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  1. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering

    PubMed Central

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph

    2015-01-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838–3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective—the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)—and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  2. Long-Range HIV Genotyping Using Viral RNA and Proviral DNA for Analysis of HIV Drug Resistance and HIV Clustering.

    PubMed

    Novitsky, Vlad; Zahralban-Steele, Melissa; McLane, Mary Fran; Moyo, Sikhulile; van Widenfelt, Erik; Gaseitsiwe, Simani; Makhema, Joseph; Essex, M

    2015-08-01

    The goal of the study was to improve the methodology of HIV genotyping for analysis of HIV drug resistance and HIV clustering. Using the protocol of Gall et al. (A. Gall, B. Ferns, C. Morris, S. Watson, M. Cotten, M. Robinson, N. Berry, D. Pillay, and P. Kellam, J Clin Microbiol 50:3838-3844, 2012, doi:10.1128/JCM.01516-12), we developed a robust methodology for amplification of two large fragments of viral genome covering about 80% of the unique HIV-1 genome sequence. Importantly, this method can be applied to both viral RNA and proviral DNA amplification templates, allowing genotyping in HIV-infected subjects with suppressed viral loads (e.g., subjects on antiretroviral therapy [ART]). The two amplicons cover critical regions across the HIV-1 genome (including pol and env), allowing analysis of mutations associated with resistance to protease inhibitors, reverse transcriptase inhibitors (nucleoside reverse transcriptase inhibitors [NRTIs] and nonnucleoside reverse transcriptase inhibitors [NNRTIs]), integrase strand transfer inhibitors, and virus entry inhibitors. The two amplicons generated span 7,124 bp, providing substantial sequence length and numbers of informative sites for comprehensive phylogenic analysis and greater refinement of viral linkage analyses in HIV prevention studies. The long-range HIV genotyping from proviral DNA was successful in about 90% of 212 targeted blood specimens collected in a cohort where the majority of patients had suppressed viral loads, including 65% of patients with undetectable levels of HIV-1 RNA loads. The generated amplicons could be sequenced by different methods, such as population Sanger sequencing, single-genome sequencing, or next-generation ultradeep sequencing. The developed method is cost-effective-the cost of the long-range HIV genotyping is under $140 per subject (by Sanger sequencing)-and has the potential to enable the scale up of public health HIV prevention interventions. PMID:26041893

  3. Comparison of Matrix-Based and Filter Paper-Based Systems for Transport of Plasma for HIV-1 RNA Quantification and Amplicon Preparation for Genotyping.

    PubMed

    Levine, Molly; Beck, Ingrid; Styrchak, Sheila; Pepper, Gregory; Frenkel, Lisa

    2016-07-01

    Two ambient-temperature, dry plasma transport systems, ViveST tubes and RNASound RNA sampling cards, and two extraction methods were compared to frozen plasma for HIV-1 RNA recovery. Significant RNA loss occurred: ViveST+MiniMag > ViveST+QIAamp > RNASound+QIAamp. RNA loss and low specimen volumes may affect the sensitivity of genotyping specimens with HIV-1 RNA of <4.70 log10 copies/ml. PMID:27076660

  4. HIV Genotypic Resistance Testing

    MedlinePlus

    ... be limited. Home Visit Global Sites Search Help? HIV Antiretroviral Drug Resistance Testing, Genotypic Share this page: Was this page helpful? Also known as: Anti-retroviral Drug Resistance Testing; ARV Resistance Testing Formal name: ...

  5. Genotypic Susceptibility Scores and HIV Type 1 RNA Responses in Treatment-Experienced Subjects with HIV Type 1 Infection

    PubMed Central

    Anderson, Jeffrey A.; Jiang, Hongyu; Ding, Xiao; Petch, Leslie; Journigan, Terri; Fiscus, Susan A.; Haubrich, Richard; Katzenstein, David; Swanstrom, Ronald; Gulick, Roy M.

    2010-01-01

    This study compared the role of genotypic susceptibility scores (GSS) as a predictor of virologic response in a group (n = 234) of HIV-infected, protease inhibitor (PI)-experienced subjects. Two scoring methods [discrete genotypic susceptibility score (dGSS) and continuous genotypic susceptibility score (cGSS)] were developed. Each drug in the subject's regimen was given a binary susceptibility score using Stanford inferred drug resistance scores to calculate the dGSS. In contrast to the dGSS, the cGSS model was designed to reflect partial susceptibility to a drug. Both GSS were independent predictors of week 16 virologic response. We also compared the GSS to a phenotypic susceptibility score (PSS) model on a subset of subjects that had both GSS and PSS performed, and found that both models were predictive of virologic response. Genotypic analyses at enrollment showed that subjects who were virologic nonresponders at week 16 revealed enrichment of several mutated codons associated with nucleoside reverse transcriptase inhibitors (NRTI) (codons 67, 69, 70, 118, 215, and 219) or PI resistance (codons 10, 24, 71, 73, and 88) compared to subjects who were virologic responders. Regression analyses revealed that protease mutations at codons 24 and 90 were most predictive of poor virologic response, whereas mutations at 82 were associated with enhanced virologic response. Certain NNRTI-associated mutations, such as K103N, were rapidly selected in the absence of NRTIs. These data indicate that GSS may be a useful tool in selecting drug regimens in HIV-1-infected subjects to maximize virologic response and improve treatment outcomes. PMID:18462083

