIV-1 positive (including 3 drug users, 9 non-drug users) and 12 HIV-1 negative (including 6 drug users, and 6 non-drug users). Histochemical labeling for NDP-an enzymatic domain co-expressed in the NOS enzyme-was employed to visualize NOSN. Drug abuse and HIV-1 infection cause independently an increase in NOSN density, but combined they result in up to a 38-fold increase in NOSN density, suggesting that the combination of these factors induces NOS expression powerfully in neurons that normally do not synthesize NDP/NOS. This is associated with an increase in the proportion of NOSN displaying dystrophic changes, indicating that NOSN undergo massive degeneration in association with NOS synthesis induction. The increase in density of NOSN in HIV-1 infected drug abusers may be among the important sources of NO mediating cerebrocortical dysfunction, and the degeneration of NOS-containing local circuit neurons in patients with HIV-1 infection or drug abuse may underlie in part their neuropsychiatric manifestations. PMID:16873197

  6. Short-peptide fusion inhibitors with high potency against wild-type and enfuvirtide-resistant HIV-1.

    PubMed

    Chong, Huihui; Yao, Xue; Qiu, Zonglin; Sun, Jianping; Zhang, Meng; Waltersperger, Sandro; Wang, Meitian; Liu, Shan-Lu; Cui, Sheng; He, Yuxian

    2013-03-01

    Peptides derived from the C-terminal heptad repeat (C peptides) of HIV-1 gp41 are potent inhibitors against virus entry. However, development of a short C peptide possessing high anti-HIV potency is considered a daunting challenge. We recently discovered that the residues Met626 and Thr627 preceding the pocket-binding domain of the C peptide adopt a unique M-T hook structure that is crucial for the design of HIV-1 fusion inhibitors. In this study, we first presented a proof-of-concept prototype that the M-T hook residues can dramatically improve the antiviral activity and thermostability of a short C peptide. We then generated a 24-mer peptide termed MT-SC22EK by incorporating the M-T hook structure to the N terminus of the poorly active short C peptide SC22EK. Amazingly, MT-SC22EK inhibited HIV-1-mediated cell fusion and infection at a level comparable to C34, T1249, SC29EK, and sifuvirtide, and it was highly active against diverse HIV-1 subtypes and variants, including those T20 (enfuvirtide) and SC29EK-resistant viruses. The high-resolution crystal structure of MT-SC22EK reveals the N-terminal M-T hook conformation folded by incorporated Met626 and Thr627 and identifies the C-terminal boundary critical for the anti-HIV activity. Collectively, our studies provide new insights into the mechanisms of HIV-1 fusion and its inhibition. PMID:23233535

  7. Neutralization resistance of virological synapse-mediated HIV-1 Infection is regulated by the gp41 cytoplasmic tail.

    PubMed

    Durham, Natasha D; Yewdall, Alice W; Chen, Ping; Lee, Rebecca; Zony, Chati; Robinson, James E; Chen, Benjamin K

    2012-07-01

    Human immunodeficiency virus type 1 (HIV-1) infection can spread efficiently from infected to uninfected T cells through adhesive contacts called virological synapses (VSs). In this process, cell-surface envelope glycoprotein (Env) initiates adhesion and viral transfer into an uninfected recipient cell. Previous studies have found some HIV-1-neutralizing patient sera to be less effective at blocking VS-mediated infection than infection with cell-free virus. Here we employ sensitive flow cytometry-based infection assays to measure the inhibitory potency of HIV-1-neutralizing monoclonal antibodies (MAb) and HIV-1-neutralizing patient sera against cell-free and VS-mediated infection. To various degrees, anti-Env MAbs exhibited significantly higher 50% inhibitory concentration (IC(50)s) against VS-mediated infection than cell-free infection. Notably, the MAb 17b, which binds a CD4-induced (CD4i) epitope on gp120, displayed a 72-fold reduced efficacy against VS-mediated inocula compared to cell-free inocula. A mutant with truncation mutation in the gp41 cytoplasmic tail (CT) which is unable to modulate Env fusogenicity in response to virus particle maturation but which can still engage in cell-to-cell infection was tested for the ability to resist neutralizing antibodies. The ΔCT mutation increased cell surface staining by neutralizing antibodies, significantly enhanced neutralization of VS-mediated infection, and had reduced or no effect on cell-free infection, depending upon the antibody. Our results suggest that the gp41 CT regulates the exposure of key neutralizing epitopes during cell-to-cell infection and plays an important role in immune evasion. Vaccine strategies should consider immunogens that reflect Env conformations exposed on the infected cell surface to enhance protection against VS-mediated HIV-1 spread. PMID:22553332

  8. The R262K substitution combined with H51Y in HIV-1 subtype B integrase confers low-level resistance against dolutegravir.

    PubMed

    Cutillas, Vincent; Mesplede, Thibault; Anstett, Kaitlin; Hassounah, Said; Wainberg, Mark A

    2015-01-01

    Clinical studies have shown that integrase strand transfer inhibitors (INSTIs) can be used effectively against HIV-1 infection. To date, no resistance substitution has been found in INSTI-naive patients treated with the new integrase inhibitor dolutegravir (DTG). In a recent selection study with DTG, using a virus bearing the H51Y substitution in integrase, the emergence of an R to K substitution at position 262 (R262K) was observed. We characterized this double mutant with respect to integrase strand transfer activity and susceptibility to DTG both biochemically and in tissue culture. We showed that the addition of R262K to H51Y decreased recombinant integrase strand transfer activity but improved integrase DNA-binding affinity, compared to wild-type or H51Y-containing enzymes. The defect in strand transfer activity did not translate into a decrease in HIV-1 infectivity. The combination of H51Y and R262K substitutions slightly decreased susceptibility to DTG (fold change = 1.87) in cell-based resistance assays. Although viral replication was not affected and enzyme efficiency was impaired by the addition of R262K to H51Y, there was an overall increase in the level of biochemical drug resistance against DTG. Our findings suggest that the R at position 262 plays an important role in DNA binding. PMID:25348535

  9. Genetic variation of the HIV-1 integrase region in newly diagnosed anti-retroviral drug-naïve patients with HIV/AIDS in Korea.

    PubMed

    Kim, J-Y; Kim, E-J; Choi, J-Y; Kwon, O-K; Kim, G J; Choi, S Y; Kim, S S

    2011-08-01

    The survival time of HIV/AIDS patients in Korea has increased since HAART (highly active anti-retroviral therapy) was introduced. However, the occurrence of drug-resistant strains requires new anti-retroviral drugs, one of which, an integrase inhibitor (INI), was approved by the US Food and Drug Administration (FDA) in 2007. INIs have been used for therapy in many countries and are about to be employed in Korea. Therefore, it is important to identify basic mutant variants prior to the introduction of INIs in order to estimate their efficacy. To monitor potential drug-resistant INI mutations in Korean HIV/AIDS patients, the polymorphism of the int gene was investigated together with the pol gene using a genotypic assay for 75 randomly selected Korean HIV-1 patients newly diagnosed in 2007. The drug-resistant mutation sequences were analysed using the Stanford HIV DB and the International AIDS Society resistance testing-USA panel (IAS-USA). Seventy strains of Korean subtype B were compared with foreign subtype-B strains, and there were no significantly different variants of the int gene region in the study population. Major mutation sites in the integrase (E92Q, F121Y, G140A/S, Y143C/R, Q148H/R/K and N155H) were not detected, and only a few minor mutation sites (L74M, V151I, E157Q, V165I, I203M, S230N and D232N) were identified in 21 strains (28%). Resistance due to mutations in the pol gene was observed in a single strain (1.3%) resistant to protease inhibitors (PIs) and in four strains (5.3%) resistant to reverse transcriptase inhibitors (RTIs). In summary, this demonstrates that INIs will be susceptible to drug naïve HIV/AIDS patients in Korea. PMID:20946407

  10. Joint X-ray/neutron crystallographic study of HIV-1 protease with clinical inhibitor amprenavir – insights for drug design

    PubMed Central

    Weber, Irene T.; Waltman, Mary Jo; Mustyakimov, Marat; Blakeley, Matthew P.; Keen, David A.; Ghosh, Arun K.; Langan, Paul; Kovalevsky, Andrey Y.

    2013-01-01

    HIV-1 protease is an important target for the development of antiviral inhibitors to treat AIDS. A room-temperature joint X-ray/neutron structure of the protease in complex with clinical drug amprenavir has been determined at 2.0 Å resolution. The structure provides direct determination of hydrogen atom positions in the enzyme active site. Analysis of the enzyme-drug interactions suggests that some hydrogen bonds may be weaker than deduced from the non-hydrogen interatomic distances. This information may be valuable for the design of improved protease inhibitors. PMID:23772563

  11. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    SciTech Connect

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; Lloyd, Krissey  E.; Hwang, Kwan-Ki; Lu, Xiaozhi; Bonsignori, Mattia; Finzi, Andrés; Vandergrift, Nathan  A.; Alam, S.  Munir; Ferrari, Guido; Shen, Xiaoying; Tomaras, Georgia  D.; Kamanga, Gift; Cohen, Myron  S.; Sam, Noel  E.; Kapiga, Saidi; Gray, Elin S.; Tumba, Nancy  L.; Morris, Lynn; Zolla-Pazner, Susan; Gorny, Miroslaw  K.; Mascola, John  R.; Hahn, Beatrice H.; Shaw, George  M.; Sodroski, Joseph  G.; Liao, Hua-Xin; Montefiori, David C.; Hraber, Peter T.; Korber, Bette T.; Haynes, Barton F.

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tier 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.

  12. Strain-Specific V3 and CD4 Binding Site Autologous HIV-1 Neutralizing Antibodies Select Neutralization-Resistant Viruses

    DOE PAGESBeta

    Moody, M.  Anthony; Gao, Feng; Gurley, Thaddeus  C.; Amos, Joshua  D.; Kumar, Amit; Hora, Bhavna; Marshall, Dawn  J.; Whitesides, John  F.; Xia, Shi-Mao; Parks, Robert; et al

    2015-09-09

    The third variable (V3) loop and the CD4 binding site (CD4bs) of the viral envelope are frequently targeted by neutralizing antibodies (nAbs) in HIV-1-infected individuals. In chronic infection, virus escape mutants repopulate the plasma and V3 and CD4bs nAbs emerge that can neutralize heterologous tier 1 easy-to-neutralize, but not tier 2 difficult-to-neutralize HIV-1 isolates. However, neutralization sensitivity of autologous plasma viruses to this type of nAb response has not been studied. We describe the development and evolution in vivo of antibodies distinguished by their target specificity for V3and CD4bs epitopes on autologous tier 2 viruses but not on heterologous tiermore » 2 viruses. A surprisingly high fraction of autologous circulating viruses was sensitive to these antibodies. These findings demonstrate a role for V3 and CD4bs antibodies in constraining the native envelope trimer in vivo to a neutralization-resistant phenotype, explaining why HIV-1 transmission generally occurs by tier 2 neutralization-resistant viruses.« less

  13. Ultrasound-assessed perirenal fat is related to increased ophthalmic artery resistance index in HIV-1 patients

    PubMed Central

    2010-01-01

    Background The introduction of highly active antiretroviral therapy (HAART) has dramatically changed the prognosis of human immunodeficiency virus (HIV) infection, with a significant decline in morbidity and mortality. Changes in body fat distribution are a common finding in individuals with HIV infection being treated with antiretrovirals, and this condition (collectively termed lipodystrophy syndrome) is associated with depletion of subcutaneous fat, increased triglycerides and insulin resistance. Obesity, particularly visceral obesity, is associated with increased risk of cardiovascular disease. Therefore, estimating visceral fat distribution is important in identifying subjects at high risk for cardiovascular disease. The aim of our study was to evaluate whether perirenal fat thickness (PRFT), a parameter of central obesity, is related to ophthalmic artery resistance index (OARI), an index of occlusive carotid artery disease in HIV-1 infected patients. Methods We enrolled 88 consecutive HIV-1-infected patients receiving highly active antiretroviral therapy for more than 12 months, in a prospective cohort study. Echographically measured PRFT and OARI, as well as serum metabolic parameters, were evaluated. PRFT and OARI were measured by 3.75 MHz convex and 7.5 MHz linear probe, respectively. Results The means of PRFT and OARI in HIV-1-infected patients with visceral obesity was considerably higher than in patients without it (p < 0.0001 and p < 0.001, respectively). Using the average OARI as the dependent variable, total serum cholesterol level, HDL, triglycerides, glycemia, sex, blood pressure, age and PRFT were independent factors associated with OARI. A PRFT of 6.1 mm was the most discriminatory value for predicting an OARI > 0.74 (sensitivity 78.9%, specificity 82.8%). Conclusions Our data indicate that ultrasound assessment of PRFT may have potential as a marker of increased endothelial damage with specific involvement of the ocular vascular region in HIV-1

  14. Replication fitness of multiple nonnucleoside reverse transcriptase-resistant HIV-1 variants in the presence of etravirine measured by 454 deep sequencing.

    PubMed

    Brumme, Chanson J; Huber, Kelly D; Dong, Winnie; Poon, Art F Y; Harrigan, P Richard; Sluis-Cremer, Nicolas

    2013-08-01

    We applied an efficient method to characterize the relative fitness levels of multiple nonnucleoside reverse transcriptase (NNRTI)-resistant HIV-1 variants by simultaneous competitive culture and 454 deep sequencing. Using this method, we show that the Y181V mutation in the HIV-1 reverse transcriptase in particular confers a clear selective advantage to the virus over 14 other NNRTI resistance mutations in the presence of etravirine in vitro. PMID:23720723

  15. HIV-1 Genital Shedding is Suppressed in the Setting of High Genital Antiretroviral Drug Concentrations Throughout the Menstrual Cycle

    PubMed Central

    Sheth, Anandi N.; Evans-Strickfaden, Tammy; Haaland, Richard; Martin, Amy; Gatcliffe, Chelsea; Adesoye, Adebola; Omondi, Michael W.; Lupo, L. Davis; Danavall, Damien; Easley, Kirk; Chen, Cheng-Yen; Pau, Chou-Pong; Hart, Clyde; Ofotokun, Igho

    2014-01-01

    Background. It is not known if fluctuations in genital tract antiretroviral drug concentrations correlate with genital virus shedding in human immunodeficiency virus (HIV)–infected women on antiretroviral therapy (ART). Methods. Among 20 HIV-infected women on ART (tenofovir [TFV], emtricitabine [FTC], and ritonavir-boosted atazanavir [ATV]) with suppressed plasma virus loads, blood and cervicovaginal samples collected twice weekly for 3 weeks were tested for antiretroviral concentrations, HIV-1 RNA, and proviral DNA. Results. Cervicovaginal:plasma antiretroviral concentration ratios were highest for FTC (11.9, 95% confidence interval [CI], 8.66–16.3), then TFV (3.52, 95% CI, 2.27–5.48), and ATV (2.39, 95% CI, 1.69–3.38). Within- and between-person variations in plasma and genital antiretroviral concentrations were observed. Low amounts of genital HIV-1 RNA (<50 copies/mL) were detected in 45% of women at 16% of visits. Genital HIV-1 DNA was detected in 70% of women at 35% of visits. Genital virus detection was associated with higher concentrations of mucosal leukocytes but not with genital antiretroviral concentrations, menstrual cycle phase, bacterial vaginosis, genital bleeding, or plasma virus detection. Conclusions. Standard doses of ART achieved higher genital than plasma concentrations across the menstrual cycle. Therapeutic ART suppresses genital virus shedding throughout the menstrual cycle, even in the presence of factors reported to increase virus shedding. PMID:24643223

  16. The effect of desolvation on the binding of inhibitors to HIV-1 protease and cyclin-dependent kinases: Causes of resistance.

    PubMed

    Fong, Clifford W

    2016-08-01

    Studies of the cyclin-dependent kinase inhibitors and HIV-1 protease inhibitors have confirmed that ligand-protein binding is dependent on desolvation effects. It has been found that a four parameter linear model incorporating desolvation energy, lipophilicity, dipole moment and molecular volume of the ligands is a good model to describe the binding between ligands and kinases or proteases. The resistance shown by MDR proteases to the anti-viral drugs is multi-faceted involving varying changes in desolvation, lipophilicity and dipole moment interaction compared to the non-resistant protease. Desolvation has been shown to be the dominant factor influencing the effect of inhibitors against the cyclin-dependent kinases, but lipophilicity and dipole moment are also significant factors. The model can differentiate between the inhibitory activity of CDK2/cycE, CDK1/cycB and CDK4/cycD enzymes. PMID:27317642

  17. Secondary mutations in viruses resistant to HIV-1 integrase inhibitors that restore viral infectivity and replication kinetics.

    PubMed

    Nakahara, Koichiro; Wakasa-Morimoto, Chiaki; Kobayashi, Masanori; Miki, Shigeru; Noshi, Takeshi; Seki, Takahiro; Kanamori-Koyama, Mikiko; Kawauchi, Shinobu; Suyama, Akemi; Fujishita, Toshio; Yoshinaga, Tomokazu; Garvey, Edward P; Johns, Brian A; Foster, Scott A; Underwood, Mark R; Sato, Akihiko; Fujiwara, Tamio

    2009-02-01

    Passage of HIV-1 in the presence of integrase inhibitors (INIs) generates resistant viruses that have mutations in the integrase region. Integrase-resistant mutations Q148K and Q148R were identified as primary mutations with the passage of HIV-1 IIIB in the presence of INIs S-1360 or S/GSK-364735, respectively. Secondary amino acid substitutions E138K or G140S were observed when passage with INI was continued. The role of these mutations was investigated with molecular clones. Relative to Q148K alone, Q148K/E138K had 2- and >6-fold increases in resistance to S-1360 and S/GSK-364735, respectively, and the double mutant had slightly better infectivity and replication kinetics. In contrast, Q148K/G140S and Q148R/E138K had nearly equivalent or slightly reduced fold resistance to the INI compared with their respective Q148 primary mutants, and had increases in infectivity and replication kinetics. Recovery of these surrogates of viral fitness coincided with the recovery of integration efficiency of viral DNA into the host cell chromosome for these double mutants. These data show that recovery of viral integration efficiency can be an important factor for the emergence and maintenance of INI-resistant mutations. PMID:19027039

  18. Development of Nevirapine Resistance in Children Exposed to the Prevention of Mother-to-Child HIV-1 Transmission Programme in Maputo, Mozambique

    PubMed Central

    Antunes, Francisco; Zindoga, Pereira; Gomes, Perpétua; Augusto, Orvalho; Mahumane, Isabel; Veloso, Luís; Valadas, Emília; Camacho, Ricardo

    2015-01-01

    Background Single-dose nevirapine (sd-NVP) has been the main option for prevention of mother-to-child transmission (PMTCT) of HIV-1 in low-resource settings. However, sd-NVP can induce the selection of HIV-1 resistant mutations in mothers and infants. In Mozambique, there are limited data regarding the profile of NVP resistance associated mutations (RAM) in the context of PMTCT. Objectives To assess the prevalence and the factors associated with NVP RAM among children born to HIV-1 infected mothers enrolled in the PMTCT programme adopted in Mozambique. Methods One hundred and fifty seven children aged 6 to 48 weeks were sequentially included (July 2011 to March 2012) at four centres in Maputo. Genotyping of RAM was performed in samples with HIV-1 RNA≥ 100 copies/μL (Viroseq). Sequencing was performed with ABI 3100 (Applied Biosystems). Logistic regression modelling was undertaken to identify the factors associated with NVP RAM. Results Seventy-nine children had their samples genotyped. Their median age was 7.0 (3–12) months and 92.4% received prophylaxis with sd-NVP at birth plus daily NVP. 35.4% of mothers received antiretrovirals (ARVs) for PMTCT. ARV RAM were detected in 43 (54.4%) of the children. 45.6% of these children had at least one NVP RAM. The most common mutations associated with NVP resistance were K103N (n = 16) and Y181C (n = 15). NVP RAM was significantly associated with mother exposure to PMTCT (crude odds ratio [OR] 30.3, 95% CI 4.93–186.34) and with mother’s CD4 count < 350 cells/mm3 (crude OR 3.08, 95% CI 1.02–9.32). In the multivariable analysis the mother’s exposure to PMTCT was the only variable significantly associated with NVP RAM (adjusted OR 48.65, 95% CI 9.33–253.66). Conclusions We found a high prevalence of NVP RAM among children who were exposed to the drug regimen for PMTCT in Mozambique. The mothers’ exposure to PMTCT significantly increased the risk of NVP RAM. PMID:26161559

  19. Pharmacotherapy of HIV-1 Infection: Focus on CCR5 Antagonist Maraviroc

    PubMed Central

    Latinovic, Olga; Kuruppu, Janaki; Davis, Charles; Le, Nhut; Heredia, Alonso

    2009-01-01

    Sustained inhibition of HIV-1, the goal of antiretroviral therapy, is often impeded by the emergence of viral drug resistance. For patients infected with HIV-1 resistant to conventional drugs from the viral reverse transcriptase and protease inhibitor classes, the recently approved entry and integration inhibitors effectively suppress HIV-1 and offer additional therapeutic options. Entry inhibitors are particularly attractive because, unlike conventional antiretrovirals, they target HIV-1 extracellularly, thereby sparing cells from both viral- and drug-induced toxicities. The fusion inhibitor enfuvirtide and the CCR5 antagonist maraviroc are the first entry inhibitors licensed for patients with drug-resistant HIV-1, with maraviroc restricted to those infected with CCR5-tropic HIV-1 (R5 HIV-1) only. Vicriviroc (another CCR5 antagonist) is in Phase III clinical trials, whereas the CCR5 antibodies PRO 140 and HGS 004 are in early stages of clinical development. Potent antiviral synergy between maraviroc and CCR5 antibodies, coupled with distinct patterns of resistance, suggest their combinations might be particularly effective in patients. In addition, given that oral administration of maraviroc achieves high drug levels in cervicovaginal fluid, combinations of maraviroc and other CCR5 inhibitors could be effective in preventing HIV-1 transmission. Moreover, since CCR5 antagonists prevent rejection of transplanted organs, maraviroc could both suppress HIV-1 and prolong organ survival for the growing number of HIV-1 patients with kidney or liver failure necessitating organ transplantation. Thus, maraviroc offers an important treatment option for patients with drug-resistant R5 HIV-1, who presently account for >50% of drug-resistance cases. PMID:19920876

  20. Crystal structures of multidrug-resistant HIV-1 protease in complex with two potent anti-malarial compounds

    SciTech Connect

    Yedidi, Ravikiran S.; Liu, Zhigang; Wang, Yong; Brunzelle, Joseph S.; Kovari, Iulia A.; Woster, Patrick M.; Kovari, Ladislau C.; Gupta, Deepak

    2012-06-19

    Two potent inhibitors (compounds 1 and 2) of malarial aspartyl protease, plasmepsin-II, were evaluated against wild type (NL4-3) and multidrug-resistant clinical isolate 769 (MDR) variants of human immunodeficiency virus type-1 (HIV-1) aspartyl protease. Enzyme inhibition assays showed that both 1 and 2 have better potency against NL4-3 than against MDR protease. Crystal structures of MDR protease in complex with 1 and 2 were solved and analyzed. Crystallographic analysis revealed that the MDR protease exhibits a typical wide-open conformation of the flaps (Gly48 to Gly52) causing an overall expansion in the active site cavity, which, in turn caused unstable binding of the inhibitors. Due to the expansion of the active site cavity, both compounds showed loss of direct contacts with the MDR protease compared to the docking models of NL4-3. Multiple water molecules showed a rich network of hydrogen bonds contributing to the stability of the ligand binding in the distorted binding pockets of the MDR protease in both crystal structures. Docking analysis of 1 and 2 showed a decrease in the binding affinity for both compounds against MDR supporting our structure-function studies. Thus, compounds 1 and 2 show promising inhibitory activity against HIV-1 protease variants and hence are good candidates for further development to enhance their potency against NL4-3 as well as MDR HIV-1 protease variants.

  1. The hunt for HIV-1 integrase inhibitors.

    PubMed

    Lataillade, Max; Kozal, Michael J

    2006-07-01

    Currently, there are three distinct mechanistic classes of antiretrovirals: inhibitors of the HIV- 1 reverse transcriptase and protease enzymes and inhibitors of HIV entry, including receptor and coreceptor binding and cell fusion. A new drug class that inhibits the HIV-1 integrase enzyme (IN) is in development and may soon be available in the clinic. IN is an attractive drug target because it is essential for a stable and productive HIV-1 infection and there is no mammalian homologue of IN. Inhibitors of integrase enzyme (INI) block the integration of viral double-stranded DNA into the host cell's chromosomal DNA. HIV-1 integration has many potential steps that can be inhibited and several new compounds that target specific integration steps have been identified by drug developers. Recently, two INIs, GS-9137 and MK-0518, demonstrated promising early clinical trial results and have been advanced into later stage trials. In this review, we describe how IN facilitates HIV-1 integration, the needed enzyme cofactors, and the resultant byproducts created during integration. Furthermore, we review the different INIs under development, their mechanism of actions, site of IN inhibition, potency, resistance patterns, and discuss the early clinical trial results. PMID:16839248

  2. Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance

    SciTech Connect

    Anastassopoulou, Cleo G.; Ketas, Thomas J.; Sanders, Rogier W.; Johan Klasse, Per; Moore, John P.