  6. HCV RNA decline in the first 24 hours exhibits high negative predictive value of sustained virologic response in HIV/HCV genotype 1 co-infected patients treated with peginterferon and ribavirin

    PubMed Central

    Laufer, N; Bolcic, F; Rolón, MJ; Martinez, A; Reynoso, R; Pérez, H; Salomón, H; Cahn, P; Quarleri, J

    2011-01-01

    Summary Background Treatment with Peg-interferon and ribavirin (PEG-IFN/RBV) for HIV patients co-infected with hepatitis C virus (HCV) genotype 1 has suboptimal rates of response. Viral kinetics has emerged as one of the best prognostic factors of treatment outcome. Methods Twenty HIV/HCV genotype 1 co-infected patients in treatment with PEG-IFN/RBV, had blood drawn at baseline, 24h, 4, 12, 24, 48, and 72 weeks. HCV-RNA levels were evaluated at each time point. ROC curves were used to evaluate the log10 HCV-RNA decay at 24h that exhibits the best predictive value of achieving response. Genomic characterization of HCV NS5A at both interferon sensitivity-determining region (ISDR) and protein-kinase binding (PKRBD) domains were performed in order to evaluate its heterogeneity and association with 24h HCV-RNA decay and SVR. Results Non-responder patients exhibited a mean of 0.7log10 (SD 0.74log10) HCV-RNA decay at 24h, whereas responder-patients presented 1.6log10 (SD 0.28log10), p=0.04. A reduction in HCV viral load from baseline to 24h of <1.4 had a negative predictive value for achieving SVR of 100% and a positive predictive value of 50%. HCV genotype 1 isolates from patients with a decrease of HCV-RNA at 24h >1.4log10, exhibited 3.1(SD 1.5) amino acids substitutions in ISDR and 4.8(SD 2.3) in PKRBD regions and 1.6(SD 0.7) and 2.4(SD1.3), respectively, in those patients presenting lower reduction in HCV-RNA. Conclusions HIV/HCV genotype 1 co-infected patients with a decrease in HCV-VL at 24h >1.4 log10 are more likely to achieve SVR when treated with PEG-IFN/RBV than those with lower levels of HCV-RNA decay. Along with other host-related and viral-related prognostic factors in HIV/HCV co-infected patients, this very early time point of evaluation could be of relevance in the management of HCV-specific treatment. PMID:21376083

  7. New cryptosporidium genotypes in HIV-infected persons.

    PubMed Central

    Pieniazek, N. J.; Bornay-Llinares, F. J.; Slemenda, S. B.; da Silva, A. J.; Moura, I. N.; Arrowood, M. J.; Ditrich, O.; Addiss, D. G.

    1999-01-01

    Using DNA sequencing and phylogenetic analysis, we identified four distinct Cryptosporidium genotypes in HIV-infected patients: genotype 1 (human), genotype 2 (bovine) Cryptosporidium parvum, a genotype identical to C. felis, and one identical to a Cryptosporidium sp. isolate from a dog. This is the first identification of human infection with the latter two genotypes. PMID:10341184

  8. Accuracy of the TRUGENE HIV-1 Genotyping Kit

    PubMed Central

    Grant, Robert M.; Kuritzkes, Daniel R.; Johnson, Victoria A.; Mellors, John W.; Sullivan, John L.; Swanstrom, Ronald; D'Aquila, Richard T.; Van Gorder, Mark; Holodniy, Mark; Lloyd, Jr., Robert M.; Reid, Caroline; Morgan, Gillian F.; Winslow, Dean L.