    2012-07-05

    A rare pathway of HIV-1 resistance to small molecule CCR5 inhibitors such as Vicriviroc (VCV) involves changes solely in the gp41 fusion peptide (FP). Here, we show that the G516V change is critical to VCV resistance in PBMC and TZM-bl cells, although it must be accompanied by either M518V or F519I to have a substantial impact. Modeling VCV inhibition data from the two cell types indicated that G516V allows both double mutants to use VCV-CCR5 complexes for entry. The model further identified F519I as an independent determinant of preference for the unoccupied, high-VCV affinity form of CCR5. From inhibitor-free reversion cultures, we also identified a substitution in the inner domain of gp120, T244A, which appears to counter the resistance phenotype created by the FP substitutions. Examining the interplay of these changes will enhance our understanding of Env complex interactions that influence both HIV-1 entry and resistance to CCR5 inhibitors.

  3. Potential Elucidation of a Novel CTL Epitope in HIV-1 Protease by the Protease Inhibitor Resistance Mutation L90M

    PubMed Central

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89–97 and PR 90–99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease. PMID:24015196

  4. HIV-1 Genetic Variability and Clinical Implications

    PubMed Central

    Santoro, Maria Mercedes; Perno, Carlo Federico

    2013-01-01

    Despite advances in antiretroviral therapy that have revolutionized HIV disease management, effective control of the HIV infection pandemic remains elusive. Beyond the classic non-B endemic areas, HIV-1 non-B subtype infections are sharply increasing in previous subtype B homogeneous areas such as Europe and North America. As already known, several studies have shown that, among non-B subtypes, subtypes C and D were found to be more aggressive in terms of disease progression. Luckily, the response to antiretrovirals against HIV-1 seems to be similar among different subtypes, but these results are mainly based on small or poorly designed studies. On the other hand, differences in rates of acquisition of resistance among non-B subtypes are already being observed. This different propensity, beyond the type of treatment regimens used, as well as access to viral load testing in non-B endemic areas seems to be due to HIV-1 clade specific peculiarities. Indeed, some non-B subtypes are proved to be more prone to develop resistance compared to B subtype. This phenomenon can be related to the presence of subtype-specific polymorphisms, different codon usage, and/or subtype-specific RNA templates. This review aims to provide a complete picture of HIV-1 genetic diversity and its implications for HIV-1 disease spread, effectiveness of therapies, and drug resistance development. PMID:23844315

  5. Toward the development of a single-round infection assay based on EGFP reporting for anti-HIV-1 drug discovery

    PubMed Central

    Soezi, Mahdieh; Memarnejadian, Arash; Aminzadeh, Saeed; Zabihollahi, Rezvan; Sadat, Seyed Mehdi; Amini, Safieh; Hekmat, Soheila; Aghasadeghi, Mohammad Reza

    2015-01-01

    Background: The rapid increase of HIV-1 strains resistant to current antiretroviral drugs is a challenge for successful AIDS therapy. This necessitates the development of novel drugs, and to this end, availability of screening systems for in vitro drug discovery is a priority. Herein, we report the modification of a previously developed system for increased sensitivity, ease of use, and cost-efficiency, based on the application of the EGFP marker. Methods: A PCR-amplified gfp gene (gfp) was cloned into pmzNL4-3, the plasmid already designed to produce single-cycle replicable virions, in frame with the reverse-transcriptase gene to construct the pmzNL4-3/GFP plasmid. GFP-mzNL4-3 pseudo-typed virions, as the first progeny viruses, were recovered from the culture supernatant of HEK293T cells co-transfected with pmzNL4-3/GFP and the helper plasmids pSPAX2 and pMD2G, which respectively encode HIV-1 Gag-Pol and vesicular stomatitis virus glycoprotein. Single-cycle replication and virion production were assessed by syncytia formation, p24 antigen assays, and electron and fluorescence microscopy. Results: The incorporation of EGFP into the viral particles allowed their quantification by fluorometry, flow-cytometry, and fluorescence microscopy; however, this modification did not affect the single-round infectivity or production rate of the GFP fluorescence-emitting virions. Conclusions: Our results certify the development of a rapid, inexpensive, and safe GFP-reporting single-cycle replicable system for anti-HIV drug discovery. Further experiments are needed to measure the validity and robustness of the assay. PMID:26989744

  6. Relatively High Prevalence of Drug Resistance Among Antiretroviral-Naive Patients from Henan, Central China

    PubMed Central

    Li, Lingnuo; Sun, Binlian; Zeng, Haiyan; Sun, Zhiwu; Sun, Guoqing

    2014-01-01

    Abstract To elucidate the prevalence of HIV-1 subtypes and transmitted drug resistance in Henan, central China, HIV-1-positive blood samples from 187 antiretroviral-naive patients were collected in our study from August 2009 to November 2010. Subtype B′ (92.0%, 172 of 187) remains the predominant HIV-1 subtype in Henan province and was prevalent in all risk populations and geographic regions. Of 98 pol sequences 67 (68.4%) harbored drug resistance mutations, and only 14 (14.3%, 14 of 98) sequences have mutations associated with significantly reduced phenotypic susceptibility to antiretroviral drugs. The unexpectedly high percentage of drug resistance in Henan province is mainly due to the prevalence of minor mutations in the protease and integrase regions, especially A71T/V and L68V/I/IM/LV. In all, we detected a relatively high prevalence of drug resistance with unique mutation distributions among antiretroviral-naive patients from Henan province. PMID:23800338

  7. In-Silico docking of HIV-1 integrase inhibitors reveals a novel drug type acting on an enzyme/DNA reaction intermediate

    PubMed Central

    Savarino, Andrea

    2007-01-01

    Background HIV-1 integrase (IN) is an emerging drug target, as IN strand transfer inhibitors (INSTIs) are proving potent antiretroviral agents in clinical trials. One credible theory sees INSTIs as docking at the cellular (acceptor) DNA-binding site after IN forms a transitional complex with viral (donor) DNA. However, mapping of the DNA and INSTI binding sites within the IN catalytic core domain (CCD) has been uncertain. Methods Structural superimpositions were conducted using the SWISS PDB and Cn3D free software. Docking simulations of INSTIs were run by a widely validated genetic algorithm (GOLD). Results Structural superimpositions suggested that a two-metal model for HIV-1 IN CCD in complex with small molecule, 1-(5-chloroindol-3-yl)-3-(tetrazoyl)-1,3-propandione-ene (5CITEP) could be used as a surrogate for an IN/viral DNA complex, because it allowed replication of contacts documented biochemically in viral DNA/IN complexes or displayed by a crystal structure of the IN-related enzyme Tn5 transposase in complex with transposable DNA. Docking simulations showed that the fitness of different compounds for the catalytic cavity of the IN/5CITEP complex significantly (P < 0.01) correlated with their 50% inhibitory concentrations (IC50s) in strand transfer assays in vitro. The amino acids involved in inhibitor binding matched those involved in drug resistance. Both metal binding and occupation of the putative viral DNA binding site by 5CITEP appeared to be important for optimal drug/ligand interactions. The docking site of INSTIs appeared to overlap with a putative acceptor DNA binding region adjacent to but distinct from the putative donor DNA binding site, and homologous to the nucleic acid binding site of RNAse H. Of note, some INSTIs such as 4,5-dihydroxypyrimidine carboxamides/N-Alkyl-5-hydroxypyrimidinone carboxamides, a highly promising drug class including raltegravir/MK-0518 (now in clinical trials), displayed interactions with IN reminiscent of those

  8. Extreme multidrug resistant HIV-1 protease with 20 mutations is resistant to novel protease inhibitors with P1′-pyrrolidinone or P2-tris-tetrahydrofuran

    PubMed Central

    Agniswamy, Johnson; Shen, Chen-Hsiang; Wang, Yuan-Fang; Ghosh, Arun K.; Rao, Kalapala Venkateswara; Xu, Chun-Xiao; Sayer, Jane M.; Louis, John M.; Weber, Irene T.

    2013-01-01

    Extreme drug resistant mutant of HIV-1 protease (PR) bearing 20 mutations (PR20) has been studied with the clinical inhibitor amprenavir (1) and two potent antiviral investigational inhibitors GRL-02031 (2) and GRL-0519 (3). Clinical inhibitors are >1000-fold less active on PR20 than on wild type enzyme, which is consistent with dissociation constants (KL) from isothermal titration calorimetry of 40 nM for 3, 178 nM for amprenavir, and 960 nM for 2. High resolution crystal structures of PR20-inhibitor complexes revealed altered interactions compared with the corresponding wild-type PR complexes in agreement with relative inhibition. Amprenavir lacks interactions due to PR20 mutations in the S2/S2′ subsites relative to PR. Inhibitors 2 and 3 lose interactions with Arg8′ in PR20 relative to the wild type enzyme since Arg8′ shifts to interact with mutated L10F side chain. Overall, inhibitor 3 compares favorably with darunavir in affinity for PR20 and shows promise for further development. PMID:23590295

  9. Drug Resistance in Leishmaniasis

    PubMed Central

    Chakravarty, Jaya; Sundar, Shyam

    2010-01-01

    The treatment options of leishmaniasis are limited and far from satisfactory. For more than 60 years, treatment of leishmaniasis has centered around pentavalent antimonials (Sbv). Widespread misuse has led to the emergence of Sbv resistance in the hyperendemic areas of North Bihar. Other antileishmanials could also face the same fate, especially in the anthroponotic cycle. The HIV/ visceral leishmaniasis (VL) coinfected patients are another potential source for the emergence of drug resistance. At present no molecular markers of resistance are available and the only reliable method for monitoring resistance of isolates is the technically demanding in vitro amastigote-macrophage model. As the armametrium of drugs for leishmaniasis is limited, it is important that effective monitoring of drug use and response should be done to prevent the spread of resistance. Regimens of simultaneous or sequential combinations should be seriously considered to limit the emergence of resistance. PMID:20606973

  10. CCR5 Gene Editing of Resting CD4+ T Cells by Transient ZFN Expression From HIV Envelope Pseudotyped Nonintegrating Lentivirus Confers HIV-1 Resistance in Humanized Mice

    PubMed Central

    Yi, Guohua; Choi, Jang Gi; Bharaj, Preeti; Abraham, Sojan; Dang, Ying; Kafri, Tal; Alozie, Ogechika; Manjunath, Manjunath N; Shankar, Premlata

    2014-01-01

    CCR5 disruption by zinc finger nucleases (ZFNs) is a promising method for HIV-1 gene therapy. However, successful clinical translation of this strategy necessitates the development of a safe and effective method for delivery into relevant cells. We used non-integrating lentivirus (NILV) for transient expression of ZFNs and pseudotyped the virus with HIV-envelope for targeted delivery to CD4+ T cells. Both activated and resting primary CD4+ T cells transduced with CCR5-ZFNs NILV showed resistance to HIV-1 infection in vitro. Furthermore, NILV transduced resting CD4+ T cells from HIV-1 seronegative individuals were resistant to HIV-1 challenge when reconstituted into NOD-scid IL2rγc null (NSG) mice. Likewise, endogenous virus replication was suppressed in NSG mice reconstituted with CCR5-ZFN–transduced resting CD4+ T cells from treatment naïve as well as ART-treated HIV-1 seropositive patients. Taken together, NILV pseudotyped with HIV envelope provides a simple and clinically viable strategy for HIV-1 gene therapy. PMID:25268698

  11. Relative resistance to HIV-1 infection of CD4 lymphocytes from persons who remain uninfected despite multiple high-risk sexual exposure.

    PubMed

    Paxton, W A; Martin, S R; Tse, D; O'Brien, T R; Skurnick, J; VanDevanter, N L; Padian, N; Braun, J F; Kotler, D P; Wolinsky, S M; Koup, R A

    1996-04-01

    Some individuals remain uninfected with human immunodeficiency virus type-1 (HIV-1) despite multiple high-risk sexual exposures. We studied a cohort of 25 subjects with histories of multiple high-risk sexual exposures to HIV-1 and found that their CD8+ lymphocytes had greater anti-HIV-1 activity than did CD8+ lymphocytes from nonexposed controls. Further studies indicated that their purified CD4+ lymphocytes were less susceptible to infection with multiple primary isolates of HIV-1 than were CD4+ lymphocytes from the nonexposed controls. This relative resistance to HIV-1 infection did not extend to T-cell line-adapted strains, was restricted by the envelope glycoprotein, was not explained by the cell surface density of CD4 molecules, but was associated with the activity of the C-C chemokines RANTES, MIP-1alpha, and MIP-1beta. This relative resistance of CD4+ lymphocytes may contribute to protection from HIV-1 in multiply exposed persons. PMID:8597950

  12. Drug targeting of HIV-1 RNA.DNA hybrid structures: thermodynamics of recognition and impact on reverse transcriptase-mediated ribonuclease H activity and viral replication.

    PubMed

    Li, Tsai-Kun; Barbieri, Christopher M; Lin, Hsin-Chin; Rabson, Arnold B; Yang, Gengcheng; Fan, Yupeng; Gaffney, Barbara L; Jones, Roger A; Pilch, Daniel S

    2004-08-01

    RNA degradation via the ribonuclease H (RNase H) activity of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) is a critical component of the reverse transcription process. In this connection, mutations of RT that inactivate RNase H activity result in noninfectious virus particles. Thus, interfering with the RNase H activity of RT represents a potential vehicle for the inhibition of HIV-1 replication. Here, we demonstrate an approach for inhibiting the RNase H activity of HIV-1 RT by targeting its RNA.DNA hybrid substrates. Specifically, we show that the binding of the 4,5-disubstituted 2-deoxystreptamine aminoglycosides, neomycin, paromomycin, and ribostamycin, to two different chimeric RNA-DNA duplexes, which mimic two distinct intermediates in the reverse transcription process, inhibits specific RT-mediated RNase H cleavage, with this inhibition being competitive in nature. UV melting and isothermal titration calorimetry studies reveal a correlation between the relative binding affinities of the three drugs for each of the chimeric RNA-DNA host duplexes and the relative extents to which the drugs inhibit RT-mediated RNase H cleavage of the duplexes. Significantly, this correlation also extends to the relative efficacies with which the drugs inhibit HIV-1 replication. In the aggregate, our results highlight a potential strategy for AIDS chemotherapy that should not be compromised by the unusual genetic diversity of HIV-1. PMID:15274628

  13. Site-specific Genome Editing in PBMCs With PLGA Nanoparticle-delivered PNAs Confers HIV-1 Resistance in Humanized Mice

    PubMed Central

    Schleifman, Erica B; McNeer, Nicole Ali; Jackson, Andrew; Yamtich, Jennifer; Brehm, Michael A; Shultz, Leonard D; Greiner, Dale L; Kumar, Priti; Saltzman, W Mark; Glazer, Peter M

    2013-01-01

    Biodegradable poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) encapsulating triplex-forming peptide nucleic acids (PNAs) and donor DNAs for recombination-mediated editing of the CCR5 gene were synthesized for delivery into human peripheral blood mononuclear cells (PBMCs). NPs containing the CCR5-targeting molecules efficiently entered PBMCs with low cytotoxicity. Deep sequencing revealed that a single treatment with the formulation resulted in a targeting frequency of 0.97% in the CCR5 gene and a low off-target frequency of 0.004% in the CCR2 gene, a 216-fold difference. NP-treated PBMCs efficiently engrafted immunodeficient NOD-scid IL-2rγ-/- mice, and the targeted CCR5 modification was detected in splenic lymphocytes 4 weeks posttransplantation. After infection with an R5-tropic strain of HIV-1, humanized mice with CCR5-NP–treated PBMCs displayed significantly higher levels of CD4+ T cells and significantly reduced plasma viral RNA loads compared with control mice engrafted with mock-treated PBMCs. This work demonstrates the feasibility of PLGA-NP–encapsulated PNA-based gene-editing molecules for the targeted modification of CCR5 in human PBMCs as a platform for conferring HIV-1 resistance. PMID:24253260

  14. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs.

    PubMed

    Alian, Akram; Griner, Sarah L; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D; Stroud, Robert M

    2009-05-19

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN-DNA complexes that form disulfide linkages between 5'-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  15. Molecular modeling studies of HIV-1 reverse transcriptase nonnucleoside inhibitors: total energy of complexation as a predictor of drug placement and activity.

    PubMed Central

    Kroeger Smith, M. B.; Rouzer, C. A.; Taneyhill, L. A.; Smith, N. A.; Hughes, S. H.; Boyer, P. L.; Janssen, P. A.; Moereels, H.; Koymans, L.; Arnold, E.

    1995-01-01

    binding and correlate with drug HOMO RHF/6-31G* energies. Modeling results are discussed with respect to the mechanism of complex formation and the design of nonnucleoside inhibitors that will be more effective against mutants of HIV-1 RT that are resistant to the currently available drugs. PMID:8535257

  16. Abacavir, an anti–HIV-1 drug, targets TDP1-deficient adult T cell leukemia

    PubMed Central

    Tada, Kohei; Kobayashi, Masayuki; Takiuchi, Yoko; Iwai, Fumie; Sakamoto, Takashi; Nagata, Kayoko; Shinohara, Masanobu; Io, Katsuhiro; Shirakawa, Kotaro; Hishizawa, Masakatsu; Shindo, Keisuke; Kadowaki, Norimitsu; Hirota, Kouji; Yamamoto, Junpei; Iwai, Shigenori; Sasanuma, Hiroyuki; Takeda, Shunichi; Takaori-Kondo, Akifumi

    2015-01-01

    Adult T cell leukemia (ATL) is an aggressive T cell malignancy caused by human T cell leukemia virus type 1 (HTLV-1) and has a poor prognosis. We analyzed the cytotoxic effects of various nucleoside analog reverse transcriptase inhibitors (NRTIs) for HIV-1 on ATL cells and found that abacavir potently and selectively kills ATL cells. Although NRTIs have minimal genotoxicities on host cells, the therapeutic concentration of abacavir induced numerous DNA double-strand breaks (DSBs) in the chromosomal DNA of ATL cells. DSBs persisted over time in ATL cells but not in other cell lines, suggesting impaired DNA repair. We found that the reduced expression of tyrosyl-DNA phosphodiesterase 1 (TDP1), a repair enzyme, is attributable to the cytotoxic effect of abacavir on ATL cells. We also showed that TDP1 removes abacavir from DNA ends in vitro. These results suggest a model in which ATL cells with reduced TDP1 expression are unable to excise abacavir incorporated into genomic DNA, leading to irreparable DSBs. On the basis of the above mechanism, we propose abacavir as a promising chemotherapeutic agent for ATL. PMID:26601161

  17. Pharmacokinetics of antiretroviral drug varies with formulation in the target population of children with HIV-1.

    PubMed

    Kasirye, P; Kendall, L; Adkison, K K; Tumusiime, C; Ssenyonga, M; Bakeera-Kitaka, S; Nahirya-Ntege, P; Mhute, T; Kekitiinwa, A; Snowden, W; Burger, D M; Gibb, D M; Walker, A S

    2012-02-01

    The bioequivalence of formulations is usually evaluated in healthy adult volunteers. In our study in 19 HIV-1-infected Ugandan children (1.8-4 years of age, weight 12 to <15 kg) receiving zidovudine, lamivudine, and abacavir solutions twice a day for ≥24 weeks, the use of scored tablets allowed comparison of plasma pharmacokinetics of oral solutions vs. tablets. Samples were collected 0, 1, 2, 4, 6, 8, and 12 h after each child's last morning dose of oral solution before changing to scored tablets of Combivir (coformulated zidovudine + lamivudine) and abacavir; this was repeated 4 weeks later. Dose-normalized area under curve (AUC)(0-12) and peak concentration (C(max)) for the tablet formulation were bioequivalent with those of the oral solution with respect to zidovudine and abacavir (e.g., dose-normalized geometric mean ratio (dnGMR) (tablet:solution) for zidovudine and abacavir AUC(0-12) were 1.01 (90% confidence interval (CI) 0.87-1.18) and 0.96 (0.83-1.12), respectively). However, lamivudine exposure was ~55% higher with the tablet formulation (AUC(0-12) dnGMR = 1.58 (1.37-1.81), C(max) dnGMR = 1.55 (1.33-1.81)). Although the clinical relevance of this finding is unclear, it highlights the impact of the formulation and the importance of conducting bioequivalence studies in target pediatric populations. PMID:22190066

  18. Impact of the growing HIV-1 epidemic on multidrug-resistant tuberculosis control in Latvia.

    PubMed

    Morozova, I; Riekstina, V; Sture, G; Wells, C; Leimane, V

    2003-09-01

    Latvia, a country with levels of multidrug-resistant (MDR) TB among the highest in the world, experienced a 58-fold increase in HIV seroprevalence among all persons tested in the country from 1996 through 2001. In addition, HIV seroprevalence among TB cases increased from 0.4% to 1.4%, and among MDR-TB cases from 0% to 5.6% from 1998 through 2001, potentially compromising gains made to date in controlling the country's MDR-TB epidemic. The following will be critical to the future of MDR-TB control in Latvia: containing HIV transmission in the country, particularly among injection drug users who comprised 72% of all HIV cases reported in the country by the end of 2001, as well as 81% of all MDR-TB cases co-infected with HIV; expanding capabilities to more rapidly detect and successfully treat patients with MDR-TB; developing mutual TB control strategies between the National TB and AIDS programs; and continuing to improve institutional infection control measures, particularly in hospitals and prisons where an increasing number of persons infected with HIV come into contact with persons with active MDR-TB. PMID:12971677

  19. Two M-T hook residues greatly improve the antiviral activity and resistance profile of the HIV-1 fusion inhibitor SC29EK

    PubMed Central

    2014-01-01

    Background Peptides derived from the C-terminal heptad repeat (CHR) of HIV-1 gp41 such as T20 (Enfuvirtide) and C34 are potent viral fusion inhibitors. We have recently found that two N-terminal residues (Met115 and Thr116) of CHR peptides form a unique M-T hook structure that can greatly enhance the binding and anti-HIV activity of inhibitors. Here, we applied two M-T hook residues to optimize SC29EK, an electrostatically constrained peptide inhibitor with a potent anti-HIV activity. Results The resulting peptide MT-SC29EK showed a dramatically increased binding affinity and could block the six-helical bundle (6-HB) formation more efficiently. As expected, MT-SC29EK potently inhibited HIV-1 entry and infection, especially against those T20- and SC29EK-resistant HIV-1 variants. More importantly, MT-SC29EK and its short form (MT-SC22EK) suffered from the difficulty to induce HIV-1 resistance during the in vitro selection, suggesting their high genetic barriers to the development of resistance. Conclusions Our studies have verified the M-T hook structure as a vital strategy to design novel HIV-1 fusion inhibitors and offered an ideal candidate for clinical development. PMID:24884671

  20. HIV-1 integrase: from biology to chemotherapeutics.

    PubMed

    Zeinalipour-Loizidou, Eriketi; Nicolaou, Christos; Nicolaides, Athanasios; Kostrikis, Leondios G

    2007-07-01

    AIDS has claimed the lives of 25 million people worldwide, an additional 40 million people are HIV-infected and new cases are being diagnosed every year. Despite the fact that HAART has moved AIDS from the category of terminal diseases to that of treatable chronic illnesses, its long-term therapeutic success may be compromised by the development of resistance to the currently used drugs. Despite the availability of RT, PR and fusion inhibitors, the development of further drugs such as inhibitors that target the third enzyme IN is essential for the clinical management of HIV-infected patients. The absence of cellular homolgues to IN and the unique nature of the reactions catalyzed by IN, make it an ideal target for drug design. Considerable progress towards designing HIV-1 IN inhibitors has been made over the last years and several lead compounds have been identified, synthesized and clinically studied. This review focuses on the existing knowledge of the biology of HIV-1 IN with emphasis on the mechanism of integration, structure and function and the technologies for measuring IN activity. This is followed by the current trends on designing HIV-1 IN inhibitors with the aid of molecular informatics and a review on the main classes of HIV-1 IN inhibitors reported this far with special emphasis on the clinical candidates. PMID:17627500

  1. Improving Viral Protease Inhibitors to Counter Drug Resistance.

    PubMed

    Kurt Yilmaz, Nese; Swanstrom, Ronald; Schiffer, Celia A

    2016-07-01

    Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design. PMID:27090931

  2. Nine Crystal Structures Determine the Substrate Envelope of the MDR HIV-1 Protease

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Brunzelle, Joseph; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-03-27

    Under drug selection pressure, emerging mutations render HIV-1 protease drug resistant, leading to the therapy failure in anti-HIV treatment. It is known that nine substrate cleavage site peptides bind to wild type (WT) HIV-1 protease in a conserved pattern. However, how the multidrug-resistant (MDR) HIV-1 protease binds to the substrate cleavage site peptides is yet to be determined. MDR769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, and 90) was selected for present study to understand the binding to its natural substrates. MDR769 HIV-1 protease was co-crystallized with nine substrate cleavage site hepta-peptides. Crystallographic studies show that MDR769 HIV-1 protease has an expanded substrate envelope with wide open flaps. Furthermore, ligand binding energy calculations indicate weaker binding in MDR769 HIV-1 protease-substrate complexes. These results help in designing the next generation of HIV-1 protease inhibitors by targeting the MDR HIV-1 protease.

  3. Molecular mechanisms of HIV-1 mother-to-child transmission and infection in neonatal target cells

    PubMed Central

    Ahmad, Nafees

    2010-01-01

    HIV-1 mother-to-child transmission (MTCT) occurs mainly at three stages, including prepartum, intrapartum and postpartum. Several maternal factors, including low CD4+ lymphocyte counts, high viral load, immune response, advanced disease status, smoking and abusing drugs have been implicated in an increased risk of HIV-1 MTCT. While use of antiretroviral therapy (ART) during pregnancy has significantly reduced the rate of MTCT, selective transmission of ART resistant mutants has been reported. Based on HIV-1 sequence comparison, the maternal HIV-1 minor genotypes with R5 phenotypes are predominantly transmitted to their infants and initially maintained in the infants with the same properties. Several HIV-1 structural, regulatory and accessory genes were highly conserved following MTCT. In addition, HIV-1 sequences from non-transmitting mothers are less heterogeneous compared with transmitting mothers, suggesting that a higher level of viral heterogeneity influences MTCT. Analysis of the immunologically relevant epitopes showed that variants evolved to escape the immune response that influenced HIV-1 MTCT. Several cytotoxic T lymphocyte (CTL) epitopes were identified in various HIV-1 genes that were conserved in HIV-1 mother-infant sequences, suggesting a role in MTCT. We have shown that HIV-1 replicates more efficiently in neonatal T-lymphocytes and monocytes/macrophages compared with adult cells, and this differential replication is influenced at the level of HIV-1 gene expression, which was due to differential expression of host factors, including transcriptional activators, signal transducers and cytokines in neonatal than adult cells. In addition, HIV-1 integration occurs in more actively transcribed genes in neonatal compared with adult cells, which may influence HIV-1 gene expression. The increased HIV-1 gene expression and replication in neonatal target cells contribute to a higher viral load and more rapid disease progression in neonates/infants than adults

  4. Antiretroviral Resistance After First-Line Antiretroviral Therapy Failure in Diverse HIV-1 Subtypes in the SECOND-LINE Study.

    PubMed

    Lam, Edward P; Moore, Cecilia L; Gotuzzo, Eduardo; Nwizu, Chidi; Kamarulzaman, Adeeba; Chetchotisakd, Ploenchan; van Wyk, Jean; Teppler, Hedy; Kumarasamy, Nagalingeswaran; Molina, Jean-Michel; Emery, Sean; Cooper, David A; Boyd, Mark A

    2016-09-01

    We investigate mutations and correlates according to HIV-1 subtype after virological failure (VF) of standard first-line antiretroviral therapy (ART) (non-nucleoside/nucleotide reverse transcriptase inhibitor [NNRTI] +2 nucleoside/nucleotide reverse transcriptase inhibitor [N(t)RTI]). SECOND-LINE study participants were assessed at baseline for HIV-1 subtype, demographics, HIV-1 history, ART exposure, viral load (VL), CD4(+) count, and genotypic ART resistance. We used backward stepwise multivariate regression (MVR) to assess associations between baseline variables and presence of ≥3 N(t)RTI mutations, ≥1 NNRTI mutation, ≥3 thymidine analog-N(t)RTI [ta-N(t)RTI] mutations (TAMs), the K65/K70 mutation, and predicted etravirine (ETV)/rilpivirine (RPV) activity. The inclusion p-value for MVR was p < .2. The exclusion p-value from stepwise elimination was p > .05. Of 541 participants, 491 (91%) had successfully characterized baseline viral isolates. Subtype distribution: B (n = 123, 25%), C (n = 202, 41%), CRF01_AE (n = 109, 22%), G (n = 25, 5%), and CRF02_AG (n = 27, 5%). Baseline CD4(+) 200-394 cells/mm(3) were associated with <3 N(t)RTI mutations (OR = 0.47; 95% CI 0.29-0.77; p = .003), absence of the K65/K70 mutation (OR = 0.43; 95% CI 0.26-0.73; p = .002), and higher ETV sensitivity (OR = 0.52; 95% CI 0.35-0.78; p = .002). Recent tenofovir (TDF) use was associated with K65/K70 mutations (OR = 8.91; 95% CI 5.00-15.85; p < .001). Subtype CRF01_AE was associated with ≥3 N(t)RTI mutations (OR = 2.34; 95% CI 1.31-4.17; p = .004) and higher RPV resistance (OR = 2.13; 95% CI 1.30-3.49; p = .003), and subtype C was associated with <3 TAMs (OR = 0.45; 95% CI 0.21-0.99; p = .015). Subtypes CRF01_AE (OR = 2.46; 95% CI 1.26-4.78; p = .008) and G (OR = 4.77; 95% CI 1.44-15.76; p = .01) were associated with K65/K70 mutations. Higher VL at confirmed first-line VF was

  5. A Newly Emerging HIV-1 Recombinant Lineage (CRF58_01B) Disseminating among People Who Inject Drugs in Malaysia

    PubMed Central

    Chow, Wei Zhen; Takebe, Yutaka; Syafina, Nur Ezreen; Prakasa, Malarvelli Soorya; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2014-01-01

    The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B–D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes—subtype B (including subtype B′, the Thai variant of subtype B), CRF01_AE, and CRF33_01B—have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B′ and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention. PMID:24465513

  6. A newly emerging HIV-1 recombinant lineage (CRF58_01B) disseminating among people who inject drugs in Malaysia.

    PubMed

    Chow, Wei Zhen; Takebe, Yutaka; Syafina, Nur Ezreen; Prakasa, Malarvelli Soorya; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2014-01-01

    The HIV epidemic is primarily characterised by the circulation of HIV-1 group M (main) comprising of 11 subtypes and sub-subtypes (A1, A2, B-D, F1, F2, G, H, J, and K) and to date 55 circulating recombinant forms (CRFs). In Southeast Asia, active inter-subtype recombination involving three main circulating genotypes--subtype B (including subtype B', the Thai variant of subtype B), CRF01_AE, and CRF33_01B--have contributed to the emergence of novel unique recombinant forms. In the present study, we conducted the molecular epidemiological surveillance of HIV-1 gag-RT genes among 258 people who inject drugs (PWIDs) in Kuala Lumpur, Malaysia, between 2009 and 2011 whereby a novel CRF candidate was recently identified. The near full-length genome sequences obtained from six epidemiologically unlinked individuals showed identical mosaic structures consisting of subtype B' and CRF01_AE, with six unique recombination breakpoints in the gag-RT, pol, and env regions. Among the high-risk population of PWIDs in Malaysia, which was predominantly infected by CRF33_01B (>70%), CRF58_01B circulated at a low but significant prevalence (2.3%, 6/258). Interestingly, the CRF58_01B shared two unique recombination breakpoints with other established CRFs in the region: CRF33_01B, CRF48_01B, and CRF53_01B in the gag gene, and CRF15_01B (from Thailand) in the env gene. Extended Bayesian Markov chain Monte Carlo sampling analysis showed that CRF58_01B and other recently discovered CRFs were most likely to have originated in Malaysia, and that the recent spread of recombinant lineages in the country had little influence from neighbouring countries. The isolation, genetic characterization, and evolutionary features of CRF58_01B among PWIDs in Malaysia signify the increasingly complex HIV-1 diversity in Southeast Asia that may hold an implication on disease treatment, control, and prevention. PMID:24465513

  7. Mitochondrial DNA Haplogroup A Decreases the Risk of Drug Addiction but Conversely Increases the Risk of HIV-1 Infection in Chinese Addicts.

    PubMed

    Zhang, A-Mei; Hu, Qiu-Xiang; Liu, Feng-Liang; Bi, Rui; Yang, Bi-Qing; Zhang, Wen; Guo, Hao; Logan, Ian; Zheng, Yong-Tang; Yao, Yong-Gang

    2016-08-01

    Drug addiction is one of the most serious social problems in the world today and addicts are always at a high risk of acquiring HIV infection. Mitochondrial impairment has been reported in both drug addicts and in HIV patients undergoing treatment. In this study, we aimed to investigate whether mitochondrial DNA (mtDNA) haplogroup could affect the risk of drug addiction and HIV-1 infection in Chinese. We analyzed mtDNA sequence variations of 577 Chinese intravenous drug addicts (289 with HIV-1 infection and 288 without) and compared with 2 control populations (n = 362 and n = 850). We quantified the viral load in HIV-1-infected patients with and without haplogroup A status and investigated the potential effect of haplogroup A defining variants m.4824A > G and m.8794C > T on the cellular reactive oxygen species (ROS) levels by using an allotopic expression assay. mtDNA haplogroup A had a protective effect against drug addiction but appeared to confer an increased risk of HIV infection in addicts. HIV-1-infected addicts with haplogroup A had a trend for a higher viral load, although the mean viral load was similar between carriers of haplogroup A and those with other haplogroup. Hela cells overexpressing allele m.8794 T showed significantly decreased ROS levels as compared to cells with the allele m.8794C (P = 0.03). Our results suggested that mtDNA haplogroup A might protect against drug addiction but increase the risk of HIV-1 infection. The contradictory role of haplogroup A might be caused by an alteration in mitochondrial function due to a particular mtDNA ancestral variant. PMID:26162319

  8. Integrase inhibitor (INI) genotypic resistance in treatment-naive and raltegravir-experienced patients infected with diverse HIV-1 clades

    PubMed Central

    Doyle, Tomas; Dunn, David T.; Ceccherini-Silberstein, Francesca; De Mendoza, Carmen; Garcia, Frederico; Smit, Erasmus; Fearnhill, Esther; Marcelin, Anne-Genevieve; Martinez-Picado, Javier; Kaiser, Rolf; Geretti, Anna Maria

    2015-01-01

    Objectives The aim of this study was to characterize the prevalence and patterns of genotypic integrase inhibitor (INI) resistance in relation to HIV-1 clade. Methods The cohort comprised 533 INI-naive subjects and 255 raltegravir recipients with viraemia who underwent integrase sequencing in routine care across Europe, including 134/533 (25.1%) and 46/255 (18.0%), respectively, with non-B clades (A, C, D, F, G, CRF01, CRF02, other CRFs, complex). Results No major INI resistance-associated mutations (RAMs) occurred in INI-naive subjects. Among raltegravir recipients with viraemia (median 3523 HIV-1 RNA copies/mL), 113/255 (44.3%) had one or more major INI RAMs, most commonly N155H (45/255, 17.6%), Q148H/R/K + G140S/A (35/255, 13.7%) and Y143R/C/H (12/255, 4.7%). In addition, four (1.6%) raltegravir recipients showed novel mutations at recognized resistance sites (E92A, S147I, N155D, N155Q) and novel mutations at other integrase positions that were statistically associated with raltegravir exposure (K159Q/R, I161L/M/T/V, E170A/G). Comparing subtype B with non-B clades, Q148H/R/K occurred in 42/209 (20.1%) versus 2/46 (4.3%) subjects (P = 0.009) and G140S/A occurred in 36/209 (17.2%) versus 1/46 (2.2%) subjects (P = 0.005). Intermediate- to high-level cross-resistance to twice-daily dolutegravir was predicted in 40/255 (15.7%) subjects, more commonly in subtype B versus non-B clades (39/209, 18.7% versus 1/46, 2.2%; P = 0.003). A glycine (G) to serine (S) substitution at integrase position 140 required one nucleotide change in subtype B and two nucleotide changes in all non-B clades. Conclusions No major INI resistance mutations occurred in INI-naive subjects. Reduced occurrence of Q148H/R/K + G140S/A was seen in non-B clades versus subtype B, and was explained by the higher genetic barrier to the G140S mutation observed in all non-B clades analysed. PMID:26311843

  9. Nevirapine Resistance in Previously Nevirapine-Unexposed HIV-1-Infected Kenyan Infants Initiating Early Antiretroviral Therapy.

    PubMed

    Chohan, Bhavna H; Tapia, Kenneth; Benki-Nugent, Sarah; Khasimwa, Brian; Ngayo, Musa; Maleche-Obimbo, Elizabeth; Wamalwa, Dalton; Overbaugh, Julie; John-Stewart, Grace

    2015-08-01

    Nevirapine (NVP) resistance occurs frequently in infants following NVP use in prevention of mother-to-child transmission (PMTCT) regimens. However, among previously NVP-unexposed infants treated with NVP-antiretroviral therapy (ART), the development and impact of NVP resistance have not been well characterized. In a prospective clinical trial providing early ART to HIV-infected infants <5 months of age in Kenya (OPH03 study), we followed NVP-unexposed infants who initiated NVP-ART for 12 months. Viral loads were assessed and resistance determined using a population-based genotypic resistance assay. Of 99 infants screened, 33 had no prior NVP exposure, 22 of whom were initiated on NVP-ART. Among 19 infants with follow-up, seven (37%) infants developed resistance: one at 3 months and six at 6 months after ART initiation. The cumulative probability of NVP resistance was 5.9% at 3 months and 43.5% at 6 months. Baseline HIV RNA levels (p=0.7) and other characteristics were not associated with developing resistance. Post-ART, higher virus levels at visits preceding the detection of resistance were significantly associated with increased detection of resistance (p=0.004). Virus levels after 6 and 12 months of ART were significantly higher in infants with resistance than those without (p=0.007, p=0.030, respectively). Among infants without previous NVP exposure, development of NVP resistance was frequent and was associated with virologic failure during the first year of ART. Earlier development of NVP resistance in infants than in adults initiating NVP-ART may be due to longer viremia following ART or inadequate NVP levels resulting from NVP lead-in dosing. The development of NVP resistance may, in part, explain the superiority of protease inhibitor-based ART in infants. PMID:25819584

  10. Drug resistance in leishmaniasis.

    PubMed

    Croft, Simon L; Sundar, Shyam; Fairlamb, Alan H

    2006-01-01

    Leishmaniasis is a complex disease, with visceral and cutaneous manifestations, and is caused by over 15 different species of the protozoan parasite genus Leishmania. There are significant differences in the sensitivity of these species both to the standard drugs, for example, pentavalent antimonials and miltefosine, and those on clinical trial, for example, paromomycin. Over 60% of patients with visceral leishmaniasis in Bihar State, India, do not respond to treatment with pentavalent antimonials. This is now considered to be due to acquired resistance. Although this class of drugs has been used for over 60 years for leishmaniasis treatment, it is only in the past 2 years that the mechanisms of action and resistance have been identified, related to drug metabolism, thiol metabolism, and drug efflux. With the introduction of new therapies, including miltefosine in 2002 and paromomycin in 2005-2006, it is essential that there be a strategy to prevent the emergence of resistance to new drugs; combination therapy, monitoring of therapy, and improved diagnostics could play an essential role in this strategy. PMID:16418526

  11. [d4U]-spacer-[HI-236] double-drug inhibitors of HIV-1 reverse-transcriptase

    PubMed Central

    Younis, Yassir; Hunter, Roger; Muhanji, Clare I; Hale, Ian; Singh, Rajinder; Bailey, Christopher M.; Sullivan, Todd S.; Anderson, Karen S.

    2010-01-01

    Four double-drug HIV NRTI / NNRTI inhibitors 15a-d of the type [d4U]-spacer-[HI-236] in which the spacer is varied as 1-butynyl (15a), propargyl-1-PEG (15b), propargyl-2-PEG (15c) and propargyl-4-PEG (15d) have been synthesized and biologically evaluated as RT inhibitors against HIV-1. The key step in their synthesis involved a Sonogashira coupling of 5-iodo d4U's benzoate with an alkynylated tethered HI-236 precursor followed by introduction of the HI-236 thiourea functionality. Biological evaluation in both cell-culture (MT-2 cells) as well as using an in vitro RT assay revealed 15a-c to be all more active than d4T. However, overall the results indicate the derivatives are acting as chain-extended NNRTIs in which for 15b-d the nucleoside component is likely situated outside of the pocket but with no evidence for any synergistic double binding between the NRTI and NNRTI sites. This is attributed, in part, to the lack of phosphorylation of the nucleoside component of the double drug as a result of kinase recognition failure, which is not improved upon with the phosphoramidate of 15d incorporating a 4-PEG spacer. PMID:20605472

  12. Poly(ethylene glycol) enclatherated pectin-mucin submicron matrices for intravaginal anti-HIV-1 drug delivery.

    PubMed

    Mashingaidze, Felix; Choonara, Yahya E; Kumar, Pradeep; du Toit, Lisa C; Maharaj, Vinesh; Buchmann, Eckhart; Pillay, Viness

    2016-04-30

    This paper explores the potential of polyethylene glycol enclatherated pectin-mucin (PEG-encl-PEC:MUC) submicron matrices (SMMs) as an intravaginal drug delivery system capable of delivering an anti-HIV-1 agent (zidovudine; AZT) over a prolonged duration. A three factor and three level (3(3)) Box-Behnken statistical design was employed to optimize the SMMs. Optimized PEG-encl-PEC:MUC SMMs prepared as a stable W/O emulsion (determined by the degree of reversible colloidal phenomena) were spherical with a mean particle size of 270.6±5.533nm and mean zeta potential of -34.4±0.539mV. The microencapsulation of AZT and the hydrogen bonding mediated shielding of AZT by SMMs was confirmed by Fourier Transform Infrared (FTIR) analysis. The thermochemical (differential scanning calorimetry and thermogravimetric analysis) data proposed that Ca(2+)-based macromolecular ionic crosslinking as well as the intermolecular interactions may be responsible for the thermal stability of the delivery system. The partially amorphous nature of drug-loaded SMMs, as confirmed by X-ray diffraction patterns, further strengthened the matricization of AZT into the pectin-mucin matrix. In vitro drug release studies from the SMMs showed approximately 91% zidovudine release in simulated vaginal fluid (SVF) and 94% in phosphate buffered saline (PBS) in 24h. The mean dissolution time (MDT) of zidovudine from the SMMs was 5.974h. The attainment of required dimensional structure and drug release profiles from SMMs highlights the potential of their inclusion into a secondary carrier system for extended and controlled intravaginal stay. PMID:26943973

  13. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro. PMID:26714703

  14. Trends of HIV-1 risk reduction among initiates into intravenous drug use 1982-1987.

    PubMed

    Vlahov, D; Anthony, J C; Celentano, D; Solomon, L; Chowdhury, N

    1991-01-01

    To assess how injection practices may have changed during the course of the AIDS epidemic, active intravenous drug users (IVDUs) recruited from the community were asked to report year of first injection as well as specific details about the first 3 months after initial injection: frequencies of injection, using sterile needles, sharing needles and other equipment. For the analysis, the users were sorted into successive cohorts of initiation (by year of first injection), and tests for trends were completed using Mantel-Haenszel statistics. Among the 421 IVDUs who reported first injection between 1982 and 1987, the use of new sterile needles to self-administer drugs increased (p less than .05) along with its corollary behavior (i.e., using equipment one is sure that no one else had used before). Conversely, there was a decrease in the proportion of those who always used equipment previously used by another IVDU (p less than .05) and a decrease in the number of needle-sharing partners (p less than .01). Over the 6 years, heroin as first drug decreased and cocaine increased (p less than .01). Although these data are from a cross-sectional interview study, they suggest a shift toward lower risk practices among new IVDUs between 1982 and 1987. The shift from heroin to cocaine is compatible with other evidence on the cocaine epidemic. PMID:2038982

  15. Inherited resistance to HIV-1 conferred by an inactivating mutation in CC chemokine receptor 5: studies in populations with contrasting clinical phenotypes, defined racial background, and quantified risk.

    PubMed Central

    Zimmerman, P. A.; Buckler-White, A.; Alkhatib, G.; Spalding, T.; Kubofcik, J.; Combadiere, C.; Weissman, D.; Cohen, O.; Rubbert, A.; Lam, G.; Vaccarezza, M.; Kennedy, P. E.; Kumaraswami, V.; Giorgi, J. V.; Detels, R.; Hunter, J.; Chopek, M.; Berger, E. A.; Fauci, A. S.; Nutman, T. B.; Murphy, P. M.

    1997-01-01

    BACKGROUND: CC chemokine receptor 5 (CCR5) is a cell entry cofactor for macrophage-tropic isolates of human immunodeficiency virus-1 (HIV-1). Recently, an inactive CCR5 allele (designated here as CCR5-2) was identified that confers resistance to HIV-1 infection in homozygotes and slows the rate of progression to AIDS in heterozygotes. The reports conflict on the effect of heterozygous CCR5-2 on HIV-1 susceptibility, and race and risk levels have not yet been fully analyzed. Here we report our independent identification of CCR5-2 and test its effects on HIV-1 pathogenesis in individuals with contrasting clinical outcomes, defined race, and quantified risk. MATERIALS AND METHODS: Mutant CCR5 alleles were sought by directed heteroduplex analysis of genomic DNA from random blood donors. Genotypic frequencies were then determined in (1) random blood donors from North America, Asia, and Africa; (2) HIV-1+ individuals; and (3) highly exposed-seronegative homosexuals with quantified risk. RESULTS: CCR5-2 was the only mutant allele found. It was common in Caucasians, less common in other North American racial groups, and not detected in West Africans or Tamil Indians. Homozygous CCR5-2 frequencies differed reciprocally in highly exposed-seronegative (4.5%, n = 111) and HIV-1-seropositive (0%, n = 614) Caucasians relative to Caucasian random blood donors (0.8%, n = 387). This difference was highly significant (p < 0.0001). By contrast, heterozygous CCR5-2 frequencies did not differ significantly in the same three groups (21.6, 22.6, and 21.7%, respectively). A 55% increase in the frequency of heterozygous CCR5-2 was observed in both of two cohorts of Caucasian homosexual male, long-term nonprogressors compared with other HIV-1+ Caucasian homosexuals (p = 0.006) and compared with Caucasian random blood donors. Moreover, Kaplan-Meier estimates indicated that CCR5-2 heterozygous seroconvertors had a 52.6% lower risk of developing AIDS than homozygous wild-type seroconvertors

  16. Trends in Genotypic HIV-1 Antiretroviral Resistance between 2006 and 2012 in South African Patients Receiving First- and Second-Line Antiretroviral Treatment Regimens

    PubMed Central

    Van Zyl, Gert U.; Liu, Tommy F.; Claassen, Mathilda; Engelbrecht, Susan; de Oliveira, Tulio; Preiser, Wolfgang; Wood, Natasha T.; Travers, Simon; Shafer, Robert W.

    2013-01-01

    Objectives South Africa’s national antiretroviral (ARV) treatment program expanded in 2010 to include the nucleoside reverse transcriptase (RT) inhibitors (NRTI) tenofovir (TDF) for adults and abacavir (ABC) for children. We investigated the associated changes in genotypic drug resistance patterns in patients with first-line ARV treatment failure since the introduction of these drugs, and protease inhibitor (PI) resistance patterns in patients who received ritonavir-boosted lopinavir (LPV/r)-containing therapy. Methods We analysed ARV treatment histories and HIV-1 RT and protease mutations in plasma samples submitted to the Tygerberg Academic Hospital National Health Service Laboratory. Results Between 2006 and 2012, 1,667 plasma samples from 1,416 ARV-treated patients, including 588 children and infants, were submitted for genotypic resistance testing. Compared with 720 recipients of a d4T or AZT-containing first-line regimen, the 153 recipients of a TDF-containing first-line regimen were more likely to have the RT mutations K65R (46% vs 4.0%; p<0.001), Y115F (10% vs. 0.6%; p<0.001), L74VI (8.5% vs. 1.8%; p<0.001), and K70EGQ (7.8% vs. 0.4%) and recipients of an ABC-containing first-line regimen were more likely to have K65R (17% vs 4.0%; p<0.001), Y115F (30% vs 0.6%; p<0.001), and L74VI (56% vs 1.8%; p<0.001). Among the 490 LPV/r recipients, 55 (11%) had ≥1 LPV-resistance mutations including 45 (9.6%) with intermediate or high-level LPV resistance. Low (20 patients) and intermediate (3 patients) darunavir (DRV) cross resistance was present in 23 (4.6%) patients. Conclusions Among patients experiencing virological failure on a first-line regimen containing two NRTI plus one NNRTI, the use of TDF in adults and ABC in children was associated with an increase in four major non- thymidine analogue mutations. In a minority of patients, LPV/r-use was associated with intermediate or high-level LPV resistance with predominantly low-level DRV cross-resistance. PMID

  17. Catalytically-active complex of HIV-1 integrase with a viral DNA substrate binds anti-integrase drugs

    PubMed Central

    Alian, Akram; Griner, Sarah L.; Chiang, Vicki; Tsiang, Manuel; Jones, Gregg; Birkus, Gabriel; Geleziunas, Romas; Leavitt, Andrew D.; Stroud, Robert M.