    2003-01-01

    Drug resistance and poor virological responses are associated with well-characterized mutations in the viral reading frames that encode the proteins that are targeted by currently available antiretroviral drugs. An integrated system was developed that includes target gene amplification, DNA sequencing chemistry (TRUGENE HIV-1 Genotyping Kit), and hardware and interpretative software (the OpenGene DNA Sequencing System) for detection of mutations in the human immunodeficiency virus type 1 (HIV-1) protease and reverse transcriptase sequences. The integrated system incorporates reverse transcription-PCR from extracted HIV-1 RNA, a coupled amplification and sequencing step (CLIP), polyacrylamide gel electrophoresis, semiautomated analysis of data, and generation of an interpretative report. To assess the accuracy and robustness of the assay system, 270 coded plasma specimens derived from nine patients were sent to six laboratories for blinded analysis. All specimens contained HIV-1 subtype B viruses. Results of 270 independent assays were compared to “gold standard” consensus sequences of the virus populations determined by sequence analysis of 16 to 20 clones of viral DNA amplicons derived from two independent PCRs using primers not used in the kit. The accuracy of the integrated system for nucleotide base identification was 98.7%, and the accuracy for codon identification at 54 sites associated with drug resistance was 97.6%. In a separate analysis of plasma spiked with infectious molecular clones, the assay reproducibly detected all 72 different drug resistance mutations that were evaluated. There were no significant differences in accuracy between laboratories, between technologists, between kit lots, or between days. This integrated assay system for the detection of HIV-1 drug resistance mutations has a high degree of accuracy and reproducibility in several laboratories. PMID:12682149

  9. Polyomavirus JCV excretion and genotype analysis in HIV-infected patients receiving highly active antiretroviral therapy

    NASA Technical Reports Server (NTRS)

    Lednicky, John A.; Vilchez, Regis A.; Keitel, Wendy A.; Visnegarwala, Fehmida; White, Zoe S.; Kozinetz, Claudia A.; Lewis, Dorothy E.; Butel, Janet S.

    2003-01-01

    OBJECTIVE: To assess the frequency of shedding of polyomavirus JC virus (JCV) genotypes in urine of HIV-infected patients receiving highly active antiretroviral therapy (HAART). METHODS: Single samples of urine and blood were collected prospectively from 70 adult HIV-infected patients and 68 uninfected volunteers. Inclusion criteria for HIV-infected patients included an HIV RNA viral load < 1000 copies, CD4 cell count of 200-700 x 106 cells/l, and stable HAART regimen. PCR assays and sequence analysis were carried out using JCV-specific primers against different regions of the virus genome. RESULTS: JCV excretion in urine was more common in HIV-positive patients but not significantly different from that of the HIV-negative group [22/70 (31%) versus 13/68 (19%); P = 0.09]. HIV-positive patients lost the age-related pattern of JCV shedding (P = 0.13) displayed by uninfected subjects (P = 0.01). Among HIV-infected patients significant differences in JCV shedding were related to CD4 cell counts (P = 0.03). Sequence analysis of the JCV regulatory region from both HIV-infected patients and uninfected volunteers revealed all to be JCV archetypal strains. JCV genotypes 1 (36%) and 4 (36%) were the most common among HIV-infected patients, whereas type 2 (77%) was the most frequently detected among HIV-uninfected volunteers. CONCLUSION: These results suggest that JCV shedding is enhanced by modest depressions in immune function during HIV infection. JCV shedding occurred in younger HIV-positive persons than in the healthy controls. As the common types of JCV excreted varied among ethnic groups, JCV genotypes associated with progressive multifocal leukoencephalopathy may reflect demographics of those infected patient populations.

  10. Significant impact of non-B HIV-1 variants genetic diversity in Gabon on plasma HIV-1 RNA quantitation.

    PubMed

    Mouinga-Ondémé, Augustin; Mabika-Mabika, Arsène; Alalade, Patrick; Mongo, Arnaud Delis; Sica, Jeanne; Liégeois, Florian; Rouet, François

    2014-01-01

    Evaluations of HIV-1 RNA viral load assays are lacking in Central Africa. The main objective of our study was to assess the reliability of HIV-1 RNA results obtained with three different assays for samples collected in Gabon. A total of 137 plasma specimens were assessed for HIV-1 RNA using the Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ® version 2.0 assays. It included HIV-1 non-B samples (n = 113) representing six subtypes, 10 CRFs and 18 URFs from patients infected with HIV-1 and treated with antiretrovirals that were found HIV-1 RNA positive (≥300 copies/ml) with the Generic HIV viral load® assay; and samples (n = 24) from untreated individuals infected with HIV-1 but showing undetectable (<300 copies/ml) results with the Biocentric kit. For samples found positive with the Generic HIV viral load® test, correlation coefficients obtained between the three techniques were relatively low (R = 0.65 between Generic HIV viral load® and Abbott RealTime HIV-1®, 0.50 between Generic HIV viral load® and Nuclisens HIV-1 EasyQ®, and 0.66 between Abbott RealTime HIV-1® and Nuclisens HIV-1 EasyQ®). Discrepancies by at least one log10 were obtained for 19.6%, 33.7%, and 20% of samples, respectively, irrespective of genotype. Most of samples (22/24) from untreated study patients, found negative with the Biocentric kit, were also found negative with the two other techniques. In Central Africa, HIV-1 genetic diversity remains challenging for viral load testing. Further studies are required in the same area to confirm the presence of HIV-1 strains that are not amplified with at least two different viral load assays.

  11. Performance Characteristics of the TRUGENE HIV-1 Genotyping Kit and the Opengene DNA Sequencing System

    PubMed Central

    Kuritzkes, Daniel R.; Grant, Robert M.; Feorino, Paul; Griswold, Marshal; Hoover, Marie; Young, Russell; Day, Stephen; Lloyd, Jr., Robert M.; Reid, Caroline; Morgan, Gillian F.; Winslow, Dean L.

    2003-01-01

    The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System are designed to sequence the protease (PR)- and reverse transcriptase (RT)-coding regions of human immunodeficiency virus type 1 (HIV-1) pol. Studies were undertaken to determine the accuracy of this assay system in detecting resistance-associated mutations and to determine the effects of RNA extraction methods, anticoagulants, specimen handling, and potentially interfering substances. Samples were plasma obtained from HIV-infected subjects or seronegative plasma to which viruses derived from wild-type and mutant infectious molecular clones (IMC) of HIV-1 were added. Extraction methods tested included standard and UltraSensitive AMPLICOR HIV-1 MONITOR, QIAGEN viral RNA extraction mini kit, and QIAGEN Ultra HIV extraction kit, and NASBA manual HIV-1 quantitative NucliSens. Sequence data from test sites were compared to a “gold standard” reference sequence to determine the percent agreement. Comparisons between test and reference sequences at the nucleotide level showed 97.5 to 100% agreement. Similar results were obtained regardless of extraction method, regardless of use of EDTA or acid citrate dextrose as anticoagulant, and despite the presence of triglycerides, bilirubin, hemoglobin, antiretroviral drugs, HIV-2, hepatitis C virus (HCV), HBV, cytomegalovirus, human T-cell leukemia virus type 1 (HTLV-1), or HTLV-2. Samples with HIV-1 RNA titers of ≥1,000 copies/ml gave consistent results. The TRUGENE HIV-1 Genotyping Kit and OpenGene DNA Sequencing System consistently generate highly accurate sequence data when tested with IMC-derived HIV and patient samples. PMID:12682150

  12. HIV Infection Affects Streptococcus mutans Levels, but Not Genotypes

    PubMed Central

    Liu, G.; Saxena, D.; Chen, Z.; Norman, R.G.; Phelan, J.A.; Laverty, M.; Fisch, G.S.; Corby, P.M.; Abrams, W.; Malamud, D.; Li, Y.

    2012-01-01

    We report a clinical study that examines whether HIV infection affects Streptococcus mutans colonization in the oral cavity. Whole stimulated saliva samples were collected from 46 HIV-seropositive individuals and 69 HIV-seronegative control individuals. The level of S. mutans colonization was determined by conventional culture methods. The genotype of S. mutans was compared between 10 HIV-positive individuals before and after highly active antiretroviral therapy (HAART) and 10 non-HIV-infected control individuals. The results were analyzed against viral load, CD4+ and CD8+ T-cell counts, salivary flow rate, and caries status. We observed that S. mutans levels were higher in HIV-infected individuals than in the non-HIV-infected control individuals (p = 0.013). No significant differences in S. mutans genotypes were found between the two groups over the six-month study period, even after HAART. There was a bivariate linear relationship between S. mutans levels and CD8+ counts (r = 0.412; p = 0.007), but not between S. mutans levels and either CD4+ counts or viral load. Furthermore, compared with non-HIV-infected control individuals, HIV-infected individuals experienced lower salivary secretion (p = 0.009) and a positive trend toward more decayed tooth surfaces (p = 0.027). These findings suggest that HIV infection can have a significant effect on the level of S. mutans, but not genotypes. PMID:22821240

  13. Genotype phenotype mapping in RNA viruses - disjunctive normal form learning.

    PubMed

    Wu, Chuang; Walsh, Andrew S; Rosenfeld, Roni

    2011-01-01

    RNA virus phenotypic changes often result from multiple alternative molecular mechanisms, where each mechanism involves changes to a small number of key residues. Accordingly, we propose to learn genotype-phenotype functions, using Disjunctive Normal Form (DNF) as the assumed functional form. In this study we develop DNF learning algorithms that attempt to construct predictors as Boolean combinations of covariates. We demonstrate the learning algorithm's consistency and efficiency on simulated sequences, and establish their biological relevance using a variety of real RNA virus datasets representing different viral phenotypes, including drug resistance, antigenicity, and pathogenicity. We compare our algorithms with previously published machine learning algorithms in terms of prediction quality: leave-one-out performance shows superior accuracy to other machine learning algorithms on the HIV drug resistance dataset and the UCIs promoter gene dataset. The algorithms are powerful in inferring the genotype-phenotype mapping from a moderate number of labeled sequences, as are typically produced in mutagenesis experiments. They can also greedily learn DNFs from large datasets. The Java implementation of our algorithms will be made publicly available.