    2009-01-01

    HIV-1 integration into the host cell genome is a multistep process catalyzed by the virally-encoded integrase (IN) protein. In view of the difficulty of obtaining a stable DNA-bound IN at high concentration as required for structure determination, we selected IN–DNA complexes that form disulfide linkages between 5′-thiolated DNA and several single mutations to cysteine around the catalytic site of IN. Mild reducing conditions allowed for selection of the most thermodynamically-stable disulfide-linked species. The most stable complexes induce tetramer formation of IN, as happens during the physiological integration reaction, and are able to catalyze the strand transfer step of retroviral integration. One of these complexes also binds strand-transfer inhibitors of HIV antiviral drugs, making it uniquely valuable among the mutants of this set for understanding portions of the integration reaction. This novel complex may help define substrate interactions and delineate the mechanism of action of known integration inhibitors. PMID:19416821

  18. Mutations Proximal to Sites of Autoproteolysis and the α-Helix That Co-evolve under Drug Pressure Modulate the Autoprocessing and Vitality of HIV-1 Protease.

    PubMed

    Louis, John M; Deshmukh, Lalit; Sayer, Jane M; Aniana, Annie; Clore, G Marius

    2015-09-01

    N-Terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multidrug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67, and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the properties mentioned above and observe no strict correlation. A 10-35-fold decrease in the cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by an only ∼4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing more drastically (>300-fold) compared to that of TFR-PR(PR20-3) because of the cumulative effect of eight noncluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the properties mentioned above governing PR regulation and function. PMID:26266692

  19. Identification of potent maturation inhibitors against HIV-1 clade C.

    PubMed

    Timilsina, Uddhav; Ghimire, Dibya; Timalsina, Bivek; Nitz, Theodore J; Wild, Carl T; Freed, Eric O; Gaur, Ritu

    2016-01-01

    Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic. PMID:27264714

  20. Identification of potent maturation inhibitors against HIV-1 clade C

    PubMed Central

    Timilsina, Uddhav; Ghimire, Dibya; Timalsina, Bivek; Nitz, Theodore J.; Wild, Carl T.; Freed, Eric O.; Gaur, Ritu

    2016-01-01

    Antiretroviral therapy has led to a profound improvement in the clinical care of HIV-infected patients. However, drug tolerability and the evolution of drug resistance have limited treatment options for many patients. Maturation inhibitors are a new class of antiretroviral agents for treatment of HIV-1. They act by interfering with the maturation of the virus by blocking the last step in Gag processing: the cleavage of the capsid-spacer peptide 1 (CA-SP1) intermediate to mature CA by the viral protease (PR). The first-in-class maturation inhibitor bevirimat (BVM) failed against a subset of HIV-1 isolates in clinical trials due to polymorphisms present in the CA-SP1 region of the Gag protein. Sequence analysis indicated that these polymorphisms are more common in non-clade B strains of HIV-1 such as HIV-1 clade C. Indeed, BVM was found to be ineffective against HIV-1 clade C molecular clones tested in this study. A number of BVM analogs were synthesized by chemical modifications at the C-28 position to improve its activity. The new BVM analogs displayed potent activity against HIV-1 clade B and C and also reduced infectivity of the virus. This study identifies novel and broadly active BVM analogs that may ultimately demonstrate efficacy in the clinic. PMID:27264714

  1. Heterosexual Transmission of Subtype C HIV-1 Selects Consensus-Like Variants without Increased Replicative Capacity or Interferon-α Resistance

    PubMed Central

    Fenton-May, Angharad E.; Dilernia, Dario A.; Kilembe, William; Allen, Susan A.; Borrow, Persephone; Hunter, Eric

    2015-01-01

    Heterosexual transmission of HIV-1 is characterized by a genetic bottleneck that selects a single viral variant, the transmitted/founder (TF), during most transmission events. To assess viral characteristics influencing HIV-1 transmission, we sequenced 167 near full-length viral genomes and generated 40 infectious molecular clones (IMC) including TF variants and multiple non-transmitted (NT) HIV-1 subtype C variants from six linked heterosexual transmission pairs near the time of transmission. Consensus-like genomes sensitive to donor antibodies were selected for during transmission in these six transmission pairs. However, TF variants did not demonstrate increased viral fitness in terms of particle infectivity or viral replicative capacity in activated peripheral blood mononuclear cells (PBMC) and monocyte-derived dendritic cells (MDDC). In addition, resistance of the TF variant to the antiviral effects of interferon-α (IFN-α) was not significantly different from that of non-transmitted variants from the same transmission pair. Thus neither in vitro viral replicative capacity nor IFN-α resistance discriminated the transmission potential of viruses in the quasispecies of these chronically infected individuals. However, our findings support the hypothesis that within-host evolution of HIV-1 in response to adaptive immune responses reduces viral transmission potential. PMID:26378795

  2. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    PubMed

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. PMID:25462990

  3. Selectivity for strand-transfer over 3'-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme-DNA interactions in the active site.

    PubMed

    Métifiot, Mathieu; Johnson, Barry C; Kiselev, Evgeny; Marler, Laura; Zhao, Xue Zhi; Burke, Terrence R; Marchand, Christophe; Hughes, Stephen H; Pommier, Yves

    2016-08-19

    Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed. PMID:27369381

  4. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible and inhibits HIV-1 infectivity

    PubMed Central

    Zhou, Jiehua; Satheesan, Sangeetha; Li, Haitang; Weinberg, Marc S.; Morris, Kevin V.; Burnett, John; Rossi, John

    2015-01-01

    SUMMARY The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T-cells and macrophages that serves as a co-receptor for macrophage-tropic HIV-1. Loss of CCR5 is associated with resistance to HIV-1. Here we combine the live cell-based SELEX with high throughput sequencing technology to generate CCR5 RNA aptamers capable of specifically targeting HIV-1 susceptible cells (as siRNA delivery agent) and inhibiting HIV-1 infectivity (as antiviral agent) via block of the CCR5 required for HIV-1 to enter cells. One of the best candidates, G-3, efficiently bound and was internalized into human CCR5 expressing cells. The G-3 specifically neutralized R5 virus infection in primary peripheral blood mononuclear cells, and in vivo generated human CD4+ T cells with a nanomolar IC50. G-3 was also capable of transferring functional siRNAs to CCR5 expressing cells. Collectively, the cell-specific, internalizing, CCR5-targeted aptamers and aptamer-siRNA conjugates offer promise for overcoming some of the current challenges of drug resistance in HIV-1 by providing cell-type- or tissue-specific delivery of various therapeutic moieties. PMID:25754473

  5. A novel genotype encoding a single amino acid insertion and five other substitutions between residues 64 and 74 of the HIV-1 reverse transcriptase confers high-level cross-resistance to nucleoside reverse transcriptase inhibitors. Abacavir CNA2007 International Study Group.

    PubMed

    Rakik, A; Ait-Khaled, M; Griffin, P; Thomas, T A; Tisdale, M; Kleim, J P

    1999-10-01

    We investigated HIV-1 reverse transcriptase (RT) polymorphisms of plasma isolates from 98 HIV-1-infected study subjects with >2 years of antiretroviral therapy who were failing their current protease inhibitor (PI)-containing regimen. In 1 patient, we detected a virus with a heavily mutated beta3-beta4 connecting loop of the HIV-1 RT fingers subdomain, consisting of a single aspartate codon insertion between positions 69 and 70 and five additional variations: 64N, K65, K66, 67G, 68Y, T69, Ins D, 70R, W71, R72, K73, 74I. Mutants with the recently described 2-aa insertions between codons 68 and 70 of RT were detected in another 3 patients. Among the four isolates with the 1- or 2-aa insertions, the novel genotype was the most refractory to therapy and displayed the highest level of phenotypic resistance to nucleoside reverse transcriptase inhibitors (NRTIs). Follow-up samples demonstrated that the novel mutant represents a stable genetic rearrangement and that the amino acid insertions can coexist with nonnucleoside analogue reverse transcriptase inhibitors (NNRTI) mutations resulting in phenotypic resistance to both NRTIs and NNRTIs. An increasing number of HIV-1 isolates containing various insertions in the beta3-beta4 hairpin of the HIV-1 RT fingers subdomain appear to emerge after prolonged therapy with different NRTIs, and these polymorphisms can confer multiple drug resistance against NRTIs. PMID:10843527

  6. Subtype diversity associated with the development of HIV-1 resistance to integrase inhibitors.

    PubMed

    Brenner, Bluma G; Lowe, Matthew; Moisi, Daniela; Hardy, Isabelle; Gagnon, Simon; Charest, Hugues; Baril, Jean Guy; Wainberg, Mark A; Roger, Michel

    2011-05-01

    We used genotypic and phylogenetic analysis to determine integrase diversity among subtypes, and studied natural polymorphisms and mutations implicated in resistance to integrase inhibitors (INI) in treatment-naïve persons (n = 220) and -experienced individuals (n = 24). Phylogenetics revealed 7 and 10% inter-subtype diversity in the integrase and reverse transcriptase (RT)/protease regions, respectively. Integrase sequencing identified a novel A/B recombinant in which all viruses in a male-sex-male (MSM) transmission cluster (n = 12) appeared to possess subtype B in integrase and subtype A in the remainder of the pol region. Natural variations and signature polymorphisms were observed at codon positions 140, 148, 151, 157, and 160 among HIV subtypes. These variations predicted higher genetic barriers to G140S and G140C in subtypes C, CRF02_AG, and A/CRF01_AE, as well as higher genetic barriers toward acquisition of V151I in subtypes CRF02_AG and A/CRF01_AE. The E157Q and E160Q mutational motif was observed in 35% of INI-naïve patients harboring subtype C infections, indicating intra-subtype variations. Thirteen patients failed raltegravir (RAL)-containing regimens within 8 ± 1 months, in association with the major Q148K/R/H and G140A/S (n = 8/24) or N155H (n = 5/24) mutational pathways. Of note, the remaining patients on RAL regimens for 14 ± 3 months harbored no or only minor integrase mutations/polymorphisms (T66I, T97A, H114P, S119P, A124S, G163R, I203M, R263K). These results demonstrate the importance of understanding subtype variability in the development of resistance to INIs. PMID:21360548

  7. Genotypic Resistance Tests Sequences Reveal the Role of Marginalized Populations in HIV-1 Transmission in Switzerland

    PubMed Central

    Shilaih, Mohaned; Marzel, Alex; Yang, Wan Lin; Scherrer, Alexandra U.; Schüpbach, Jörg; Böni, Jürg; Yerly, Sabine; Hirsch, Hans H.; Aubert, Vincent; Cavassini, Matthias; Klimkait, Thomas; Vernazza, Pietro L.; Bernasconi, Enos; Furrer, Hansjakob; Günthard, Huldrych F.; Kouyos, Roger; Battegay, Manuel; Braun, Dominique; Bucher, Heiner; Burton-Jeangros, Claudine; Calmy, Alexandra; Dollenmaier, Günter; Egger, Matthias; Elzi, Luigia; Fehr, Jan; Fellay, Jaque; Fux, Christoph; Gorgievski, Meri; Haerry, David; Hasse, Barbara; Hoffmann, Matthias; Hösli, Irene; Kahlert, Christian; Kaiser, Laurent; Keiser, Olivia; Kovari, Helen; Ledergerber, Bruno; Martinetti, Gladys; de Tejada, Begoña Martinez; Marzolini, Catia; Metzner, Karin; Müller, Nicolas; Nadal, David; Nicca, Dunja; Pantaleo, Giuseppe; Rauch, Andre; Regenass, Stephan; Rudin, Christoph; Schöni-Affolter, Franziska; Schmid, Patrick; Speck, Roberto; Stöckle, Marcel; Tarr, Philip; Trkola, Alexandra; Weber, Reiner

    2016-01-01

    Targeting hard-to-reach/marginalized populations is essential for preventing HIV-transmission. A unique opportunity to identify such populations in Switzerland is provided by a database of all genotypic-resistance-tests from Switzerland, including both sequences from the Swiss HIV Cohort Study (SHCS) and non-cohort sequences. A phylogenetic tree was built using 11,127 SHCS and 2,875 Swiss non-SHCS sequences. Demographics were imputed for non-SHCS patients using a phylogenetic proximity approach. Factors associated with non-cohort outbreaks were determined using logistic regression. Non-B subtype (univariable odds-ratio (OR): 1.9; 95% confidence interval (CI): 1.8–2.1), female gender (OR: 1.6; 95% CI: 1.4–1.7), black ethnicity (OR: 1.9; 95% CI: 1.7–2.1) and heterosexual transmission group (OR:1.8; 95% CI: 1.6–2.0), were all associated with underrepresentation in the SHCS. We found 344 purely non-SHCS transmission clusters, however, these outbreaks were small (median 2, maximum 7 patients) with a strong overlap with the SHCS’. 65% of non-SHCS sequences were part of clusters composed of >= 50% SHCS sequences. Our data suggests that marginalized-populations are underrepresented in the SHCS. However, the limited size of outbreaks among non-SHCS patients in-care implies that no major HIV outbreak in Switzerland was missed by the SHCS surveillance. This study demonstrates the potential of sequence data to assess and extend the scope of infectious-disease surveillance. PMID:27297284

  8. Genotypic Resistance Tests Sequences Reveal the Role of Marginalized Populations in HIV-1 Transmission in Switzerland.

    PubMed

    Shilaih, Mohaned; Marzel, Alex; Yang, Wan Lin; Scherrer, Alexandra U; Schüpbach, Jörg; Böni, Jürg; Yerly, Sabine; Hirsch, Hans H; Aubert, Vincent; Cavassini, Matthias; Klimkait, Thomas; Vernazza, Pietro L; Bernasconi, Enos; Furrer, Hansjakob; Günthard, Huldrych F; Kouyos, Roger

    2016-01-01

    Targeting hard-to-reach/marginalized populations is essential for preventing HIV-transmission. A unique opportunity to identify such populations in Switzerland is provided by a database of all genotypic-resistance-tests from Switzerland, including both sequences from the Swiss HIV Cohort Study (SHCS) and non-cohort sequences. A phylogenetic tree was built using 11,127 SHCS and 2,875 Swiss non-SHCS sequences. Demographics were imputed for non-SHCS patients using a phylogenetic proximity approach. Factors associated with non-cohort outbreaks were determined using logistic regression. Non-B subtype (univariable odds-ratio (OR): 1.9; 95% confidence interval (CI): 1.8-2.1), female gender (OR: 1.6; 95% CI: 1.4-1.7), black ethnicity (OR: 1.9; 95% CI: 1.7-2.1) and heterosexual transmission group (OR:1.8; 95% CI: 1.6-2.0), were all associated with underrepresentation in the SHCS. We found 344 purely non-SHCS transmission clusters, however, these outbreaks were small (median 2, maximum 7 patients) with a strong overlap with the SHCS'. 65% of non-SHCS sequences were part of clusters composed of >= 50% SHCS sequences. Our data suggests that marginalized-populations are underrepresented in the SHCS. However, the limited size of outbreaks among non-SHCS patients in-care implies that no major HIV outbreak in Switzerland was missed by the SHCS surveillance. This study demonstrates the potential of sequence data to assess and extend the scope of infectious-disease surveillance. PMID:27297284

  9. Antimalarial drug resistance: An overview.

    PubMed

    Antony, Hiasindh Ashmi; Parija, Subhash Chandra

    2016-01-01

    Malaria is a major public health burden throughout the world. Resistance to the antimalarial drugs has increased the mortality and morbidity rate that is achieved so far through the malaria control program. Monitoring the drug resistance to the available antimalarial drugs helps to implement effective drug policy, through the in vivo efficacy studies, in vitro drug susceptibility tests and detection of molecular markers. It is important to understand the mechanism of the antimalarial drugs, as it is one of the key factors in the emergence and spread of drug resistance. This review summarizes the commonly used antimalarial drugs, their mechanism of action and the genetic markers validated so far for the detection of drug-resistant parasites. PMID:26998432

  10. Antimalarial drug resistance: An overview

    PubMed Central

    Antony, Hiasindh Ashmi; Parija, Subhash Chandra

    2016-01-01

    Malaria is a major public health burden throughout the world. Resistance to the antimalarial drugs has increased the mortality and morbidity rate that is achieved so far through the malaria control program. Monitoring the drug resistance to the available antimalarial drugs helps to implement effective drug policy, through the in vivo efficacy studies, in vitro drug susceptibility tests and detection of molecular markers. It is important to understand the mechanism of the antimalarial drugs, as it is one of the key factors in the emergence and spread of drug resistance. This review summarizes the commonly used antimalarial drugs, their mechanism of action and the genetic markers validated so far for the detection of drug-resistant parasites. PMID:26998432

  11. Sequence and peptide-binding motif for a variant of HLA-A*0214 (A*02142) in an HIV-1-resistant individual from the Nairobi Sex Worker cohort.

    PubMed

    Luscher, M A; MacDonald, K S; Bwayo, J J; Plummer, F A; Barber, B H

    2001-02-01

    As part of the ongoing study of natural HIV-1 resistance in the women of the Nairobi Sex Workers' study, we have examined a resistance-associated HLA class I allele at the molecular level. Typing by polymerase chain reaction using sequence-specific primers determined that this molecule is closely related to HLA-A*0214, one of a family of HLA-A2 supertype alleles which correlate with HIV-1 resistance in this population. Direct nucleotide sequencing shows that this molecule differs from A*0214, having a silent nucleotide substitution. We therefore propose to designate it HLA-A*02142. We have determined the peptide-binding motif of HLA-A*0214/02142 by peptide elution and bulk Edman degradative sequencing. The resulting motif, X-[Q,V]-X-X-X-K-X-X-[V,L], includes lysine as an anchor at position 6. The data complement available information on the peptide-binding characteristics of this molecule, and will be of use in identifying antigenic peptides from HIV-1 and other pathogens. PMID:11261925

  12. Antiretroviral Therapy (ART) Use, Human Immunodeficiency Virus (HIV)-1 RNA Suppression, and Medical Causes of Hospitalization Among HIV-Infected Intravenous Drug Users in the Late ART Era

    PubMed Central

    Vallecillo, Gabriel; Mojal, Sergio; Torrens, Marta; Muga, Roberto

    2014-01-01

    Background.  Antiretroviral therapy (ART) has reduced the rates and changed the causes of hospital admission. However, human immunodeficiency virus-positive intravenous drug users (HIV-IDU) continue to have increased hospitalizations and discharge diagnosis are less defined in the late ART era. Our aim was to examine ART use, HIV-1 RNA suppression, and hospital discharge diagnoses among HIV-IDU admitted to an urban hospital. Methods.  A retrospective analysis was made of HIV-IDU admitted for medical causes for the first time (2006–2010). Surgical, obstetric, or mental (except HIV-associated neurocognitive disorder) diagnoses were excluded. Clinical characteristics, number of admissions, and primary discharge diagnoses were determined for each patient. Results.  Three hundred and seventy-five admissions were recorded among 197 hospitalized HIV-IDU. Lifetime prevalence of ART use was 83.2% (164 of 197) and the rate of HIV-1 RNA <50 copies/mL was 38.1% (75 of 197). Primary discharge diagnosis groups were as follows: bacterial infections (59.2%), chronic end-organ damage (16.8%), complications derived from injected drug use (16.8%), malignancies (9.1%), and opportunistic infections (6.6%). Chronic end-organ damage was diagnosed more frequently in patients with HIV-1 RNA <50 copies/mL (36% vs 4.9%; P < .000), and complications derived from injected drug use (23.8% vs 5.3%; P < .0008) and acquired immune deficiency syndrome (AIDS) opportunistic infections (19.8% vs 1.3% P < .019) were usually diagnosed in patients with HIV-1 RNA detectable viral load. Conclusions.  Human immunodeficiency virus-positive intravenous drug users are admitted to hospitals mainly for non-AIDS-related illnesses; however, sustained HIV-1 RNA viral load suppression is poor and determines hospital discharge diagnoses. Providers need to be aware of the management of HIV-related comorbidities and reinforce strategies to improve ART retention in this population. PMID:25734084

  13. Antiretroviral therapy and drug resistance in human immunodeficiency virus type 2 infection.

    PubMed

    Menéndez-Arias, Luis; Alvarez, Mar

    2014-02-01

    One to two million people worldwide are infected with the human immunodeficiency virus type 2 (HIV-2), with highest prevalences in West African countries, but also present in Western Europe, Asia and North America. Compared to HIV-1, HIV-2 infection undergoes a longer asymptomatic phase and progresses to AIDS more slowly. In addition, HIV-2 shows lower transmission rates, probably due to its lower viremia in infected individuals. There is limited experience in the treatment of HIV-2 infection and several antiretroviral drugs used to fight HIV-1 are not effective against HIV-2. Effective drugs against HIV-2 include nucleoside analogue reverse transcriptase (RT) inhibitors (e.g. zidovudine, tenofovir, lamivudine, emtricitabine, abacavir, stavudine and didanosine), protease inhibitors (saquinavir, lopinavir and darunavir), and integrase inhibitors (raltegravir, elvitegravir and dolutegravir). Maraviroc, a CCR5 antagonist blocking coreceptor binding during HIV entry, is active in vitro against CCR5-tropic HIV-2 but more studies are needed to validate its use in therapeutic treatments against HIV-2 infection. HIV-2 strains are naturally resistant to a few antiretroviral drugs developed to suppress HIV-1 propagation such as nonnucleoside RT inhibitors, several protease inhibitors and the fusion inhibitor enfuvirtide. Resistance selection in HIV-2 appears to be faster than in HIV-1. In this scenario, the development of novel drugs specific for HIV-2 is an important priority. In this review, we discuss current anti-HIV-2 therapies and mutational pathways leading to drug resistance. PMID:24345729

  14. Burden of Nonnucleoside Reverse Transcriptase Inhibitor Resistance in HIV-1-Infected Patients: A Systematic Review and Meta-Analysis

    PubMed Central

    Sudharshan, Lavanya; Nedrow, Katherine; Bhanegaonkar, Abhijeet; Simpson, Kit N.; Haider, Seema; Chambers, Richard; Craig, Charles; Stephens, Jennifer

    2014-01-01

    Abstract The prevalence of HIV drug resistance varies with geographic location, year, and treatment exposure. This study generated yearly estimates of nonnucleoside reverse transcriptase inhibitor (NNRTI) resistance in treatment-naive (TN) and treatment-experienced (TE) patients in the United States (US), Europe (EU), and Canada. Studies reporting NNRTI resistance identified in electronic databases and 11 conferences were analyzed in three groups: (1) TN patients in one of four geographic regions [US, Canada, EU countries with larger surveillance networks (“EU1”), and EU countries with fewer data (“EU2”)]; (2) TE patients from any region; and (3) TN patients failing NNRTI-based treatments in clinical trials. Analysis data included 158 unique studies from 22 countries representing 84 cohorts of TN patients, 21 cohorts of TE patients, and 8 trials reporting resistance at failure. From 1995 to 2000, resistance prevalence in TN patients increased in US and EU1 from 3.1% to 7.5% and 0.8% to 3.6%, respectively. Resistance in both regions stabilized in 2006 onward. Little resistance was identified in EU2 before 2000, and increased from 2006 (5.0%) to 2010 (13.7%). One TN Canadian study was identified and reported resistance of 8.1% in 2006. Half of TN clinical trial patients had resistance after treatment failure at weeks 48–144. Resistance in TE patients increased from 1998 (10.1%) to 2001 (44.0%), then decreased after 2004. Trends in NNRTI resistance among TN patients show an increased burden in the US and some EU countries compared to others. These findings signify a need for alternate first-line treatments in some regions. PMID:24925216

  15. Novel approaches to inhibiting HIV-1 replication.

    PubMed

    Adamson, Catherine S; Freed, Eric O

    2010-01-01

    Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010. PMID:19782103

  16. Mechanisms of drug resistance: quinolone resistance

    PubMed Central

    Hooper, David C.; Jacoby, George A.