  14. Genotype identification of Enterocytozoon bieneusi isolates from stool samples of HIV-infected Tunisian patients

    PubMed Central

    Chabchoub, N.; Abdelmalek, R.; Breton, J.; Kanoun, F.; Thellier, M.; Bouratbine, A.; Aoun, K.

    2012-01-01

    The microsporidian species Enterocytozoon bieneusi is a major cause of chronic diarrhea and malabsorption in patients with AIDS. Genotyping was performed on seven E. bieneusi strains for the first time in Tunisia. All the strains were isolated from stool samples of humans with immunodeficiency virus (HIV) infection. Analysis of the ribosomal RNA gene internal transcribed spacer (rDNA ITS) allowed the identification of three distinct genotypes previously described in other studies. Genotypes D and B were characterized in four and two respectively. The Peruvian genotype (Peru 8) was detected in the last isolate. These results indicate a genetic diversity in E. bieneusi strains from HIV Tunisian patients and suggest the coexistence of both zoonotic and anthroponotic route of transmission. PMID:22550625

  15. Genotype identification of Enterocytozoon bieneusi isolates from stool samples of HIV-infected Tunisian patients.

    PubMed

    Chabchoub, N; Abdelmalek, R; Breton, J; Kanoun, F; Thellier, M; Bouratbine, A; Aoun, K

    2012-05-01

    The microsporidian species Enterocytozoon bieneusi is a major cause of chronic diarrhea and malabsorption in patients with AIDS. Genotyping was performed on seven E. bieneusi strains for the first time in Tunisia. All the strains were isolated from stool samples of humans with immunodeficiency virus (HIV) infection. Analysis of the ribosomal RNA gene internal transcribed spacer (rDNA ITS) allowed the identification of three distinct genotypes previously described in other studies. Genotypes D and B were characterized in four and two respectively. The Peruvian genotype (Peru 8) was detected in the last isolate. These results indicate a genetic diversity in E. bieneusi strains from HIV Tunisian patients and suggest the coexistence of both zoonotic and anthroponotic route of transmission.

  16. IFN-stimulated gene expression is independent of the IFNL4 genotype in chronic HIV-1 infection.

    PubMed

    Monteleone, Katia; Scheri, Giuseppe Corano; Statzu, Maura; Selvaggi, Carla; Falasca, Francesca; Giustini, Noemi; Mezzaroma, Ivano; Turriziani, Ombretta; d'Ettorre, Gabriella; Antonelli, Guido; Scagnolari, Carolina

    2016-11-01

    This study aimed to evaluate the association between the IFNL4 rs368234815 (ΔG/TT) dinucleotide polymorphism and the IFN response during chronic HIV-1 infection. We carried out genotyping analysis and measured the expression of IFN-stimulated genes (ISGs) (myxovirus resistance protein A [MxA], ISG15, ISG56, apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like [APOBEC] 3F and APOBEC3G) on peripheral blood mononuclear cells collected from naïve and HAART-treated HIV-1-infected patients. There were no statistically significant differences in endogenous ISGs mRNA levels among HIV-1-positive patients bearing different IFNL4 genotypes, suggesting that ISG expression is independent of the IFNL4 genotype in HIV-1 infection. PMID:27558125

  17. Dried blood spot in the genotyping, quantification and storage of HCV RNA: a systematic literature review

    PubMed Central

    Greenman, Jamie; Roberts, Teri; Cohn, Jennifer; Messac, Luke

    2015-01-01

    The entry of new all-oral direct acting antiviral therapy for hepatitis C provides an opportunity to scale up HCV care in low- and middle-income countries. In HIV, use of dried blood spots (DBS) has facilitated the diagnosis and management of HIV in resource-poor settings. DBS may be used in a similar way to facilitate diagnosis and management of HCV. Here, we present a systematic review of the literature of DBS for HCV RNA detection and genotyping. Using an a priori review protocol, three databases were searched for studies published up to August 2013 that reported the use of dried blood and serum spots in genotyping, detection and measurement of HCV RNA, as well as the rate of degradation of HCV RNA when stored in DBS at room temperature. Nine papers were eligible for inclusion; eight studied DBS and one dried serum. Two studies measured concordance between genotype and subtype determined by DBS and whole plasma and both found 100% concordance. Four studies measured endpoint detection limits of HCV RNA-positive samples by DBS and found positive predictive values of 100% down to 250, 334, 2500 and 24160 IU/mL. Two studies found deterioration of HCV RNA in DBS samples stored at room temperature, while two others failed to detect such deterioration. These results support the potential use of DBS for genotyping and HCV RNA detection. Studies of the use of DBS for HCV RNA viral load measurement and of the rate of degradation of HCV RNA when stored in DBS at ambient temperatures remain inconclusive. PMID:25367722

  18. Prevalence and Risk Factors of HCV/HIV Co-Infection and HCV Genotype Distribution in North-Eastern Poland

    PubMed Central

    Grzeszczuk, Anna; Wandalowicz, Alicja Danuta; Jaroszewicz, Jerzy; Flisiak, Robert

    2015-01-01

    Background: HIV/HCV co-infection predisposes to accelerated liver damage and increased both liver-related and unrelated morbidity and mortality in patients with HIV infection. Objectives: The aim of this study was to evaluate the prevalence of HCV infection, seropositivity, risk factors and genotype distribution among treated HIV positive patients. Furthermore, the occurrence and causes of deaths were analyzed. Patients and Methods: Adult HIV-1 infected patients, with at least one antiHCV result, treated in one of Polish HIV/AIDS reference centers, participated in this cross-sectional study. Results: Four hundred and fifty seven patients with a median age of 38 years (ranged 23 - 72), and predominantly male (76.6%) were enrolled in the study. Anti-HCV antibodies were detected in 325 individuals (71.1%). HCV RNA was detected in 207 of the 233 patients tested (88%). The HCV genotype analysis (n = 193) demonstrated almost equal distribution with slight genotype 1 domination as 37.3%, mainly 1b, followed by genotypes 3 as 32.1% and 4 as 30.6%. No association was found between HCV genotype and route of HIV acquisition. In univariate analysis, higher HCV seropositivity was related to male sex, intravenous drug use (IDU), mode of HIV transmission, history of drug and alcohol abuse and imprisonment. In multivariate analysis, only being injection drug user (P = 0.0001), imprisonment (P = 0.310) and younger age at the HIV diagnosis per each year (P = 0.025) were identified as risk factors for HCV infection. Sixty three deaths were reported; no association was found between HCV seropositivity and death prevalence. Conclusions: HIV/HCV co-infection is an important medical problem in North-Eastern Poland. A history of incarceration and younger age at HIV diagnosis were additional to IDU risk factors for HCV seropositivity in this cohort. PMID:26300929

  19. MicroRNA-155 Reinforces HIV Latency*

    PubMed Central

    Ruelas, Debbie S.; Chan, Jonathan K.; Oh, Eugene; Heidersbach, Amy J.; Hebbeler, Andrew M.; Chavez, Leonard; Verdin, Eric; Rape, Michael; Greene, Warner C.

    2015-01-01

    The presence of a small number of infected but transcriptionally dormant cells currently thwarts a cure for the more than 35 million individuals infected with HIV. Reactivation of these latently infected cells may result in three fates: 1) cell death due to a viral cytopathic effect, 2) cell death due to immune clearance, or 3) a retreat into latency. Uncovering the dynamics of HIV gene expression and silencing in the latent reservoir will be crucial for developing an HIV-1 cure. Here we identify and characterize an intracellular circuit involving TRIM32, an HIV activator, and miR-155, a microRNA that may promote a return to latency in these transiently activated reservoir cells. Notably, we demonstrate that TRIM32, an E3 ubiquitin ligase, promotes reactivation from latency by directly modifying IκBα, leading to a novel mechanism of NF-κB induction not involving IκB kinase activation. PMID:25873391

  20. A Randomised Trial Comparing Genotypic and Virtual Phenotypic Interpretation of HIV Drug Resistance: The CREST Study

    PubMed Central

    Hales, Gillian; Birch, Chris; Crowe, Suzanne; Workman, Cassy; Hoy, Jennifer F; Law, Matthew G; Kelleher, Anthony D; Lincoln, Douglas; Emery, Sean

    2006-01-01

    Objectives: The aim of this study was to compare the efficacy of different HIV drug resistance test reports (genotype and virtual phenotype) in patients who were changing their antiretroviral therapy (ART). Design: Randomised, open-label trial with 48-week followup. Setting: The study was conducted in a network of primary healthcare sites in Australia and New Zealand. Participants: Patients failing current ART with plasma HIV RNA > 2000 copies/mL who wished to change their current ART were eligible. Subjects were required to be > 18 years of age, previously treated with ART, have no intercurrent illnesses requiring active therapy, and to have provided written informed consent. Interventions: Eligible subjects were randomly assigned to receive a genotype (group A) or genotype plus virtual phenotype (group B) prior to selection of their new antiretroviral regimen. Outcome Measures: Patient groups were compared for patterns of ART selection and surrogate outcomes (plasma viral load and CD4 counts) on an intention-to-treat basis over a 48-week period. Results: Three hundred and twenty seven patients completing > one month of followup were included in these analyses. Resistance tests were the primary means by which ART regimens were selected (group A: 64%, group B: 62%; p = 0.32). At 48 weeks, there were no significant differences between the groups for mean change from baseline plasma HIV RNA (group A: 0.68 log copies/mL, group B: 0.58 log copies/mL; p = 0.23) and mean change from baseline CD4+ cell count (group A: 37 cells/mm3, group B: 50 cells/mm3; p = 0.28). Conclusions: In the absence of clear demonstrated benefits arising from the use of the virtual phenotype interpretation, this study suggests resistance testing using genotyping linked to a reliable interpretive algorithm is adequate for the management of HIV infection. PMID:16878178