    2015-01-01

    Quinolone antimicrobials are synthetic and widely used in clinical medicine. Resistance emerged with clinical use and became common in some bacterial pathogens. Mechanisms of resistance include two categories of mutation and acquisition of resistance-conferring genes. Resistance mutations in one or both of the two drug target enzymes, DNA gyrase and DNA topoisomerase IV, are commonly in a localized domain of the GyrA and ParE subunits of the respective enzymes and reduce drug binding to the enzyme-DNA complex. Other resistance mutations occur in regulatory genes that control the expression of native efflux pumps localized in the bacterial membrane(s). These pumps have broad substrate profiles that include quinolones as well as other antimicrobials, disinfectants, and dyes. Mutations of both types can accumulate with selection pressure and produce highly resistant strains. Resistance genes acquired on plasmids can confer low-level resistance that promotes the selection of mutational high-level resistance. Plasmid-encoded resistance is due to Qnr proteins that protect the target enzymes from quinolone action, one mutant aminoglycoside-modifying enzyme that also modifies certain quinolones, and mobile efflux pumps. Plasmids with these mechanisms often encode additional antimicrobial resistances and can transfer multidrug resistance that includes quinolones. Thus, the bacterial quinolone resistance armamentarium is large. PMID:26190223

  17. Mechanisms of drug resistance: quinolone resistance.

    PubMed

    Hooper, David C; Jacoby, George A

    2015-09-01

    Quinolone antimicrobials are synthetic and widely used in clinical medicine. Resistance emerged with clinical use and became common in some bacterial pathogens. Mechanisms of resistance include two categories of mutation and acquisition of resistance-conferring genes. Resistance mutations in one or both of the two drug target enzymes, DNA gyrase and DNA topoisomerase IV, are commonly in a localized domain of the GyrA and ParE subunits of the respective enzymes and reduce drug binding to the enzyme-DNA complex. Other resistance mutations occur in regulatory genes that control the expression of native efflux pumps localized in the bacterial membrane(s). These pumps have broad substrate profiles that include quinolones as well as other antimicrobials, disinfectants, and dyes. Mutations of both types can accumulate with selection pressure and produce highly resistant strains. Resistance genes acquired on plasmids can confer low-level resistance that promotes the selection of mutational high-level resistance. Plasmid-encoded resistance is due to Qnr proteins that protect the target enzymes from quinolone action, one mutant aminoglycoside-modifying enzyme that also modifies certain quinolones, and mobile efflux pumps. Plasmids with these mechanisms often encode additional antimicrobial resistances and can transfer multidrug resistance that includes quinolones. Thus, the bacterial quinolone resistance armamentarium is large. PMID:26190223

  18. Novel Approaches to Inhibiting HIV-1 Replication

    PubMed Central

    Adamson, Catherine S.; Freed, Eric O.

    2009-01-01

    Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. PMID:19782103

  19. HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells.

    PubMed

    Jerebtsova, Marina; Kumari, Namita; Xu, Min; de Melo, Gustavo Brito Alvim; Niu, Xiaomei; Jeang, Kuan-Teh; Nekhai, Sergei

    2012-07-26

    A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs) through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages. PMID:22934150

  20. HIV-1 Resistant CDK2-Knockdown Macrophage-Like Cells Generated from 293T Cell-Derived Human Induced Pluripotent Stem Cells

    PubMed Central

    Jerebtsova, Marina; Kumari, Namita; Xu, Min; de Melo, Gustavo Brito Alvim; Niu, Xiaomei; Jeang, Kuan-Teh; Nekhai, Sergei

    2012-01-01

    A major challenge in studies of human diseases involving macrophages is low yield and heterogeneity of the primary cells and limited ability of these cells for transfections and genetic manipulations. To address this issue, we developed a simple and efficient three steps method for somatic 293T cells reprogramming into monocytes and macrophage-like cells. First, 293T cells were reprogrammed into induced pluripotent stem cells (iPSCs) through a transfection-mediated expression of two factors, Oct-4 and Sox2, resulting in a high yield of iPSC. Second, the obtained iPSC were differentiated into monocytes using IL-3 and M-CSF treatment. And third, monocytes were differentiated into macrophage-like cells in the presence of M-CSF. As an example, we developed HIV-1-resistant macrophage-like cells from 293T cells with knockdown of CDK2, a factor critical for HIV-1 transcription. Our study provides a proof-of-principle approach that can be used to study the role of host cell factors in HIV-1 infection of human macrophages. PMID:22934150

  1. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    SciTech Connect

    Liu, Zhigang; Wang, Yong; Yedidi, Ravikiran S.; Dewdney, Tamaria G.; Reiter, Samuel J.; Brunzelle, Joseph S.; Kovari, Iulia A.; Kovari, Ladislau C.

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  2. Mutational anatomy of an HIV-1 protease variant conferring cross-resistance to protease inhibitors in clinical trials. Compensatory modulations of binding and activity.

    PubMed

    Schock, H B; Garsky, V M; Kuo, L C

    1996-12-13

    Site-specific substitutions of as few as four amino acids (M46I/L63P/V82T/I84V) of the human immunodeficiency virus type 1 (HIV-1) protease engenders cross-resistance to a panel of protease inhibitors that are either in clinical trials or have recently been approved for HIV therapy (Condra, J. H., Schleif, W. A., Blahy, O. M. , Gadryelski, L. J., Graham, D. J., Quintero, J. C., Rhodes, A., Robbins, H. L., Roth, E., Shivaprakash, M., Titus, D., Yang, T., Teppler, H., Squires, K. E., Deutsch, P. J., and Emini, E. A. (1995) Nature 374, 569-571). These four substitutions are among the prominent mutations found in primary HIV isolates obtained from patients undergoing therapy with several protease inhibitors. Two of these mutations (V82T/I84V) are located in, while the other two (M46I/L63P) are away from, the binding cleft of the enzyme. The functional role of these mutations has now been delineated in terms of their influence on the binding affinity and catalytic efficiency of the protease. We have found that the double substitutions of M46I and L63P do not affect binding but instead endow the enzyme with a catalytic efficiency significantly exceeding (110-360%) that of the wild-type enzyme. In contrast, the double substitutions of V82T and I84V are detrimental to the ability of the protease to bind and, thereby, to catalyze. When combined, the four amino acid replacements institute in the protease resistance against inhibitors and a significantly higher catalytic activity than one containing only mutations in its active site. The results suggest that in raising drug resistance, these four site-specific mutations of the protease are compensatory in function; those in the active site diminish equilibrium binding (by increasing Ki), and those away from the active site enhance catalysis (by increasing kcat/KM). This conclusion is further supported by energy estimates in that the Gibbs free energies of binding and catalysis for the quadruple mutant are quantitatively

  3. Decreasing population selection rates of resistance mutation K65R over time in HIV-1 patients receiving combination therapy including tenofovir

    PubMed Central

    Theys, K.; Snoeck, J.; Vercauteren, J.; Abecasis, A. B.; Vandamme, A.-M.; Camacho, R. J.

    2013-01-01

    Objectives The use of tenofovir is highly associated with the emergence of mutation K65R, which confers broad resistance to nucleoside/nucleotide analogue reverse transcriptase inhibitors (NRTIs), especially when tenofovir is combined with other NRTIs also selecting for K65R. Although recent HIV-1 treatment guidelines discouraging these combinations resulted in reduced K65R selection with tenofovir, updated information on the impact of currently recommended regimens on the population selection rate of K65R is presently lacking. Methods In this study, we evaluated changes over time in the selection rate of resistance mutation K65R in a large population of 2736 HIV-1-infected patients failing combination antiretroviral treatment between 2002 and 2010. Results The K65R resistance mutation was detected in 144 patients, a prevalence of 5.3%. A large majority of observed K65R cases were explained by the use of tenofovir, reflecting its wide use in clinical practice. However, changing patterns over time in NRTIs accompanying tenofovir resulted in a persistent decreasing probability of K65R selection by tenofovir-based therapy. The currently recommended NRTI combination tenofovir/emtricitabine was associated with a low probability of K65R emergence. For any given dual NRTI combination including tenofovir, higher selection rates of K65R were consistently observed with a non-nucleoside reverse transcriptase inhibitor than with a protease inhibitor as the third agent. Discussion Our finding of a stable time trend of K65R despite elevated use of tenofovir illustrates increased potency of current HIV-1 therapy including tenofovir. PMID:23027713

  4. Clinical, virological and biochemical evidence supporting the association of HIV-1 reverse transcriptase polymorphism R284K and thymidine analogue resistance mutations M41L, L210W and T215Y in patients failing tenofovir/emtricitabine therapy

    PubMed Central

    2012-01-01

    Background Thymidine analogue resistance mutations (TAMs) selected under treatment with nucleoside analogues generate two distinct genotypic profiles in the HIV-1 reverse transcriptase (RT): (i) TAM1: M41L, L210W and T215Y, and (ii) TAM2: D67N, K70R and K219E/Q, and sometimes T215F. Secondary mutations, including thumb subdomain polymorphisms (e.g. R284K) have been identified in association with TAMs. We have identified mutational clusters associated with virological failure during salvage therapy with tenofovir/emtricitabine-based regimens. In this context, we have studied the role of R284K as a secondary mutation associated with mutations of the TAM1 complex. Results The cross-sectional study carried out with >200 HIV-1 genotypes showed that virological failure to tenofovir/emtricitabine was strongly associated with the presence of M184V (P < 10-10) and TAMs (P < 10-3), while K65R was relatively uncommon in previously-treated patients failing antiretroviral therapy. Clusters of mutations were identified, and among them, the TAM1 complex showed the highest correlation coefficients. Covariation of TAM1 mutations and V118I, V179I, M184V and R284K was observed. Virological studies showed that the combination of R284K with TAM1 mutations confers a fitness advantage in the presence of zidovudine or tenofovir. Studies with recombinant HIV-1 RTs showed that when associated with TAM1 mutations, R284K had a minimal impact on zidovudine or tenofovir inhibition, and in their ability to excise the inhibitors from blocked DNA primers. However, the mutant RT M41L/L210W/T215Y/R284K showed an increased catalytic rate for nucleotide incorporation and a higher RNase H activity in comparison with WT and mutant M41L/L210W/T215Y RTs. These effects were consistent with its enhanced chain-terminated primer rescue on DNA/DNA template-primers, but not on RNA/DNA complexes, and can explain the higher fitness of HIV-1 having TAM1/R284K mutations. Conclusions Our study shows the association

  5. Bayesian analysis of complex interacting mutations in HIV drug resistance and cross-resistance.

    PubMed

    Kozyryev, Ivan; Zhang, Jing

    2015-01-01

    A successful treatment of AIDS world-wide is severely hindered by the HIV virus' drug resistance capability resulting from complicated mutation patterns of viral proteins. Such a system of mutations enables the virus to survive and reproduce despite the presence of various antiretroviral drugs by disrupting their binding capability. Although these interacting mutation patterns are extremely difficult to efficiently uncover and interpret, they contribute valuable information to personalized therapeutic regimen design. The use of Bayesian statistical modeling provides an unprecedented opportunity in the field of anti-HIV therapy to understand detailed interaction structures of drug resistant mutations. Multiple Bayesian models equipped with Markov Chain Monte Carlo (MCMC) methods have been recently proposed in this field (Zhang et al. in PNAS 107:1321, 2010 [1]; Zhang et al. in J Proteome Sci Comput Biol 1:2, 2012 [2]; Svicher et al. in Antiviral Res 93(1):86-93, 2012 [3]; Svicher et al. in Antiviral Therapy 16(7):1035-1045, 2011 [4]; Svicher et al. in Antiviral Ther 16(4):A14-A14, 2011 [5]; Svicher et al. in Antiviral Ther 16(4):A85-A85, 2011 [6]; Alteri et al. in Signature mutations in V3 and bridging sheet domain of HIV-1 gp120 HIV-1 are specifically associated with dual tropism and modulate the interaction with CCR5 N-Terminus, 2011 [7]). Probabilistically modeling mutations in the HIV-1 protease or reverse transcriptase (RT) isolated from drug-treated patients provides a powerful statistical procedure that first detects mutation combinations associated with single or multiple-drug resistance, and then infers detailed dependence structures among the interacting mutations in viral proteins (Zhang et al. in PNAS 107:1321, 2010 [1]; Zhang et al. in J Proteome Sci Comput Biol 1:2, 2012 [2]). Combined with molecular dynamics simulations and free energy calculations, Bayesian analysis predictions help to uncover genetic and structural mechanisms in the HIV treatment

  6. More about the Viking hypothesis of origin of the delta32 mutation in the CCR5 gene conferring resistance to HIV-1 infection.

    PubMed

    Lucotte, Gérard; Dieterlen, Florent

    2003-11-01

    The chemokine receptor CCR5 constitutes the major coreceptor for the HIV-1, because a mutant allele of the CCR5 gene named delta32 was shown to provide to homozygotes a strong resistance against infection. In the present study the frequency of the delta32 allele was collected in 36 European populations and in Cyprus, and the highest allele frequencies were found in Nordic countries. We constructed an allele map of delta32 frequencies in Europe; the map is in accordance to the Vikings hypothesis of the origin of the mutation and his dissemination during the eighth to the tenth centuries. PMID:14636691

  7. [Resistance to the antimalarial drugs].

    PubMed

    Venanzi, E; López-Vélez, R

    2016-09-01

    Malaria is one of the most widespread infectious diseases around the world with 214 million cases and 438,000 deaths in 2015. In the early twentieth century it was described for the first time the resistance to quinine and, since then, drug resistance to antimalarial drugs has spread up to represent a global challenge in the fight and control of malaria. Understanding the mechanisms, geography and monitoring tools that we can act against resistance to antimalarial drugs is critical to prevent its expansion. PMID:27608319

  8. The triple threat of HIV-1 protease inhibitors.

    PubMed

    Potempa, Marc; Lee, Sook-Kyung; Wolfenden, Richard; Swanstrom, Ronald

    2015-01-01

    Newly released human immunodeficiency virus type 1 (HIV-1) particles obligatorily undergo a maturation process to become infectious. The HIV-1 protease (PR) initiates this step, catalyzing the cleavage of the Gag and Gag-Pro-Pol structural polyproteins. Proper organization of the mature virus core requires that cleavage of these polyprotein substrates proceeds in a highly regulated, specific series of events. The vital role the HIV-1 PR plays in the viral life cycle has made it an extremely attractive target for inhibition and has accordingly fostered the development of a number of highly potent substrate-analog inhibitors. Though the PR inhibitors (PIs) inhibit only the HIV-1 PR, their effects manifest at multiple different stages in the life cycle due to the critical importance of the PR in preparing the virus for these subsequent events. Effectively, PIs masquerade as entry inhibitors, reverse transcription inhibitors, and potentially even inhibitors of post-reverse transcription steps. In this chapter, we review the triple threat of PIs: the intermolecular cooperativity in the form of a cooperative dose-response for inhibition in which the apparent potency increases with increasing inhibition; the pleiotropic effects of HIV-1 PR inhibition on entry, reverse transcription, and post-reverse transcription steps; and their potency as transition state analogs that have the potential for further improvement that could lead to an inability of the virus to evolve resistance in the context of single drug therapy. PMID:25778681

  9. Cross-resistance profile determination of two second-generation HIV-1 integrase inhibitors using a panel of recombinant viruses derived from raltegravir-treated clinical isolates.

    PubMed

    Van Wesenbeeck, L; Rondelez, E; Feyaerts, M; Verheyen, A; Van der Borght, K; Smits, V; Cleybergh, C; De Wolf, H; Van Baelen, K; Stuyver, L J

    2011-01-01

    The integrase inhibitor raltegravir (RAL) is currently used for the treatment of both treatment-naïve and treatment-experienced HIV-1-infected patients. Elvitegravir (EVG) is in late phases of clinical development. Since significant cross-resistance between RAL and EVG is observed, there is a need for second-generation integrase inhibitors (INIs) with a higher genetic barrier and limited cross-resistance to RAL/EVG. A panel of HIV-1 integrase recombinants, derived from plasma samples from raltegravir-treated patients (baseline and follow-up samples), were used to study the cross-resistance profile of two second-generation integrase inhibitors, MK-2048 and compound G. Samples with Q148H/R mutations had elevated fold change values with all compounds tested. Although samples with the Y143R/C mutation had reduced susceptibility to RAL, they remained susceptible to MK-2048 and compound G. Samples with the N155H mutation had no reduced susceptibility to compound G. In conclusion, our results allowed ranking of the INIs on the basis of the antiviral activities using recombinant virus stocks from RAL-treated patient viruses. The order according to decreasing susceptibility is compound G, MK-2048, and EVG. PMID:20956600

  10. How drug resistance takes shape

    PubMed Central

    Jeselsohn, Rinath

    2016-01-01

    Mutations in a hormone receptor can lead to therapeutic resistance by making it less able to bind and respond to hormone blocking drugs and by making it active, even when the hormome is not present. PMID:27010172

  11. Protease inhibitors effectively block cell-to-cell spread of HIV-1 between T cells

    PubMed Central

    2013-01-01

    Background The Human Immunodeficiency Virus type-1 (HIV-1) spreads by cell-free diffusion and by direct cell-to-cell transfer, the latter being a significantly more efficient mode of transmission. Recently it has been suggested that cell-to-cell spread may permit ongoing virus replication in the presence of antiretroviral therapy (ART) based on studies performed using Reverse Transcriptase Inhibitors (RTIs). Protease Inhibitors (PIs) constitute an important component of ART; however whether this class of inhibitors can suppress cell-to-cell transfer of HIV-1 is unexplored. Here we have evaluated the inhibitory effect of PIs during cell-to-cell spread of HIV-1 between T lymphocytes. Results Using quantitative assays in cell line and primary cell systems that directly measure the early steps of HIV-1 infection we find that the PIs Lopinavir and Darunavir are equally potent against both cell-free and cell-to-cell spread of HIV-1. We further show that a protease resistant mutant maintains its resistant phenotype during cell-to-cell spread and is transmitted more efficiently than wild-type virus in the presence of drug. By contrast we find that T cell-T cell spread of HIV-1 is 4–20 fold more resistant to inhibition by the RTIs Nevirapine, Zidovudine and Tenofovir. Notably, varying the ratio of infected and uninfected cells in co-culture impacted on the degree of inhibition, indicating that the relative efficacy of ART is dependent on the multiplicity of infection. Conclusions We conclude that if the variable effects of antiviral drugs on cell-to-cell virus dissemination of HIV-1 do indeed impact on viral replication and maintenance of viral reservoirs this is likely to be influenced by the antiviral drug class, since PIs appear particularly effective against both modes of HIV-1 spread. PMID:24364896

  12. Fragment Based Strategies for Discovery of Novel HIV-1 Reverse Transcriptase and Integrase Inhibitors.

    PubMed

    Latham, Catherine F; La, Jennifer; Tinetti, Ricky N; Chalmers, David K; Tachedjian, Gilda

    2016-01-01

    Human immunodeficiency virus (HIV) remains a global health problem. While combined antiretroviral therapy has been successful in controlling the virus in patients, HIV can develop resistance to drugs used for treatment, rendering available drugs less effective and limiting treatment options. Initiatives to find novel drugs for HIV treatment are ongoing, although traditional drug design approaches often focus on known binding sites for inhibition of established drug targets like reverse transcriptase and integrase. These approaches tend towards generating more inhibitors in the same drug classes already used in the clinic. Lack of diversity in antiretroviral drug classes can result in limited treatment options, as cross-resistance can emerge to a whole drug class in patients treated with only one drug from that class. A fresh approach in the search for new HIV-1 drugs is fragment-based drug discovery (FBDD), a validated strategy for drug discovery based on using smaller libraries of low molecular weight molecules (<300 Da) screened using primarily biophysical assays. FBDD is aimed at not only finding novel drug scaffolds, but also probing the target protein to find new, often allosteric, inhibitory binding sites. Several fragment-based strategies have been successful in identifying novel inhibitory sites or scaffolds for two proven drug targets for HIV-1, reverse transcriptase and integrase. While any FBDD-generated HIV-1 drugs have yet to enter the clinic, recent FBDD initiatives against these two well-characterised HIV-1 targets have reinvigorated antiretroviral drug discovery and the search for novel classes of HIV-1 drugs. PMID:26324045

  13. Incarceration is associated with used syringe lending among active injection drug users with detectable plasma HIV-1 RNA: a longitudinal analysis

    PubMed Central

    2013-01-01

    Background Informed by recent studies demonstrating the central role of plasma HIV-1 RNA viral load (VL) on HIV transmission, interventions to employ HIV antiretroviral treatment as prevention (TasP) are underway. To optimize these efforts, evidence is needed to identify factors associated with both non-suppressed VL and HIV risk behaviours. Thus, we sought to assess the possible role played by exposure to correctional facilities on VL non-suppression and used syringe lending among HIV-seropositive people who use injection drugs (PWID). Methods We used data from the ACCESS study, a community-recruited prospective cohort. We used longitudinal multivariate mixed-effects analyses to estimate the relationship between incarceration and plasma HIV-1 RNA > 500 copies/mL among antiretroviral therapy (ART)-exposed active PWID and, during periods of non-suppression, the relationship between incarceration and used syringe lending. Results Between May 1996 and March 2012, 657 ART-exposed PWID were recruited. Incarceration was independently associated with higher odds of VL non-suppression (Adjusted Odds Ratio [AOR] = 1.54, 95% Confidence Interval [95% CI]: 1.10, 2.16). In a separate multivariate model restricted to periods of VL non-suppression, incarceration was independently associated with lending used syringes (AOR = 1.81, 95% CI: 1.03, 3.18). Conclusions The current findings demonstrate that incarceration is associated with used syringe lending among active PWID with detectable plasma HIV-1 RNA. Our results provide a possible pathway for the commonly observed association between incarceration and increased risk of HIV transmission. Our results suggest that alternatives to incarceration of non-violent PWID and evidence-based combination HIV prevention interventions for PWID within correctional facilities are urgently needed. PMID:24289651

  14. A copper(I)-catalyzed 1,2,3-triazole azide-alkyne click compound, is a potent inhibitor of a multidrug-resistant HIV-1 protease variant

    PubMed Central

    Giffin, Michael J.; Heaslet, Holly; Brik, Ashraf; Lin, Ying-Chuan; Cauvi, Gabrielle; Wong, Chi-Huey; McRee, Duncan E.; Elder, John H.; Stout, C. David; Torbett, Bruce E.

    2009-01-01

    Treatment with protease inhibitors, a component of Highly Active Anti-retroviral Therapy (HAART), often results in viral resistance. Structural and biochemical characterization of a 6X mutant arising from in vitro selection with compound 1, a C2-symmetric diol protease inhibitor, has been previously described. We now show that compound 2, a copper(I)-catalyzed 1,2,3-triazole derived compound previously shown to be potently effective against wild-type protease (IC50 = 6.0 nM), has low nM activity (IC50 = 15.7 nM) against the multidrug-resistant 6X HIV-1 protease mutant. Compound 2 displays similar efficacy against wild-type and 6X HIV-1 in viral replication assays. While structural studies of compound 1 bound to wild type and mutant protease revealed a progressive change in binding mode in the mutants, the 1.3 Å resolution 6X protease–compound 2 crystal structure reveals nearly identical interactions for 2 as in the wild-type protease complex with very little change in compound 2 or protease conformation. PMID:18823110

  15. Persistent HIV-1 replication maintains the tissue reservoir during therapy

    PubMed Central

    Bedford, Trevor; Kim, Eun-Young; Archer, John; Pond, Sergei L. Kosakovsky; Chung, Yoon-Seok; Penugonda, Sudhir; Chipman, Jeffrey; Fletcher, Courtney V.; Schacker, Timothy W.; Malim, Michael H.; Rambaut, Andrew; Haase, Ashley T.; McLean, Angela R.; Wolinsky, Steven M.

    2015-01-01

    Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site of viral production, storage of viral particles in immune complexes, and viral persistence. Whilst combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. A spatial dynamic model of persistent viral replication and spread explains why the development of drug resistance is not a foregone conclusion under conditions where drug concentrations are insufficient to completely block virus replication. These data provide fresh insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and refill the viral reservoir despite potent antiretroviral therapy. PMID:26814962

  16. Persistent HIV-1 replication maintains the tissue reservoir during therapy.

    PubMed

    Lorenzo-Redondo, Ramon; Fryer, Helen R; Bedford, Trevor; Kim, Eun-Young; Archer, John; Kosakovsky Pond, Sergei L; Chung, Yoon-Seok; Penugonda, Sudhir; Chipman, Jeffrey G; Fletcher, Courtney V; Schacker, Timothy W; Malim, Michael H; Rambaut, Andrew; Haase, Ashley T; McLean, Angela R; Wolinsky, Steven M

    2016-02-01

    Lymphoid tissue is a key reservoir established by HIV-1 during acute infection. It is a site associated with viral production, storage of viral particles in immune complexes, and viral persistence. Although combinations of antiretroviral drugs usually suppress viral replication and reduce viral RNA to undetectable levels in blood, it is unclear whether treatment fully suppresses viral replication in lymphoid tissue reservoirs. Here we show that virus evolution and trafficking between tissue compartments continues in patients with undetectable levels of virus in their bloodstream. We present a spatial and dynamic model of persistent viral replication and spread that indicates why the development of drug resistance is not a foregone conclusion under conditions in which drug concentrations are insufficient to completely block virus replication. These data provide new insights into the evolutionary and infection dynamics of the virus population within the host, revealing that HIV-1 can continue to replicate and replenish the viral reservoir despite potent antiretroviral therapy. PMID:26814962

  17. Pyrazolo[1,5-a]pyrimidine-based macrocycles as novel HIV-1 inhibitors: a patent evaluation of WO2015123182.

    PubMed

    Sun, Lin; Gao, Ping; Zhan, Peng; Liu, Xinyong

    2016-09-01

    The emergence of drug resistance in Combination Antiretroviral Therapy (cART) confirms a continuing need to investigate novel HIV-1 inhibitors with unexplored mechanisms of action. Recently, a series of pyrazolopyrimidine-based macrocyclic compounds were reported as inhibitors of HIV-1 replication disclosed in the patent WO2015123182. Most of the disclosed compounds possessed in vitro antiviral potency in single-digit nanomolar range, which were determined by MT-2 cell assay. Then, the structural diversity, pharmacophore similarity of HIV-1 IN-LEDGF/p75 inhibitors, and implications for drug design were analyzed. In the end of this article, a glimpse of some macrocycles as potent antiviral agents (drug candidates) was provided. Some strategies and technologies enabling macrocycle design were also described. We expect that further development of these macrocyclic compounds will offer new anti-HIV-1 drug candidates. PMID:27398994

  18. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    SciTech Connect

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang; Bencze, Krisztina Z.; Koupparis, Kyriacos; O’Connor, Carrie E.; Kovari, Iulia A.; Spaller, Mark R.; Kovari, Ladislau C.