  1. Epidemiological Distribution and Genotype Characterization of the Hepatitis C Virus Among HIV Patients in Kashan, Iran

    PubMed Central

    Afzali, Hasan; Momen-Heravi, Mansooreh; Farokhzad, Asefeh

    2016-01-01

    Background Parenteral transmission is a common route of transmission for both human immunodeficiency virus (HIV) and hepatitis C virus (HCV); therefore, hepatitis C viral infection is highly prevalent among people infected with HIV. Objectives This study was designed to examine the epidemiology and describe the clinical manifestation as well as the HCV genotypes in patients from the city of Kashan, Iran, who are coinfected with HIV and HCV. Patients and Methods This descriptive study was conducted in 2014 in the city of Kashan. The population consisted of all the HIV-infected patients who were referred to the behavioral counseling center and jail in Kashan. Demographic information and HCV- and HIV-related risk behaviors were obtained through the use of an interviewer-assisted questionnaire. After the participants gave written informed consent to participate, 10 cc venous blood samples were collected. The serum samples were screened for HCV infection using an enzyme-linked immunosorbent assay (ELISA). In the event of a positive test for HCV, the RNA was then amplified by polymerase chain reaction (PCR) amplification. The HCV subtypes were determined via the direct sequencing of the amplicons. All data analysis was performed using SPSS version 16.0 for the descriptive statistics, and then the chi-square test and Pearson coefficient were performed for additional analysis. Results The results of the analysis indicated that 54 (85%) of the 63 HIV-infected patients were males who were also HCV positive and who had less than a high school level education. There was a significant association between HCV infection and both occupation (P < 0.0001) and level of education (P < 0.05). All the HIV/HCV coinfected cases had a history of illicit drug use, while 92.6% had a history of imprisonment and 40.7% had high risk sexual contacts. Overall, genotype 1 was found in 75.9% of HCV patients, while genotype 3 was found in 24.1%. Some 94.4% of HCV patients had subtype A. There were

  2. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 8 2012-04-01 2012-04-01 false In vitro human immunodeficiency virus (HIV) drug... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification. The in vitro HIV drug resistance genotype assay is a device that consists of nucleic acid...

  3. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 8 2013-04-01 2013-04-01 false In vitro human immunodeficiency virus (HIV) drug... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification. The in vitro HIV drug resistance genotype assay is a device that consists of nucleic acid...

  4. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 8 2014-04-01 2014-04-01 false In vitro human immunodeficiency virus (HIV) drug... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification. The in vitro HIV drug resistance genotype assay is a device that consists of nucleic acid...

  5. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 8 2011-04-01 2011-04-01 false In vitro human immunodeficiency virus (HIV) drug... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification. The in vitro HIV drug resistance genotype assay is a device that consists of nucleic acid...

  6. 21 CFR 866.3950 - In vitro human immunodeficiency virus (HIV) drug resistance genotype assay.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 8 2010-04-01 2010-04-01 false In vitro human immunodeficiency virus (HIV) drug... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay. (a) Identification. The in vitro HIV drug resistance genotype assay is a device that consists of nucleic acid...

  7. The role of a metastable RNA secondary structure in hepatitis delta virus genotype III RNA editing

    PubMed Central

    Linnstaedt, Sarah D.; Kasprzak, Wojciech K.; Shapiro, Bruce A.; Casey, John L.

    2006-01-01

    RNA editing plays a critical role in the life cycle of hepatitis delta virus (HDV). The host editing enzyme ADAR1 recognizes specific RNA secondary structure features around the amber/W site in the HDV antigenome and deaminates the amber/W adenosine. A previous report suggested that a branched secondary structure is necessary for editing in HDV genotype III. This branched structure, which is distinct from the characteristic unbranched rod structure required for HDV replication, was only partially characterized, and knowledge concerning its formation and stability was limited. Here, we examine the secondary structures, conformational dynamics, and amber/W site editing of HDV genotype III RNA using a miniaturized HDV genotype III RNA in vitro. Computational analysis of this RNA using the MPGAfold algorithm indicated that the RNA has a tendency to form both metastable and stable unbranched secondary structures. Moreover, native polyacrylamide gel electrophoresis demonstrated that this RNA forms both branched and unbranched rod structures when transcribed in vitro. As predicted, the branched structure is a metastable structure that converts readily to the unbranched rod structure. Only branched RNA was edited at the amber/W site by ADAR1 in vitro. The structural heterogeneity of HDV genotype III RNA is significant because not only are both conformations of the RNA functionally important for viral replication, but the ratio of the two forms could modulate editing by determining the amount of substrate RNA available for modification. PMID:16790843