    2013-09-06

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  19. Drug resistance in eukaryotic microorganisms.

    PubMed

    Fairlamb, Alan H; Gow, Neil A R; Matthews, Keith R; Waters, Andrew P

    2016-01-01

    Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies. PMID:27572976

  20. Microplate-Based Assay for Identifying Small Molecules That Bind a Specific Intersubunit Interface within the Assembled HIV-1 Capsid

    PubMed Central

    Halambage, Upul D.; Wong, Jason P.; Melancon, Bruce J.; Lindsley, Craig W.

    2015-01-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid–targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity. PMID:26077250

  1. Microplate-based assay for identifying small molecules that bind a specific intersubunit interface within the assembled HIV-1 capsid.

    PubMed

    Halambage, Upul D; Wong, Jason P; Melancon, Bruce J; Lindsley, Craig W; Aiken, Christopher

    2015-09-01

    Despite the availability of >30 effective drugs for managing HIV-1 infection, no current therapy is curative, and long-term management is challenging owing to the emergence and spread of drug-resistant mutants. Identification of drugs against novel HIV-1 targets would expand the current treatment options and help to control resistance. The highly conserved HIV-1 capsid protein represents an attractive target because of its multiple roles in replication of the virus. However, the low antiviral potencies of the reported HIV-1 capsid-targeting inhibitors render them unattractive for therapeutic development. To facilitate the identification of more-potent HIV-1 capsid inhibitors, we developed a scintillation proximity assay to screen for small molecules that target a biologically active and specific intersubunit interface in the HIV-1 capsid. The assay, which is based on competitive displacement of a known capsid-binding small-molecule inhibitor, exhibited a signal-to-noise ratio of >9 and a Z factor of >0.8. In a pilot screen of a chemical library containing 2,400 druglike compounds, we obtained a hit rate of 1.8%. This assay has properties that are suitable for screening large compound libraries to identify novel HIV-1 capsid ligands with antiviral activity. PMID:26077250

  2. Antimicrobial (Drug) Resistance Prevention

    MedlinePlus

    ... Action Plan for Combating Antibiotic-Resistant Bacteria (PDF) ​​​​​​ Javascript Error Your browser JavaScript is turned off causing certain features of the ... incorrectly. Please visit your browser settings and turn JavaScript on. Read more information on enabling JavaScript. Skip ...

  3. Discordant responses to cART in HIV-1 patients in the era of high potency antiretroviral drugs: clinical evaluation, classification, management prospects.

    PubMed

    Cenderello, Giovanni; De Maria, Andrea

    2016-01-01

    The goal of antiretroviral treatment (ART) in HIV-1 patients is immune reconstitution following control of viral replication. CD4+ cell number/proportions are a crude but essential correlate of immune reconstitution. Despite suppression of HIV replication, a fraction of ART-treated patients still fails to fully reconstitute CD4+ T cell numbers (immunological nonresponders, INRs). New drugs, regimens and treatment strategies led to increased efficacy, lower side effects and higher virological success rates in clinical practice. The multitude of described immune defects and clinical events accompanying INR opposed to the marginal effect of antiretroviral intensification or immunotherapy trials underline the need for continuing efforts at understanding the mechanisms that underlie INR. Here, we reassess INR definition, frequency, and the achievements of active clinical and translational research suggesting a shared definition for insufficient, partial and complete CD4+ cell number recovery thus improving homogeneity in patient selection and mechanism identification. PMID:26513236

  4. Characterization of the HIV-1 TAR RNA-Tat peptide and drug interactions by on-line acoustic wave sensor

    NASA Astrophysics Data System (ADS)

    Tassew, Nardos Gobena

    This thesis presents the application of the thickness shear-mode (TSM) acoustic wave sensor to the study of RNA-protein and RNA-drug interactions at the solid-liquid interface. The binding of the human immunodeficiency virus-type 1 Tat protein to the trans-activation responsive RNA element (TAR) has been studied using this sensor. Data from such measurements show that the sensor is able to discriminate between different Tat peptides derived from the parent protein based on size. The effects of mutations introduced at specific sites in the protein and RNA on the TAR-Tat binding have also been examined in detail. Reduced level of response in acoustic parameters due to mutations was observed indicating that the decrease in binding in response to site specific mutations can be acoustically detected. Data from acoustic wave sensor measurements indicate that the TAR-Tat binding is also affected by ionic strength. Both the frequency and motional resistance signals show periodic responses when varying concentrations of salt are introduced on a TAR-modified surface. The binding of the two molecules seems to be a function of the response of the nucleic acid to salt concentrations. The kinetics of binding of Tat peptides to TAR RNA and to a bulge mutant analogue (MTAR) is also examined from the rate of change of the series resonant frequency. Results from such analysis illustrate longer Tat peptides formed more stable complexes with TAR RNA and exhibited increased discrimination between mutant and wild type TAR. The binding of two aminoglycoside antibiotics, neomycin and streptomycin, to TAR RNA and their effectiveness in preventing TAR-Tat complex formation has been studied in detail. Binding affinity is directly correlated with the inhibitory potency of these molecules and the TSM sensor shows that neomycin exhibits at least a ten fold greater affinity to TAR and that it is also a more potent inhibitor than streptomycin. The results from this research involving TAR-Tat and

  5. Measuring glutathione redox potential of HIV-1-infected macrophages.

    PubMed

    Bhaskar, Ashima; Munshi, MohamedHusen; Khan, Sohrab Zafar; Fatima, Sadaf; Arya, Rahul; Jameel, Shahid; Singh, Amit

    2015-01-01

    Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (E(GSH); Grx1-roGFP2) and measured subcellular changes in E(GSH) during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial E(GSH) (approximately -310 mV), active viral replication induces substantial oxidative stress (E(GSH) more than -240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular E(GSH) between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in E(GSH) is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular E(GSH). Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV. PMID:25406321

  6. Measuring Glutathione Redox Potential of HIV-1-infected Macrophages*

    PubMed Central

    Bhaskar, Ashima; Munshi, MohamedHusen; Khan, Sohrab Zafar; Fatima, Sadaf; Arya, Rahul; Jameel, Shahid; Singh, Amit

    2015-01-01

    Redox signaling plays a crucial role in the pathogenesis of human immunodeficiency virus type-1 (HIV-1). The majority of HIV redox research relies on measuring redox stress using invasive technologies, which are unreliable and do not provide information about the contributions of subcellular compartments. A major technological leap emerges from the development of genetically encoded redox-sensitive green fluorescent proteins (roGFPs), which provide sensitive and compartment-specific insights into redox homeostasis. Here, we exploited a roGFP-based specific bioprobe of glutathione redox potential (EGSH; Grx1-roGFP2) and measured subcellular changes in EGSH during various phases of HIV-1 infection using U1 monocytic cells (latently infected U937 cells with HIV-1). We show that although U937 and U1 cells demonstrate significantly reduced cytosolic and mitochondrial EGSH (approximately −310 mV), active viral replication induces substantial oxidative stress (EGSH more than −240 mV). Furthermore, exposure to a physiologically relevant oxidant, hydrogen peroxide (H2O2), induces significant deviations in subcellular EGSH between U937 and U1, which distinctly modulates susceptibility to apoptosis. Using Grx1-roGFP2, we demonstrate that a marginal increase of about ∼25 mV in EGSH is sufficient to switch HIV-1 from latency to reactivation, raising the possibility of purging HIV-1 by redox modulators without triggering detrimental changes in cellular physiology. Importantly, we show that bioactive lipids synthesized by clinical drug-resistant isolates of Mycobacterium tuberculosis reactivate HIV-1 through modulation of intracellular EGSH. Finally, the expression analysis of U1 and patient peripheral blood mononuclear cells demonstrated a major recalibration of cellular redox homeostatic pathways during persistence and active replication of HIV. PMID:25406321

  7. Hepatitis C virus infection in a large cohort of homosexually active men: independent associations with HIV-1 infection and injecting drug use but not sexual behaviour.

    PubMed Central

    Bodsworth, N J; Cunningham, P; Kaldor, J; Donovan, B

    1996-01-01

    OBJECTIVE: To determine the prevalence and risk factors for hepatitis C virus (HCV) infection in a cohort of homosexually active men, with particular reference to assessing sexual transmission. DESIGN: Prevalence based on cross-sectional testing for HCV (c100 protein) antibody in a cohort using sera stored between 1984 and 1989, and assessment of risk factors using a case-control analysis based on questionnaire data from HCV positive and negative subjects. SUBJECTS/SETTING: 1038 homosexually active men who were participating in a prospective study established to identify risk factors for AIDS. They had been recruited through private and public primary care and sexually transmissible disease (STD) services in central Sydney. MAIN OUTCOME MEASURES: Prevalence of HCV antibody and its association with human immunodeficiency virus type 1 (HIV-1) infection and other STDs, number of sexual partners, sexual practices and recreational drug use. RESULTS: Overall, 7.6% of subjects tested were seropositive for HCV antibody. In univariate analysis, HCV infection was significantly associated with injecting drug use (IDU) (OR = 8.18, p < 0.0001) and HIV infection (OR = 3.14, p < 0.0001) and with self reported history of syphilis (OR = 1.88, p = 0.016), anogenital herpes (OR = 1.93, p = 0.017), gonorrhoea (OR = 2.43, p = 0.009) and hepatitis B (OR = 1.92, p = 0.010). In case control analysis, similar sexual behaviours (partner numbers and practices) were reported by HCV positive and HCV negative subjects except that HCV negative subjects more frequently reported engaging than HCV positive subject in unprotected receptive anal intercourse without ejaculation (OR = 0.61, p = 0.034), unprotected insertive (OR = 0.59, p = 0.039) and receptive (OR = 0.56, p = 0.016) oro-anal intercourse (rimming) and insertive fisting (OR = 0.48, p = 0.034). In multiple logistic regression analyses, only HIV-1 infection (OR = 3.18, p < 0.0001) and IDU in the previous six months (OR = 7.24, p < 0

  8. Extensively drug-resistant tuberculosis.

    PubMed

    Jassal, Mandeep; Bishai, William R

    2009-01-01

    Extensively drug-resistant (XDR) tuberculosis is defined as disease caused by Mycobacterium tuberculosis with resistance to at least isoniazid and rifampicin, any fluoroquinolone, and at least one of three injectable second-line drugs (amikacin, capreomycin, or kanamycin). The definition has applicable clinical value and has allowed for more uniform surveillance in varied international settings. Recent surveillance data have indicated that the prevalence of tuberculosis drug resistance has risen to the highest rate ever recorded. The gold standard for drug-susceptibility testing has been the agar proportion method; however, this technique requires several weeks for results to be determined. More sensitive and specific diagnostic tests are still unavailable in resource-limited settings. Clinical manifestations, although variable in different settings and among different strains, have in general shown that XDR tuberculosis is associated with greater morbidity and mortality than non-XDR tuberculosis. The treatment of XDR tuberculosis should include agents to which the organism is susceptible, and should continue for a minimum of 18-24 months. However, treatment continues to be limited in tuberculosis-endemic countries largely because of weaknesses in national tuberculosis health-care models. The ultimate strategy to control drug-resistant tuberculosis is one that implements a comprehensive approach incorporating innovation from the political, social, economic, and scientific realms. PMID:18990610

  9. Structure of HIV-1 nonnucleoside reverse transcriptase inhibitors derivatives of N-benzyl-benzimidazole with different substituents in position 4

    NASA Astrophysics Data System (ADS)

    Ziółkowska, Natasza E.; Michejda, Christopher J.; Bujacz, Grzegorz D.

    2010-01-01

    The constant development of new drugs against HIV-1 is necessary due to global expansion of AIDS and HIV-1 drug resistance. Nonnucleoside reverse transcriptase inhibitors of HIV-1 (NNRTIs) are potentially effective and nontoxic drugs in AIDS therapy. The crystal structures of six nonnucleoside inhibitors of HIV-1 reverse transcriptase (RT) derivatives of N-benzyl-benzimidazole are reported here. The investigated compounds belong to the group of so called "butterfly like" inhibitors with characteristic two π-electron moieties with an angled orientation. The structural data show the influence of the substituents of the benzimidazole ring on the geometry of the molecule and correlation between the structure of the inhibitor and its biological activity.

  10. Psychoneuroimmunology and HIV-1.

    ERIC Educational Resources Information Center

    Antoni, Michael H.; And Others

    1990-01-01

    Presents evidence describing benefits of behavioral interventions such as aerobic exercise training on both psychological and immunological functioning among high risk human immunodeficiency virus-Type 1 (HIV-1) seronegative and very early stage seropositive homosexual men. HIV-1 infection is cast as chronic disease for which early…

  11. Antiviral Drug Resistance: Mechanisms and Clinical Implications

    PubMed Central

    Chou, Sunwen

    2010-01-01

    Summary Antiviral drug resistance is an increasing concern in immunocompromised patient populations, where ongoing viral replication and prolonged drug exposure lead to the selection of resistant strains. Rapid diagnosis of resistance can be made by associating characteristic viral mutations with resistance to various drugs as determined by phenotypic assays. Management of drug resistance includes optimization of host factors and drug delivery, selection of alternative therapies based on knowledge of mechanisms of resistance, and the development of new antivirals. This article discusses drug resistance in herpesviruses and hepatitis B. PMID:20466277

  12. Persistent Production of an Integrase-Deleted HIV-1 Variant with No Resistance Mutation and Wild-Type Proviral DNA in a Treated Patient

    PubMed Central

    Cotte, Laurent; Saison, Julien; Ramière, Christophe; Ronfort, Corinne; Venet, Fabienne; Tardy, Jean-Claude; Monneret, Guillaume; André, Patrice

    2015-01-01

    Abstract An HIV-infected patient presenting an unexpected viral escape under combined antiretroviral treatment is described. The virus isolated from plasma contained a large deletion in the HIV-1 integrase gene but no known resistance mutation. Nested polymerase chain reactions (PCRs) with patient virus integrase-specific primers and probes were developed and used to detect the mutant from plasma, blood, rectal biopsies, and sperm. The variant progressively emerged during a period of therapy-induced virosuppression, and persisted at a low but detectable level for at least 5 years. Surprisingly, proviral DNA from lymphocytes, rectal cells, and sperm cells was, and remained, mainly wild type. Cellular HIV RNA with the deletion was detected only once from the rectum. The origin and mechanisms underlying this so far not described production at a detectable level are largely hypothetical. This observation raised concern about the ability of defective viruses to spread. PMID:25333615

  13. In Vitro Characterization of GS-8374, a Novel Phosphonate-Containing Inhibitor of HIV-1 Protease with a Favorable Resistance Profile ▿ †

    PubMed Central

    Callebaut, Christian; Stray, Kirsten; Tsai, Luong; Williams, Matt; Yang, Zheng-Yu; Cannizzaro, Carina; Leavitt, Stephanie A.; Liu, Xiaohong; Wang, Kelly; Murray, Bernard P.; Mulato, Andrew; Hatada, Marcos; Priskich, Tina; Parkin, Neil; Swaminathan, Swami; Lee, William; He, Gong-Xin; Xu, Lianhong; Cihlar, Tomas

    2011-01-01

    GS-8374 is a novel bis-tetrahydrofuran HIV-1 protease (PR) inhibitor (PI) with a unique diethylphosphonate moiety. It was selected from a series of analogs containing various di(alkyl)phosphonate substitutions connected via a linker to the para position of a P-1 phenyl ring. GS-8374 inhibits HIV-1 PR with high potency (Ki = 8.1 pM) and with no known effect on host proteases. Kinetic and thermodynamic analysis of GS-8374 binding to PR demonstrated an extremely slow off rate for the inhibitor and favorable contributions of both the enthalpic and entropic components to the total free binding energy. GS-8374 showed potent antiretroviral activity in T-cell lines, primary CD4+ T cells (50% effective concentration [EC50] = 3.4 to 11.5 nM), and macrophages (EC50 = 25.5 nM) and exhibited low cytotoxicity in multiple human cell types. The antiviral potency of GS-8374 was only moderately affected by human serum protein binding, and its combination with multiple approved antiretrovirals showed synergistic effects. When it was tested in a PhenoSense assay against a panel of 24 patient-derived viruses with high-level PI resistance, GS-8374 showed lower mean EC50s and lower fold resistance than any of the clinically approved PIs. Similar to other PIs, in vitro hepatic microsomal metabolism of GS-8374 was efficiently blocked by ritonavir, suggesting a potential for effective pharmacokinetic boosting in vivo. In summary, results from this broad in vitro pharmacological profiling indicate that GS-8374 is a promising candidate to be further assessed as a new antiretroviral agent with potential for clinical efficacy in both treatment-naïve and -experienced patients. PMID:21245449

  14. HIV-1 assembly in macrophages

    PubMed Central

    2010-01-01

    The molecular mechanisms involved in the assembly of newly synthesized Human Immunodeficiency Virus (HIV) particles are poorly understood. Most of the work on HIV-1 assembly has been performed in T cells in which viral particle budding and assembly take place at the plasma membrane. In contrast, few studies have been performed on macrophages, the other major target of HIV-1. Infected macrophages represent a viral reservoir and probably play a key role in HIV-1 physiopathology. Indeed macrophages retain infectious particles for long periods of time, keeping them protected from anti-viral immune response or drug treatments. Here, we present an overview of what is known about HIV-1 assembly in macrophages as compared to T lymphocytes or cell lines. Early electron microscopy studies suggested that viral assembly takes place at the limiting membrane of an intracellular compartment in macrophages and not at the plasma membrane as in T cells. This was first considered as a late endosomal compartment in which viral budding seems to be similar to the process of vesicle release into multi-vesicular bodies. This view was notably supported by a large body of evidence involving the ESCRT (Endosomal Sorting Complex Required for Transport) machinery in HIV-1 budding, the observation of viral budding profiles in such compartments by immuno-electron microscopy, and the presence of late endosomal markers associated with macrophage-derived virions. However, this model needs to be revisited as recent data indicate that the viral compartment has a neutral pH and can be connected to the plasma membrane via very thin micro-channels. To date, the exact nature and biogenesis of the HIV assembly compartment in macrophages remains elusive. Many cellular proteins potentially involved in the late phases of HIV-1 cycle have been identified; and, recently, the list has grown rapidly with the publication of four independent genome-wide screens. However, their respective roles in infected cells

  15. Coreceptor Usage, Diversity, and Divergence in Drug-Naive and Drug-Exposed Individuals from Malawi, Infected with HIV-1 Subtype C for More Than 20 Years

    PubMed Central

    Seager, Ishla; Travers, Simon A.; Leeson, Michael D.; Crampin, Amelia C.; French, Neil; Glynn, Judith R.

    2014-01-01

    Abstract There are few cohorts of individuals who have survived infection with HIV-1 for more than 20 years, reported and followed in the literature, and even fewer from Africa. Here we present data on a cohort of subtype C-infected individuals from rural northern Malawi. By sequencing multiple clones from long-term survivors at different time points, and using multiple genotyping approaches, we show that 5 of the 11 individuals are predicted as CXCR4 using (by ≥3/5 predictors) but only one individual is predicted as CXCR4 using by all five algorithms. Using any one genotyping approach overestimates the number of predicted CXCR4 sequences. Patterns of diversity and divergence were variable between the HIV-1 long-term survivors with some individuals showing very small amounts of variation and change, and others showing a greater amount; both patterns are consistent with what has been described in the literature. PMID:24925099

  16. Pharmacological cyclin-dependent kinase inhibitors as HIV-1 antiviral therapeutics.

    PubMed

    de la Fuente, Cynthia; Maddukuri, Anil; Kehn, Kylene; Baylor, Shanese Y; Deng, Longwen; Pumfery, Anne; Kashanchi, Fatah

    2003-04-01

    Human immunodeficiency virus type 1 (HIV-1) can infect quiescent cells; however, viral production is restricted to actively proliferating cells. Recent evidence has indicated that HIV-1 viral proteins, Vpr and Tat, perturb the cell cycle to optimize HIV-1 replication. Vpr arrests the cell cycle at G2 by inactivating the cyclin B/cdk1 complex. Tat regulates the cell cycle by altering factors involved in proliferation and differentiation (i.e. the cdk inhibitor p21/waf1) and associating with cyclin/cdk complexes (i.e. cyclin E/cdk2, cyclin H/cdk7, and cyclin T/cdk9). These studies indicate the importance of host cellular factors, such as cyclin/cdk complexes, in regulating HIV-1 replication and therefore represent novel targets for antiviral therapeutics. Recently, the efficacy of pharmalogical cdk inhibitors (PCIs) in abrogating viral replication has been under development. To date there are 25-30 PCIs that have been synthesized against known cdks, several of which have been shown to inhibit HIV-1 and other AIDS-associated viruses in vitro and in vivo. Targeting these critical cyclin/cdk complexes needed for viral propagation may solve the problems inherent in current HAART therapy, including the emergence of drug-resistant viruses. Thus, PCIs have the potential to become novel therapeutic antiviral drugs that can inhibit HIV-1 transcription and opens the possibility of new avenues of treatment. PMID:15043199

  17. Synthesis of a Vpr-Binding Derivative for Use as a Novel HIV-1 Inhibitor

    PubMed Central

    Hagiwara, Kyoji; Ishii, Hideki; Murakami, Tomoyuki; Takeshima, Shin-nosuke; Chutiwitoonchai, Nopporn; Kodama, Eiichi N.; Kawaji, Kumi; Kondoh, Yasumitsu; Honda, Kaori; Osada, Hiroyuki; Tsunetsugu-Yokota, Yasuko; Suzuki, Masaaki; Aida, Yoko

    2015-01-01

    The emergence of multidrug-resistant viruses compromises the efficacy of anti-human immunodeficiency virus type 1 (HIV-1) therapy and limits treatment options. Therefore, new targets that can be used to develop novel antiviral agents need to be identified. We previously identified a potential parent compound, hematoxylin, which suppresses the nuclear import of HIV-1 via the Vpr-importin α interaction and inhibits HIV-1 replication in a Vpr-dependent manner by blocking nuclear import of the pre-integration complex. However, it was unstable. Here, we synthesized a stable derivative of hematoxylin that bound specifically and stably to Vpr and inhibited HIV-1 replication in macrophages. Furthermore, like hematoxylin, the derivative inhibited nuclear import of Vpr in an in vitro nuclear import assay, but had no effect on Vpr-induced G2/M phase cell cycle arrest or caspase activity. Interestingly, this derivative bound strongly to amino acid residues 54–74 within the C-terminal α-helical domain (αH3) of Vpr. These residues are highly conserved among different HIV strains, indicating that this region is a potential target for drug-resistant HIV-1 infection. Thus, we succeeded in developing a stable hematoxylin derivative that bound directly to Vpr, suggesting that specific inhibitors of the interaction between cells and viral accessory proteins may provide a new strategy for the treatment of HIV-1 infection. PMID:26701275

  18. Compartmentalization of drug resistance-associated mutations in a treatment-naive HIV-infected female.

    PubMed

    Tirado, Grissell; Jove, Gloria; Kumar, Rakesh; Noel, Richard J; Reyes, Evelyn; Sepulveda, Gladys; Yamamura, Yasuhiro; Kumar, Anil

    2004-06-01

    Development of a drug-resistant variant of HIV-1 has been one of the major concerns contributing to the transmission of the virus. A 40-year-old woman presented to the clinic with micosis and oral candidiasis. The subject was referred for HIV-1 diagnosis. Subsequent investigations revealed a very low CD4 T cell count (48 cell/microl blood) and high plasma HIV-1 RNA load (4.33 x 10(5) copy/ml). A 1.3-kb pol fragment was sequenced in virus collected from plasma and the vaginal compartment. Plasma virus had no mutation in reverse transcriptase and one mutation in protease (L63P). On the other hand vaginal virus contained L63P and M184V mutations in protease and reverse transcriptase, respectively. These mutations were accompanied by several other mutations in previously identified CTL epitopic regions of the two genes. In the absence of antiretroviral treatment, a drug-resistant mutant was thought to develop because of immune pressure. This is the first report describing the role of immune pressure in the development of a drug-resistant virus. PMID:15242547

  19. Suppression of HIV-1 Infectivity by Human Glioma Cells.

    PubMed

    Hoque, Sheikh Ariful; Tanaka, Atsushi; Islam, Salequl; Ahsan, Gias Uddin; Jinno-Oue, Atsushi; Hoshino, Hiroo

    2016-05-01

    HIV-1 infection to the central nervous system (CNS) is very common in AIDS patients. The predominant cell types infected in the brain are monocytes and macrophages, which are surrounded by several HIV-1-resistant cell types, such as astrocytes, oligodendrocytes, neurons, and microvascular cells. The effect of these HIV-1-resistant cells on HIV-1 infection is largely unknown. In this study, we examined the stability of HIV-1 cultured with several human glioblastoma cell lines, for example, NP-2, U87MG, T98G, and A172, to determine whether these HIV-1-resistant brain cells could enhance or suppress HIV-1 infection and thus modulate HIV-1 infection in the CNS. The HIV-1 titer was determined using the MAGIC-5A indicator cell line as well as naturally occurring CD4(+) T cells. We found that the stability of HIV-1 incubated with NP-2 or U87MG cells at 37°C was significantly shorter (half-life, 2.5-4 h) compared to that of HIV-1 incubated with T98G or A172 cells or in culture medium without cells (half-life, 8-18 h). The spent culture media (SCM) of NP-2 and U87MG cells had the ability to suppress both R5- and X4-HIV-1 infection by inhibiting HIV-1 attachment to target cells. This inhibitory effect was eliminated by the treatment of the SCM with chondroitinase ABC but not heparinase, suggesting that the inhibitory factor(s) secreted by NP-2 and U87MG cells was chiefly mediated by chondroitin sulfate (CS) or CS-like moiety. Thus, this study reveals that some but not all glioma cells secrete inhibitory molecules to HIV-1 infection that may contribute in lowering HIV-1 infection in the CNS in vivo. PMID:26650729

  20. Super Learning: An Application to the Prediction of HIV-1 Drug Resistance*

    PubMed Central

    Sinisi, Sandra E.; Polley, Eric C.; Petersen, Maya L.; Rhee, Soo-Yon; van der Laan, Mark J.

    2008-01-01

    Many alternative data-adaptive algorithms can be used to learn a predictor based on observed data. Examples of such learners include decision trees, neural networks, support vector regression, least angle regression, logic regression, and the Deletion/Substitution/Addition algorithm. The optimal learner for prediction will vary depending on the underlying data-generating distribution. In this article we introduce the “super learner”, a prediction algorithm that applies any set of candidate learners and uses cross-validation to select between them. Theory shows that asymptotically the super learner performs essentially as well as or better than any of the candidate learners. In this article we present the theory behind the super learner, and illustrate its performance using simulations. We further apply the super learner to a data example, in which we predict the phenotypic antiretroviral susceptibility of HIV based on viral genotype. Specifically, we apply the super learner to predict susceptibility to a specific protease inhibitor, nelfinavir, using a set of database-derived non-polymorphic treatment-selected mutations. PMID:17402922

  1. Super learning: an application to the prediction of HIV-1 drug resistance.

    PubMed

    Sinisi, Sandra E; Polley, Eric C; Petersen, Maya L; Rhee, Soo-Yon; van der Laan, Mark J

    2007-01-01

    Many alternative data-adaptive algorithms can be used to learn a predictor based on observed data. Examples of such learners include decision trees, neural networks, support vector regression, least angle regression, logic regression, and the Deletion/Substitution/Addition algorithm. The optimal learner for prediction will vary depending on the underlying data-generating distribution. In this article we introduce the "super learner", a prediction algorithm that applies any set of candidate learners and uses cross-validation to select between them. Theory shows that asymptotically the super learner performs essentially as well as or better than any of the candidate learners. In this article we present the theory behind the super learner, and illustrate its performance using simulations. We further apply the super learner to a data example, in which we predict the phenotypic antiretroviral susceptibility of HIV based on viral genotype. Specifically, we apply the super learner to predict susceptibility to a specific protease inhibitor, nelfinavir, using a set of database-derived non-polymorphic treatment-selected mutations. PMID:17402922

  2. NMR characterization of HIV-1 reverse transcriptase binding to various non-nucleoside reverse transcriptase inhibitors with different activities

    PubMed Central

    Thammaporn, Ratsupa; Yagi-Utsumi, Maho; Yamaguchi, Takumi; Boonsri, Pornthip; Saparpakorn, Patchreenart; Choowongkomon, Kiattawee; Techasakul, Supanna; Kato, Koichi; Hannongbua, Supa

    2015-01-01

    Human immunodeficiency virus type 1 reverse transcriptase (HIV-1 RT) is an important target for antiviral therapy against acquired immunodeficiency syndrome. However, the efficiency of available drugs is impaired most typically by drug-resistance mutations in this enzyme. In this study, we applied a nuclear magnetic resonance (NMR) spectroscopic technique to the characterization of the binding of HIV-1 RT to various non-nucleoside reverse transcriptase inhibitors (NNRTIs) with different activities, i.e., nevirapine, delavirdine, efavirenz, dapivirine, etravirine, and rilpivirine. 1H-13C heteronuclear single-quantum coherence (HSQC) spectral data of HIV-1 RT, in which the methionine methyl groups of the p66 subunit were selectively labeled with 13C, were collected in the presence and absence of these NNRTIs. We found that the methyl 13C chemical shifts of the M230 resonance of HIV-1 RT bound to these drugs exhibited a high correlation with their anti-HIV-1 RT activities. This methionine residue is located in proximity to the NNRTI-binding pocket but not directly involved in drug interactions and serves as a conformational probe, indicating that the open conformation of HIV-1 RT was more populated with NNRTIs with higher inhibitory activities. Thus, the NMR approach offers a useful tool to screen for novel NNRTIs in developing anti-HIV drugs. PMID:26510386

  3. Discovery of potent HIV-1 non-nucleoside reverse transcriptase inhibitors from arylthioacetanilide structural motif.

    PubMed

    Li, Wenxin; Li, Xiao; De Clercq, Erik; Zhan, Peng; Liu, Xinyong

    2015-09-18

    The poor pharmacokinetics, side effects and particularly the rapid emergence of drug resistance compromise the efficiency of the clinically used anti-HIV drugs. Therefore, the discovery of novel and effective NNRTIs is still an extremely primary mission. Arylthioacetanilide family is one of the highly active HIV-1 NNRTIs against wide-type (WT) HIV-1 and a wide range of drug-resistant mutant strains. Especially, VRX-480773 and RDEA806 have been chosen as candidates for further clinical studies. In this article, we review the discovery and development of the arylthioacetanilides, and, especially, pay much attention to the structural modifications, SARs conclusions and molecular modeling. Moreover, several medicinal chemistry strategies to overcome drug resistance involved in the optimization process of arylthioacetanilides are highlighted, providing valuable clues for further investigations. PMID:26276432

  4. A phylogenetic and Markov model approach for the reconstruction of mutational pathways of drug resistance

    PubMed Central

    Buendia, Patricia; Cadwallader, Brice; DeGruttola, Victor

    2009-01-01

    Motivation: Modern HIV-1, hepatitis B virus and hepatitis C virus antiviral therapies have been successful at keeping viruses suppressed for prolonged periods of time, but therapy failures attributable to the emergence of drug resistant mutations continue to be a distressing reminder that no therapy can fully eradicate these viruses from their host organisms. To better understand the emergence of drug resistance, we combined phylogenetic and statistical models of viral evolution in a 2-phase computational approach that reconstructs mutational pathways of drug resistance. Results: The first phase of the algorithm involved the modeling of the evolution of the virus within the human host environment. The inclusion of longitudinal clonal sequence data was a key aspect of the model due to the progressive fashion in which multiple mutations become linked in the same genome creating drug resistant genotypes. The second phase involved the development of a Markov model to calculate the transition probabilities between the different genotypes. The proposed method was applied to data from an HIV-1 Efavirenz clinical trial study. The obtained model revealed the direction of evolution over time with greater detail than previous models. Our results show that the mutational pathways facilitate the identification of fast versus slow evolutionary pathways to drug resistance. Availability: Source code for the algorithm is publicly available at http://biorg.cis.fiu.edu/vPhyloMM/ Contact: pbuendia@miami.edu PMID:19654117

  5. HIV-1 A1 Subtype Epidemic in Italy Originated from Africa and Eastern Europe and Shows a High Frequency of Transmission Chains Involving Intravenous Drug Users

    PubMed Central

    Lai, Alessia; Bozzi, Giorgio; Franzetti, Marco; Binda, Francesca; Simonetti, Francesco R.; De Luca, Andrea; Micheli, Valeria; Meraviglia, Paola; Bagnarelli, Patrizia; Di Biagio, Antonio; Monno, Laura; Saladini, Francesco; Zazzi, Maurizio; Zehender, Gianguglielmo; Ciccozzi, Massimo; Balotta, Claudia

    2016-01-01

    Background Subtype A accounts for only 12% of HIV-1 infections worldwide but predominates in Russia and Former Soviet Union countries of Eastern Europe. After an early propagation via heterosexual contacts, this variant spread explosively among intravenous drug users. A distinct A1 variant predominates in Greece and Albania, which penetrated directly from Africa. Clade A1 accounts for 12.5% of non-B subtypes in Italy, being the most frequent after F1 subtype. Aim Aim of this study was to investigate the circulation of A1 subtype in Italy and trace its origin and diffusion through phylogenetic and phylodynamic approaches. Results The phylogenetic analysis of 113 A1 pol sequences included in the Italian ARCA database, indicated that 71 patients (62.8%) clustered within 5 clades. A higher probability to be detected in clusters was found for patients from Eastern Europe and Italy (88.9% and 60.4%, respectively) compared to those from Africa (20%) (p < .001). Higher proportions of clustering sequences were found in intravenous drug users with respect to heterosexuals (85.7% vs. 59.3%, p = .056) and in women with respect to men (81.4% vs. 53.2%, p < .006). Subtype A1 dated phylogeny indicated an East African origin around 1961. Phylogeographical reconstruction highlighted 3 significant groups. One involved East European and some Italian variants, the second encompassed some Italian and African strains, the latter included the majority of viruses carried by African and Italian subjects and all viral sequences from Albania and Greece. Conclusions Subtype A1 originated in Central Africa and spread among East European countries in 1982. It entered Italy through three introduction events: directly from East Africa, from Albania and Greece, and from the area encompassing Moldavia and Ukraine. As in previously documented A1 epidemics of East European countries, HIV-1 A1 subtype spread in Italy in part through intravenous drug users. However, Eastern European women contributed

  6. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules

    PubMed Central

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S. Y.

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This “shock” approach is then followed by “kill” of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  7. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules.

    PubMed

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  8. Drug resistance in Giardia duodenalis.

    PubMed

    Ansell, Brendan R E; McConville, Malcolm J; Ma'ayeh, Showgy Y; Dagley, Michael J; Gasser, Robin B; Svärd, Staffan G; Jex, Aaron R

    2015-11-01

    Giardia duodenalis is a microaerophilic parasite of the human gastrointestinal tract and a major contributor to diarrheal and post-infectious chronic gastrointestinal disease world-wide. Treatment of G. duodenalis infection currently relies on a small number of drug classes. Nitroheterocyclics, in particular metronidazole, have represented the front line treatment for the last 40 years. Nitroheterocyclic-resistant G. duodenalis have been isolated from patients and created in vitro, prompting considerable research into the biomolecular mechanisms of resistance. These compounds are redox-active and are believed to damage proteins and DNA after being activated by oxidoreductase enzymes in metabolically active cells. In this review, we explore the molecular phenotypes of nitroheterocyclic-resistant G. duodenalis described to date in the context of the protist's unusual glycolytic and antioxidant systems. We propose that resistance mechanisms are likely to extend well beyond currently described resistance-associated enzymes (i.e., pyruvate ferredoxin oxidoreductases and nitroreductases), to include NAD(P)H- and flavin-generating pathways, and possibly redox-sensitive epigenetic regulation. Mechanisms that allow G. duodenalis to tolerate oxidative stress may lead to resistance against both oxygen and nitroheterocyclics, with implications for clinical control. The present review highlights the potential for systems biology tools and advanced bioinformatics to further investigate the multifaceted mechanisms of nitroheterocyclic resistance in this important pathogen. PMID:25922317

  9. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

    PubMed

    Hightower, Kendra E; Wang, Ruolan; Deanda, Felix; Johns, Brian A; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H; Carter, H Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; Kobayashi, Masanori; Underwood, Mark R

    2011-10-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile. PMID:21807982

  10. Flap Conformations in HIV-1 Protease are Altered by Mutations

    NASA Astrophysics Data System (ADS)

    Fanucci, Gail; Blackburn, Mandy; Veloro, Angelo; Galiano, Luis; Fangu, Ding; Simmerling, Carlos

    2009-03-01

    HIV-1 protease (PR) is an enzyme that is a major drug target in the treatment of AIDS. Although the structure and function of HIV-1 PR have been studied for over 20 years, questions remain regarding the conformations and dynamics of the β-hairpin turns (flaps) that cover the active site cavity. Distance measurements with pulsed EPR spectroscopy of spin labeled constructs of HIV-1 PR have been used to characterize the flap conformations in the apo and inhibitor bound states. From the most probably distances and the breadth of the distance distribution profiles from analysis of the EPR data, insights regarding the flap conformations and flexibility are gained. The EPR results clearly show how drug pressure selected mutations alter the average conformation of the flaps and the degree of opening of the flaps. Molecular dynamics simulations successfully regenerate the experimentally determined distance distribution profiles, and more importantly, provide structural models for full interpretation of the EPR results. By combining experiment and theory to understand the role that altered flap flexibility/conformations play in the mechanism of drug resistance, key insights are gained toward the rational development of new inhibitors of this important enzyme.

  11. YAP and the drug resistance highway

    PubMed Central

    Keren-Paz, Alona; Emmanuel, Rafi

    2016-01-01

    Deciphering mechanisms of drug resistance is crucial to winning the battle against cancer. A new study points to an unexpected function of YAP in drug resistance and illuminates its potential role as a therapeutic target. PMID:25711863

  12. YAP and the drug resistance highway.

    PubMed

    Keren-Paz, Alona; Emmanuel, Rafi; Samuels, Yardena

    2015-03-01

    Deciphering mechanisms of drug resistance is crucial to winning the battle against cancer. A new study points to an unexpected function of YAP in drug resistance and illuminates its potential role as a therapeutic target. PMID:25711863

  13. Pulsed EPR Characterization of HIV-1 Protease Conformational Sampling and Inhibitor-Induced Population Shifts

    PubMed Central

    Liu, Zhanglong; Casey, Thomas M.; Blackburn, Mandy E.; Huang, Xi; Pham, Linh; de Vera, Ian Mitchelle S.; Carter, Jeffrey D.; Kear-Scott, Jamie L.; Veloro, Angelo M.; Galiano, Luis; Fanucci, Gail E.

    2015-01-01

    The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed “curled/tucked”, “closed”, “semi-open” and “wide-open” conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function. PMID:26489725

  14. Pulsed EPR characterization of HIV-1 protease conformational sampling and inhibitor-induced population shifts.

    PubMed

    Liu, Zhanglong; Casey, Thomas M; Blackburn, Mandy E; Huang, Xi; Pham, Linh; de Vera, Ian Mitchelle S; Carter, Jeffrey D; Kear-Scott, Jamie L; Veloro, Angelo M; Galiano, Luis; Fanucci, Gail E

    2016-02-17

    The conformational landscape of HIV-1 protease (PR) can be experimentally characterized by pulsed-EPR double electron-electron resonance (DEER). For this characterization, nitroxide spin labels are attached to an engineered cysteine residue in the flap region of HIV-1 PR. DEER distance measurements from spin-labels contained within each flap of the homodimer provide a detailed description of the conformational sampling of apo-enzyme as well as induced conformational shifts as a function of inhibitor binding. The distance distribution profiles are further interpreted in terms of a conformational ensemble scheme that consists of four unique states termed "curled/tucked", "closed", "semi-open" and "wide-open" conformations. Reported here are the DEER results for a drug-resistant variant clinical isolate sequence, V6, in the presence of FDA approved protease inhibitors (PIs) as well as a non-hydrolyzable substrate mimic, CaP2. Results are interpreted in the context of the current understanding of the relationship between conformational sampling, drug resistance, and kinetic efficiency of HIV-1PR as derived from previous DEER and kinetic data for a series of HIV-1PR constructs that contain drug-pressure selected mutations or natural polymorphisms. Specifically, these collective results support the notion that inhibitor-induced closure of the flaps correlates with inhibitor efficiency and drug resistance. This body of work also suggests DEER as a tool for studying conformational sampling in flexible enzymes as it relates to function. PMID:26489725

  15. Drug Resistance Mechanisms in Mycobacterium tuberculosis

    PubMed Central

    Palomino, Juan Carlos; Martin, Anandi

    2014-01-01

    Tuberculosis (TB) is a serious public health problem worldwide. Its situation is worsened by the presence of multidrug resistant (MDR) strains of Mycobacterium tuberculosis, the causative agent of the disease. In recent years, even more serious forms of drug resistance have been reported. A better knowledge of the mechanisms of drug resistance of M. tuberculosis and the relevant molecular mechanisms involved will improve the available techniques for rapid drug resistance detection and will help to explore new targets for drug activity and development. This review article discusses the mechanisms of action of anti-tuberculosis drugs and the molecular basis of drug resistance in M. tuberculosis. PMID:27025748

  16. Novel HIV-1 Non-nucleoside Reverse Transcriptase Inhibitor Agents: Optimization of Diarylanilines with High Potency against Wild-Type and Rilpivirine-Resistant E138K Mutant Virus.

    PubMed

    Liu, Na; Wei, Lei; Huang, Li; Yu, Fei; Zheng, Weifan; Qin, Bingjie; Zhu, Dong-Qin; Morris-Natschke, Susan L; Jiang, Shibo; Chen, Chin-Ho; Lee, Kuo-Hsiung; Xie, Lan

    2016-04-28

    Three series (6, 13, and 14) of new diarylaniline (DAAN) analogues were designed, synthesized, and evaluated for anti-HIV potency, especially against the E138K viral strain with a major mutation conferring resistance to the new-generation non-nucleoside reverse transcriptase inhibitor drug rilpivirine (1b). Promising new compounds were then assessed for physicochemical and associated pharmaceutical properties, including aqueous solubility, log P value, and metabolic stability, as well as predicted lipophilic parameters of ligand efficiency, ligand lipophilic efficiency, and ligand efficiency-dependent lipophilicity indices, which are associated with ADME property profiles. Compounds 6a, 14c, and 14d showed high potency against the 1b-resistant E138K mutated viral strain as well as good balance between anti-HIV-1 activity and desirable druglike properties. From the perspective of optimizing future NNRTI compounds as clinical trial candidates, computational modeling results provided valuable information about how the R(1) group might provide greater efficacy against the E138K mutant. PMID:27070547

  17. HIV-1 Eradication: Early Trials (and Tribulations).

    PubMed

    Spivak, Adam M; Planelles, Vicente

    2016-01-01

    Antiretroviral therapy (ART) has rendered HIV-1 infection a manageable illness for those with access to treatment. However, ART does not lead to viral eradication owing to the persistence of replication-competent, unexpressed proviruses in long-lived cellular reservoirs. The potential for long-term drug toxicities and the lack of access to ART for most people living with HIV-1 infection have fueled scientific interest in understanding the nature of this latent reservoir. Exploration of HIV-1 persistence at the cellular and molecular level in resting memory CD4(+) T cells, the predominant viral reservoir in patients on ART, has uncovered potential strategies to reverse latency. We review recent advances in pharmacologically based 'shock and kill' HIV-1 eradication strategies, including comparative analysis of early clinical trials. PMID:26691297

  18. Virtual Screening of Indonesian Herbal Database as HIV-1 Protease Inhibitor

    PubMed Central

    Yanuar, Arry; Suhartanto, Heru; Mun׳im, Abdul; Anugraha, Bram Hik; Syahdi, Rezi Riadhi

    2014-01-01

    HIV-1 (Human immunodeficiency virus type 1)׳s infection is considered as one of most harmful disease known by human, the survivability rate of the host reduced significantly when it developed into AIDS. HIV drug resistance is one of the main problems of its treatment and several drug designs have been done to find new leads compound as the cure. In this study, in silico virtual screening approach was used to find lead molecules from the library or database of natural compounds as HIV-1 protease inhibitor. Virtual screening against Indonesian Herbal Database with AutoDock was performed on HIV-1 protease. From the virtual screening, top ten compounds obtained were 8-Hydroxyapigenin 8-(2",4"-disulfatoglucuronide), Isoscutellarein 4'-methyl ether, Amaranthin, Torvanol A, Ursonic acid, 5-Carboxypyranocyanidin 3-O-(6"-O-malonyl-beta-glucopyranoside), Oleoside, Jacoumaric acid, Platanic acid and 5-Carboxypyranocyanidin 3-O-beta-glucopyranoside. PMID:24616554

  19. Genetic Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF74_01B) Identified among Intravenous Drug Users in Malaysia: Recombination History and Phylogenetic Linkage with Previously Defined Recombinant Lineages.

    PubMed

    Cheong, Hui Ting; Chow, Wei Zhen; Takebe, Yutaka; Chook, Jack Bee; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2015-01-01

    In many parts of Southeast Asia, the HIV-1 epidemic has been driven by the sharing of needles and equipment among intravenous drug users (IDUs). Over the last few decades, many studies have proven time and again that the diversity of HIV-1 epidemics can often be linked to the route of infection transmission. That said, the diversity and complexity of HIV-1 molecular epidemics in the region have been increasing at an alarming rate, due in part to the high tendency of the viral RNA to recombine. This scenario was exemplified by the discovery of numerous circulating recombinant forms (CRFs), especially in Thailand and Malaysia. In this study, we characterized a novel CRF designated CRF74_01B, which was identified in six epidemiologically unlinked IDUs in Kuala Lumpur, Malaysia. The near-full length genomes were composed of CRF01_AE and subtype B', with eight breakpoints dispersed in the gag-pol and nef regions. Remarkably, this CRF shared four and two recombination hotspots with the previously described CRF33_01B and the less prevalent CRF53_01B, respectively. Genealogy-based Bayesian phylogenetic analysis of CRF74_01B genomic regions showed that it is closely related to both CRF33_01B and CRF53_01B. This observation suggests that CRF74_01B was probably a direct descendent from specific lineages of CRF33_01B, CRF53_01B and subtype B' that could have emerged in the mid-1990s. Additionally, it illustrated the active recombination processes between prevalent HIV-1 subtypes and recombinants in Malaysia. In summary, we report a novel HIV-1 genotype designated CRF74_01B among IDUs in Kuala Lumpur, Malaysia. The characterization of the novel CRF74_01B is of considerable significance towards the understanding of the genetic diversity and population dynamics of HIV-1 circulating in the region. PMID:26196131

  20. Genetic Characterization of a Novel HIV-1 Circulating Recombinant Form (CRF74_01B) Identified among Intravenous Drug Users in Malaysia: Recombination History and Phylogenetic Linkage with Previously Defined Recombinant Lineages

    PubMed Central

    Cheong, Hui Ting; Chow, Wei Zhen; Takebe, Yutaka; Chook, Jack Bee; Chan, Kok Gan; Al-Darraji, Haider Abdulrazzaq Abed; Koh, Clayton; Kamarulzaman, Adeeba; Tee, Kok Keng

    2015-01-01

    In many parts of Southeast Asia, the HIV-1 epidemic has been driven by the sharing of needles and equipment among intravenous drug users (IDUs). Over the last few decades, many studies have proven time and again that the diversity of HIV-1 epidemics can often be linked to the route of infection transmission. That said, the diversity and complexity of HIV-1 molecular epidemics in the region have been increasing at an alarming rate, due in part to the high tendency of the viral RNA to recombine. This scenario was exemplified by the discovery of numerous circulating recombinant forms (CRFs), especially in Thailand and Malaysia. In this study, we characterized a novel CRF designated CRF74_01B, which was identified in six epidemiologically unlinked IDUs in Kuala Lumpur, Malaysia. The near-full length genomes were composed of CRF01_AE and subtype B', with eight breakpoints dispersed in the gag-pol and nef regions. Remarkably, this CRF shared four and two recombination hotspots with the previously described CRF33_01B and the less prevalent CRF53_01B, respectively. Genealogy-based Bayesian phylogenetic analysis of CRF74_01B genomic regions showed that it is closely related to both CRF33_01B and CRF53_01B. This observation suggests that CRF74_01B was probably a direct descendent from specific lineages of CRF33_01B, CRF53_01B and subtype Bʹ that could have emerged in the mid-1990s. Additionally, it illustrated the active recombination processes between prevalent HIV-1 subtypes and recombinants in Malaysia. In summary, we report a novel HIV-1 genotype designated CRF74_01B among IDUs in Kuala Lumpur, Malaysia. The characterization of the novel CRF74_01B is of considerable significance towards the understanding of the genetic diversity and population dynamics of HIV-1 circulating in the region. PMID:26196131

  1. Identification and characterization of HIV-1 latent viral reservoirs in peripheral blood.

    PubMed

    Chargin, Amanda; Yin, Fangfang; Song, Min; Subramaniam, Srividyabhuvaneswari; Knutson, Grace; Patterson, Bruce K

    2015-01-01

    Plasma viral load and CD4 counts are effective for clinical monitoring, but they do not give a full representation of HIV-1 quasispecies in cellular reservoirs, the major repository of replication-competent HIV-1 in infected individuals. We sought to develop a diagnostic system that might stimulate the replication-competent HIV-1 reservoirs for enhanced clinical monitoring, including selection of antiretroviral regimens. Whole-blood samples from 45 HIV-infected individuals were collected into 1 ViraStim HIV-1 activation tube and 1 EDTA tube. Samples were tested for viral load and cell type-specific HIV-1 replication. Further, 7 matched activated/nonactivated samples were sequenced using the Trugene HIV-1 genotyping kit. The percentage of patients with replication-competent virus in peripheral blood mononuclear cells (PBMCs) varied, depending on the baseline plasma viral load in the EDTA tubes. Six out of 24 patients with a starting plasma viral load of <20 copies/ml (cp/ml), 6 out of 8 patients with starting viral loads of >20 and <1,000 cp/ml, and 8 out of 13 patients with starting viral loads of >1,000 all showed increases in viral replication of >5-fold. These increases came from cellular reservoirs in blood as determined by simultaneous ultrasensitive subpopulation staining/hybridization in situ (SUSHI). When resistance genotypes in plasma from activation tubes were compared to those from EDTA tubes for 7 patients, all patients showed additional mutations in the activation tube, while 3 patients demonstrated additional genotypic resistance determinants. We show that HIV-1 viral replication can be stimulated directly from infected whole blood. The sequencing results showed that 3 of 7 cases demonstrated additional drug resistance following stimulation. PMID:25339403

  2. Cancer Metabolism and Drug Resistance

    PubMed Central

    Rahman, Mahbuba; Hasan, Mohammad Rubayet

    2015-01-01

    Metabolic alterations, driven by genetic and epigenetic factors, have long been known to be associated with the etiology of cancer. Furthermore, accumulating evidence suggest that cancer metabolism is intimately linked to drug resistance, which is currently one of the most important challenges in cancer treatment. Altered metabolic pathways help cancer cells to proliferate at a rate higher than normal, adapt to nutrient limited conditions, and develop drug resistance phenotypes. Application of systems biology, boosted by recent advancement of novel high-throughput technologies to obtain cancer-associated, transcriptomic, proteomic and metabolomic data, is expected to make a significant contribution to our understanding of metabolic properties related to malignancy. Indeed, despite being at a very early stage, quantitative data obtained from the omics platforms and through applications of 13C metabolic flux analysis (MFA) in in vitro studies, researchers have already began to gain insight into the complex metabolic mechanisms of cancer, paving the way for selection of molecular targets for therapeutic interventions. In this review, we discuss some of the major findings associated with the metabolic pathways in cancer cells and also discuss new evidences and achievements on specific metabolic enzyme targets and target-directed small molecules that can potentially be used as anti-cancer drugs. PMID:26437434

  3. A novel chimeric protein-based HIV-1 fusion inhibitor targeting gp41 glycoprotein with high potency and stability.

    PubMed

    Pan, Chungen; Cai, Lifeng; Lu, Hong; Lu, Lu; Jiang, Shibo

    2011-08-12

    T20 (enfuvirtide, Fuzeon) is the first generation HIV-1 fusion inhibitor approved for salvage therapy of HIV-1-infected patients refractory to current antiretroviral drugs. However, its application is limited by the high cost of peptide synthesis, rapid proteolysis, and poor efficacy against emerging drug-resistant strains. Here we reported the design of a novel chimera protein-based fusion inhibitor targeting gp41, TLT35, that uses a flexible 35-mer linker to couple T20 and T1144, the first and next generation HIV-1 fusion inhibitors, respectively. TLT35, which was expressed in Escherichia coli with good yield, showed low nm activity against HIV-1-mediated cell-cell fusion and infection by laboratory-adapted HIV-1 strains (X4 or R5), including T20-resistant variants and primary HIV-1 isolates of clades A to G and group O (R5 or X4R5). TLT35 was stable in human sera and in peripheral blood mononuclear cell culture and was more resistant to proteolysis than either T20 or T1144 alone. Circular dichroism spectra showed that TLT35 folded into a thermally stable conformation with high α-helical content and T(m) value in aqueous solution. It formed a highly stable complex with gp41 N-terminal heptad repeat peptide and blocked formation of the gp41 six-helix-bundle core. These merits combined with an anticipated low production cost for expression of TLT35 in E. coli make this novel protein-based fusion inhibitor a promising candidate for further development as an anti-HIV-1 microbicide or therapeutic for the prevention and treatment of HIV-1 infection. PMID:21690094

  4. Suppression of Drug Resistance in Dengue Virus

    PubMed Central

    Mateo, Roberto; Nagamine, Claude M.

    2015-01-01

    ABSTRACT Dengue virus is a major human pathogen responsible for 400 million infections yearly. As with other RNA viruses, daunting challenges to antiviral design exist due to the high error rates of RNA-dependent RNA synthesis. Indeed, treatment of dengue virus infection with a nucleoside analog resulted in the expected genetic selection of resistant viruses in tissue culture and in mice. However, when the function of the oligomeric core protein was inhibited, no detectable selection of drug resistance in tissue culture or in mice was detected, despite the presence of drug-resistant variants in the population. Suppressed selection of drug-resistant virus correlated with cooligomerization of the targeted drug-susceptible and drug-resistant core proteins. The concept of “dominant drug targets,” in which inhibition of oligomeric viral assemblages leads to the formation of drug-susceptible chimeras, can therefore be used to prevent the outgrowth of drug resistance during dengue virus infection. PMID:26670386

  5. Off-label use of maraviroc in HIV-1-infected paediatric patients in clinical practice.

    PubMed

    Palladino, Claudia; Gómez, María Luisa Navarro; Soler-Palacín, Pere; González-Tomé, María Isabel; De Ory, Santiago J; Espiau, María; Hoyos, Santiago Pérez; León-Leal, Juan Antonio; Méndez, María; Moreno-Pérez, David; Guasch, Claudia Fortuny; Sierra, Antoni Mur; Guruceta, Itziar Pocheville; Guillén, Santiago Moreno; Briz, Verónica

    2015-10-23

    Maraviroc (MVC) is not approved for HIV-1-infected paediatric patients. This is the first assessment of the use of MVC-based salvage therapy in vertically HIV-1-infected paediatric patients in clinical settings. The results suggest that MVC-based salvage therapy is useful in children and adolescents with extensive resistance profile leading to maintained virological suppression in up to 88% of the patients with CCR5-tropic virus. The likelihood of treatment success might increase when MVC is combined with other active drugs. PMID:26544580

  6. Southern African Treatment Resistance Network (SATuRN) RegaDB HIV drug resistance and clinical management database: supporting patient management, surveillance and research in southern Africa.

    PubMed

    Manasa, Justen; Lessells, Richard; Rossouw, Theresa; Naidu, Kevindra; Van Vuuren, Cloete; Goedhals, Dominique; van Zyl, Gert; Bester, Armand; Skingsley, Andrew; Stott, Katharine; Danaviah, Siva; Chetty, Terusha; Singh, Lavanya; Moodley, Pravi; Iwuji, Collins; McGrath, Nuala; Seebregts, Christopher J; de Oliveira, Tulio

    2014-01-01

    Substantial amounts of data have been generated from patient management and academic exercises designed to better understand the human immunodeficiency virus (HIV) epidemic and design interventions to control it. A number of specialized databases have been designed to manage huge data sets from HIV cohort, vaccine, host genomic and drug resistance studies. Besides databases from cohort studies, most of the online databases contain limited curated data and are thus sequence repositories. HIV drug resistance has been shown to have a great potential to derail the progress made thus far through antiretroviral therapy. Thus, a lot of resources have been invested in generating drug resistance data for patient management and surveillance purposes. Unfortunately, most of the data currently available relate to subtype B even though >60% of the epidemic is caused by HIV-1 subtype C. A consortium of clinicians, scientists, public health experts and policy markers working in southern Africa came together and formed a network, the Southern African Treatment and Resistance Network (SATuRN), with the aim of increasing curated HIV-1 subtype C and tuberculosis drug resistance data. This article describes the HIV-1 data curation process using the SATuRN Rega database. The data curation is a manual and time-consuming process done by clinical, laboratory and data curation specialists. Access to the highly curated data sets is through applications that are reviewed by the SATuRN executive committee. Examples of research outputs from the analysis of the curated data include trends in the level of transmitted drug resistance in South Africa, analysis of the levels of acquired resistance among patients failing therapy and factors associated with the absence of genotypic evidence of drug resistance among patients failing therapy. All these studies have been important for informing first- and second-line therapy. This database is a free password-protected open source database available on

  7. HIV Drug Resistance Surveillance Among Jamaican Men Who Have Sex with Men Should Be Prioritized for Reducing HIV Transmission.

    PubMed

    Collins-Fairclough, Aneisha M; Dennis, Ann M; Nelson, Julie A E; Weir, Sharon S; Figueroa, J Peter

    2015-08-01

    The prevalence of human immunodeficiency virus type 1 (HIV-1) is highest among men who have sex with men (MSM) in Jamaica but no genotypic data are available on the virus strains that are responsible for the epidemic among this key population. HIV-1 polymerase (pol) genes from 65 MSM were sequenced and used to predict drug resistance mutations. An HIV drug resistance prevalence of 28% (minimum 13%) was observed among this cohort, with the most frequent mutations conferring resistance to efavirenz, nevirapine, and lamivudine. Phylogenetic analysis of the sequences revealed 10 times the number of linked HIV infections among this cohort than respondent reporting. HIV treatment and prevention efforts in Jamaica could benefit significantly from Pol genotyping of the HIV strains infecting socially vulnerable MSM prior to initiating antiretroviral therapy (ART), as this would guide suppressive ART and unearth HIV transmission clusters to enable more effective delivery of treatment and prevention programs. PMID:26133540

  8. HIV Drug Resistance Surveillance Among Jamaican Men Who Have Sex with Men Should Be Prioritized for Reducing HIV Transmission

    PubMed Central

    Dennis, Ann M.; Nelson, Julie A.E.; Weir, Sharon S.; Figueroa, J. Peter

    2015-01-01

    Abstract The prevalence of human immunodeficiency virus type 1 (HIV-1) is highest among men who have sex with men (MSM) in Jamaica but no genotypic data are available on the virus strains that are responsible for the epidemic among this key population. HIV-1 polymerase (pol) genes from 65 MSM were sequenced and used to predict drug resistance mutations. An HIV drug resistance prevalence of 28% (minimum 13%) was observed among this cohort, with the most frequent mutations conferring resistance to efavirenz, nevirapine, and lamivudine. Phylogenetic analysis of the sequences revealed 10 times the number of linked HIV infections among this cohort than respondent reporting. HIV treatment and prevention efforts in Jamaica could benefit significantly from Pol genotyping of the HIV strains infecting socially vulnerable MSM prior to initiating antiretroviral therapy (ART), as this would guide suppressive ART and unearth HIV transmission clusters to enable more effective delivery of treatment and prevention programs. PMID:26133540

  9. HIV drug resistance interpreted by cumulative versus last genotypes in HIV-infected patients with multiple treatment failures.

    PubMed

    Punyacam, Punthiya; Iemwimangsa, Nareenart; Chantratita, Wasun; Sukasem, Chonlaphat; Sungkanuparph, Somnuek

    2012-04-01

    Genotypic resistance test has been recommended to evaluate HIV drug resistance and guide the effective regimens of antiretroviral therapy (ART) in HIV-infected patients with treatment failure. In patients with multiple treatment failures, drug resistance-associated mutations may disappear due to the loss of selective drug pressure after switching regimens. A cohort study was conducted among HIV-infected patients who had ≥2 genotypic resistance tests during 2003-2011. HIV-1 pol nucleotide sequencing of reverse transcriptase and protease region was carried out using TRUGENE HIV-1 Genotypic Assay. Sequencing data was analyzed using Stanford rule-based interpretation algorithms. Of 54 patients with mean age of 30.1 years, 46.3% were males. HIV-1 subtype A/E was observed in 88.9% of patients. At the latest failure, 55.3% were receiving protease inhibitor-based regimens. Median CD4 and HIV RNA were 167 cells/mm(3) and 22,359 copies/mL. During a median duration of ART of 38.6 months, 72.2%, 22.2%, and 5.6% had 5, 3, and 2 genotype tests, respectively. When compared between using cumulative (CG) and last genotypes (LG), CG interpreted resistance to any drug 59.3% higher than LG did. For NRTI, NNRTI, and PI drug classes, CG interpreted as resistance 42.6%, 27.8%, and 7.4% higher than LG, respectively. The most common drugs that CG interpreted resistance with the higher rate than LG were lamivudine/emtricitabine, nevirapine, efavirenz, etravirine and abacavir. In conclusion, CG interprets HIV drug resistance at a higher rate than LG and may be more accurate to use for selecting the next effective regimen of ART among HIV-infected patients with multiple treatment failures. PMID:22497699

  10. Lentivector Knockdown of CCR5 in Hematopoietic Stem and Progenitor Cells Confers Functional and Persistent HIV-1 Resistance in Humanized Mice

    PubMed Central

    Myburgh, Renier; Ivic, Sandra; Pepper, Michael S.; Gers-Huber, Gustavo; Li, Duo; Audigé, Annette; Rochat, Mary-Aude; Jaquet, Vincent; Regenass, Stephan; Manz, Markus G.; Salmon, Patrick; Krause, Karl-Heinz

    2015-01-01

    ABSTRACT Gene-engineered CD34+ hematopoietic stem and progenitor cells (HSPCs) can be used to generate an HIV-1-resistant immune system. However, a certain threshold of transduced HSPCs might be required for transplantation into mice for creating an HIV-resistant immune system. In this study, we combined CCR5 knockdown by a highly efficient microRNA (miRNA) lentivector with pretransplantation selection of transduced HSPCs to obtain a rather pure population of gene engineered CD34+ cells. Low-level transduction of HSPCs and subsequent sorting by flow cytometry yielded >70% transduced cells. Mice transplanted with these cells showed functional and persistent resistance to a CCR5-tropic HIV strain: viral load was significantly decreased over months, and human CD4+ T cells were preserved. In one mouse, viral mutations, resulting presumably in a CXCR4-tropic strain, overcame HIV resistance. Our results suggest that HSPC-based CCR5 knockdown may lead to efficient control of HIV in vivo. We overcame a major limitation of previous HIV gene therapy in humanized mice in which only a proportion of the cells in chimeric mice in vivo are anti-HIV engineered. Our strategy underlines the promising future of gene engineering HIV-resistant CD34+ cells that produce a constant supply of HIV-resistant progeny. IMPORTANCE Major issues in experimental long-term in vivo HIV gene therapy have been (i) low efficacy of cell transduction at the time of transplantation and (ii) transduction resulting in multiple copies of heterologous DNA in target cells. In this study, we demonstrated the efficacy of a transplantation approach with a selection step for transduced cells that allows transplantation of an enriched population of HSPCs expressing a single (low) copy of a CCR5 miRNA. Efficient maintenance of CD4+ T cells and a low viral titer resulted only when at least 70% of the HIV target cells were genetically modified. These findings imply that clinical protocols of HIV gene therapy require a

  11. Serum adiponectin in HIV-1 and hepatitis C virus mono- and co-infected Kenyan injection drug users

    PubMed Central

    Ndombi, Eric M; Budambula, Valentine; Webale, Mark K; Musumba, Francis O; Wesongah, Jesca O; Mibei, Erick; Ahmed, Aabid A; Lihana, Raphael; Were, Tom

    2015-01-01

    Adiponectin is an important marker of anthropometric profiles of adipose tissue. However, association of adiponectin and adiposity in HIV mono- and co-infected and hepatitis (HCV) injection drug users (IDUs) has not been elucidated. Therefore, the relationship of total adiponectin levels with anthropometric indices of adiposity was examined in HIV mono-infected (anti-retroviral treatment, ART-naive, n=16 and -experienced, n=34); HCV mono-infected, n=36; HIV and HCV co-infected (ART-naive, n=5 and -experienced, n=13); uninfected, n=19 IDUs; and healthy controls, n=16 from coastal Kenya. Anthropometric indices of adiposity were recorded and total circulating adiponectin levels were measured in serum samples using enzyme-linked immunosorbent assay. Adiponectin levels differed significantly amongst the study groups (P<0.0001). Post-hoc analyses revealed decreased levels in HIV mono-infected ART-naive IDUs in comparison to uninfected IDUs (P<0.05) and healthy controls (P<0.05). However, adiponectin levels were elevated in HCV mono-infected IDUs relative to HIV mono-infected ART-naive (P<0.001) and -experienced (P<0.001) as well as HIV and HCV co-infected ART-naive (P<0.05) IDUs. Furthermore, adiponectin correlated with weight (ρ=0.687; P=0.003) and BMI (ρ=0.598; P=0.014) in HIV mono-infected ART-naive IDUs; waist circumference (ρ=−0.626; P<0.0001), hip (ρ=−0.561; P=0.001) circumference, and bust-to-waist ratio (ρ=0.561; P=0.001) in HIV mono-infected ART-experienced IDUs; waist girth (ρ=0.375; P=0.024) in HCV mono-infected IDUs; and waist-to-hip ratio (ρ=−0.872; P=0.048) in HIV and HCV co-infected ART-naive IDUs. Altogether, these results suggest suppression of adiponectin production in treatment-naive HIV mono-infected IDUs and that circulating adiponectin is a useful surrogate marker of altered adiposity in treatment-naive and -experienced HIV and HCV mono- and co-infected IDUs. PMID:26306727

  12. Activities, crystal structures and molecular dynamics of dihydro-1H-isoindole derivatives, inhibitors of HIV-1 integrase

    PubMed Central

    Métifiot, Mathieu; Maddali, Kasthuraiah; Johnson, Barry C.; Hare, Stephen; Smith, Steven J.; Zhao, XueZhi; Marchand, Christophe; Burke, Terrence R.; Hughes, Stephen H.; Cherepanov, Peter; Pommier, Yves

    2013-01-01

    Based on a series of lactam and phthalimide derivatives that inhibit HIV-1 integrase, we developed a new derivative, XZ-259, with biochemical and antiviral activities comparable to raltegravir. We determined the crystal structures of XZ-259 and four other derivatives in complex with the prototype foamy virus intasome. The compounds bind at the integrase-Mg2+-DNA interface of the integrase active site. In biochemical and antiviral assays, XZ-259 inhibits raltegravir-resistant HIV-1 integrases harboring the Y143R mutation. Molecular modeling is also presented suggesting that XZ-259 can bind in the HIV-1 intasome with its dimethyl sulfonamide group adopting two opposite orientations. Molecular dynamics analyses of the HIV-1 intasome highlight the importance of the viral DNA in drug potency. PMID:23075516

  13. Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening.

    PubMed

    La, Jennifer; Latham, Catherine F; Tinetti, Ricky N; Johnson, Adam; Tyssen, David; Huber, Kelly D; Sluis-Cremer, Nicolas; Simpson, Jamie S; Headey, Stephen J; Chalmers, David K; Tachedjian, Gilda

    2015-06-01

    Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment. PMID:26038551

  14. Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

    PubMed Central

    La, Jennifer; Latham, Catherine F.; Tinetti, Ricky N.; Johnson, Adam; Tyssen, David; Huber, Kelly D.; Sluis-Cremer, Nicolas; Simpson, Jamie S.; Headey, Stephen J.; Chalmers, David K.; Tachedjian, Gilda

    2015-01-01

    Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment. PMID:26038551

  15. 8-Modified-2′-Deoxyadenosine Analogues Induce Delayed Polymerization Arrest during HIV-1 Reverse Transcription

    PubMed Central

    Sleiman, Marwan; Smyth, Redmond; Ben Gaied, Nouha; Barhoum, Patrick; Laumond, Géraldine; Bec, Guillaume; Götte, Matthias; Mak, Johnson; Aubertin, Anne-Marie; Burger, Alain; Marquet, Roland

    2011-01-01

    The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2′-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2′-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication. PMID:22087320

  16. Structure-activity relationship studies on clinically relevant HIV-1 NNRTIs.

    PubMed

    Rawal, R K; Murugesan, V; Katti, S B

    2012-01-01

    In addition to the nucleoside reverse transcriptase inhibitors (NRTIs), protease inhibitors (PIs) and integrase inhibitors (INIs), nonnucleoside reverse transcriptase inhibitors (NNRTIs) have contributed significantly in the treatment of HIV-1 infections. More than 60 structurally different classes of compounds have been identified as NNRTIs, which are specifically inhibiting HIV-1 reverse transcriptase (RT). Five NNRTIs (nevirapine, delavirdine, efavirenz, etravirine and rilpivirine) have been approved by US Food and Drug Administration (FDA) for clinical use. The NNRTIs bind with a specific 'pocket' site of HIV-1 RT (allosteric site) that is closely associated with the NRTI binding site. Due to mutations of the amino acid residues surrounding the NNRTI-binding site, NNRTIs are notorious for rapidly eliciting resistance. Though, the emergence of resistant HIV strains can be circumvented if the NNRTIs are used either alone or in combination with NRTIs (AZT, 3TC, ddI, ddC, TVD or d4T) and PIs (Indinavir, nelfinavir, saquinavir, ritonavir and lopinavir etc.) as shown by both a decrease in plasma HIV-1 RNA levels and increased CD4 T-cells. Here we are going to discuss recent advances in structure activity relationship studies on nevirapine, delavirdine, efavirenz, etravirine, rilpivirine and 4-thiazolidinones (privileged scaffold) HIV-1 NNRTIs. PMID:22998569

  17. Restricting HIV-1 pathways for escape using rationally designed anti–HIV-1 antibodies

    PubMed Central

    Klein, Florian; Horwitz, Joshua A.; Halper-Stromberg, Ariel; Sather, D. Noah; Marcovecchio, Paola M.; Lee, Terri; West, Anthony P.; Gao, Han; Seaman, Michael S.; Stamatatos, Leonidas; Nussenzweig, Michel C.; Bjorkman, Pamela J.

    2013-01-01

    Recently identified broadly neutralizing antibodies (bNAbs) that potently neutralize most HIV-1 strains are key to potential antibody-based therapeutic approaches to combat HIV/AIDS in the absence of an effective vaccine. Increasing bNAb potencies and resistance to common routes of HIV-1 escape through mutation would facilitate their use as therapeutics. We previously used structure-based design to create the bNAb NIH45-46G54W, which exhibits superior potency and/or breadth compared with other bNAbs. We report new, more effective NIH45-46G54W variants designed using analyses of the NIH45-46–gp120 complex structure and sequences of NIH45-46G54W–resistant HIV-1 strains. One variant, 45-46m2, neutralizes 96% of HIV-1 strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti–HIV-1 antibody to date. A description of its mechanism is presented based on a 45-46m2–gp120 crystal structure. A second variant, 45-46m7, designed to thwart HIV-1 resistance to NIH45-46G54W arising from mutations in a gp120 consensus sequence, targets a common route of HIV-1 escape. In combination, 45-46m2 and 45-46m7 reduce the possible routes for the evolution of fit viral escape mutants in HIV-1YU-2–infected humanized mice, with viremic control exhibited when a third antibody, 10–1074, was added to the combination. PMID:23712429

  18. Solvation effects are responsible for the reduced inhibitor affinity of some HIV-1 PR mutants.

    PubMed Central

    Sussman, F.; Villaverde, M. C.; Davis, A.

    1997-01-01

    The formulation of HIV-1 PR inhibitors as anti-viral drugs has been hindered by the appearance of protease strains that present drug resistance to these compounds. The mechanism by which the HIV-1 PR mutants lower their affinity for the inhibitor is not yet fully understood. We have applied a modified Poisson-Boltzmann method to the evaluation of the molecular interactions that contribute to the lowering of the inhibitor affinity to some polar mutants at position 82. These strains present drug resistance behavior and hence are ideally suited for these studies. Our results indicate that the reduction in binding affinity is due to the solvation effects that penalize the binding to the more polar mutants. The inhibitor binding ranking of the different mutants can be explained from the analysis of the different components of our free energy scoring function. PMID:9144773

  19. Susceptibility Testing by Polymerase Chain Reaction DNA Quantitation: A Method to Measure Drug Resistance of Human Immunodeficiency Virus Type 1 Isolates

    NASA Astrophysics Data System (ADS)

    Eron, Joseph J.; Gorczyca, Paul; Kaplan, Joan C.; D'Aquila, Richard T.

    1992-04-01

    Polymerase chain reaction (PCR) DNA quantitation (PDQ) susceptibility testing rapidly and directly measures nucleoside sensitivity of human immunodeficiency virus type 1 (HIV-1) isolates. PCR is used to quantitate the amount of HIV-1 DNA synthesized after in vitro infection of peripheral blood mononuclear cells. The relative amounts of HIV-1 DNA in cell lysates from cultures maintained at different drug concentrations reflect drug inhibition of virus replication. The results of PDQ susceptibility testing of 2- or 3-day cultures are supported by