  8. Tat RNA silencing suppressor activity contributes to perturbation of lymphocyte miRNA by HIV-1

    PubMed Central

    2011-01-01

    Background MicroRNA (miRNA)-mediated RNA silencing is integral to virtually every cellular process including cell cycle progression and response to virus infection. The interplay between RNA silencing and HIV-1 is multifaceted, and accumulating evidence posits a strike-counterstrike interface that alters the cellular environment to favor virus replication. For instance, miRNA-mediated RNA silencing of HIV-1 translation is antagonized by HIV-1 Tat RNA silencing suppressor activity. The activity of HIV-1 accessory proteins Vpr/Vif delays cell cycle progression, which is a process prominently modulated by miRNA. The expression profile of cellular miRNA is altered by HIV-1 infection in both cultured cells and clinical samples. The open question stands of what, if any, is the contribution of Tat RNA silencing suppressor activity or Vpr/Vif activity to the perturbation of cellular miRNA by HIV-1. Results Herein, we compared the perturbation of miRNA expression profiles of lymphocytes infected with HIV-1NL4-3 or derivative strains that are deficient in Tat RNA silencing suppressor activity (Tat K51A substitution) or ablated of the vpr/vif open reading frames. Microarrays recapitulated the perturbation of the cellular miRNA profile by HIV-1 infection. The miRNA expression trends overlapped ~50% with published microarray results on clinical samples from HIV-1 infected patients. Moreover, the number of miRNA perturbed by HIV-1 was largely similar despite ablation of Tat RSS activity and Vpr/Vif; however, the Tat RSS mutation lessened HIV-1 downregulation of twenty-two miRNAs. Conclusions Our study identified miRNA expression changes attributable to Tat RSS activity in HIV-1NL4-3. The results accomplish a necessary step in the process to understand the interface of HIV-1 with host RNA silencing activity. The overlap in miRNA expression trends observed between HIV-1 infected CEMx174 lymphocytes and primary cells supports the utility of cultured lymphocytes as a tractable model

  9. An update to the HIV-TRePS system: the development of new computational models that do not require a genotype to predict HIV treatment outcomes

    PubMed Central

    Revell, Andrew D.; Wang, Dechao; Wood, Robin; Morrow, Carl; Tempelman, Hugo; Hamers, Raph; Alvarez-Uria, Gerardo; Streinu-Cercel, Adrian; Ene, Luminita; Wensing, Annemarie; Reiss, Peter; van Sighem, Ard I.; Nelson, Mark; Emery, Sean; Montaner, Julio S. G.; Lane, H. Clifford; Larder, Brendan A.; Reiss, Peter; van Sighem, Ard; Montaner, Julio; Harrigan, Richard; Rinke de Wit, Tobias; Hamers, Raph; Sigaloff, Kim; Agan, Brian; Marconi, Vincent; Wegner, Scott; Sugiura, Wataru; Zazzi, Maurizio; Streinu-Cercel, Adrian; Alvarez-Uria, Gerardo; Gatell, Jose; Lazzari, Elisa; Gazzard, Brian; Nelson, Mark; Pozniak, Anton; Mandalia, Sundhiya; Ruiz, Lidia; Clotet, Bonaventura; Staszewski, Schlomo; Torti, Carlo; Lane, Cliff; Metcalf, Julie; Perez-Elias, Maria-Jesus; Carr, Andrew; Norris, Richard; Hesse, Karl; Vlahakis, Emanuel; Tempelman, Hugo; Barth, Roos; Morrow, Carl; Wood, Robin; Ene, Luminita; Dragovic, Gordana; Emery, Sean; Cooper, David; Torti, Carlo; Baxter, John; Monno, Laura; Torti, Carlo; Gatell, Jose; Clotet, Bonventura; Picchio, Gaston; deBethune, Marie-Pierre; Perez-Elias, Maria-Jesus

    2014-01-01

    Objectives The optimal individualized selection of antiretroviral drugs in resource-limited settings is challenging because of the limited availability of drugs and genotyping. Here we describe the development of the latest computational models to predict the response to combination antiretroviral therapy without a genotype, for potential use in such settings. Methods Random forest models were trained to predict the probability of a virological response to therapy (<50 copies HIV RNA/mL) following virological failure using the following data from 22 567 treatment-change episodes including 1090 from southern Africa: baseline viral load and CD4 cell count, treatment history, drugs in the new regimen, time to follow-up and follow-up viral load. The models were assessed during cross-validation and with an independent global test set of 1000 cases including 100 from southern Africa. The models' accuracy [area under the receiver-operating characteristic curve (AUC)] was evaluated and compared with genotyping using rules-based interpretation systems for those cases with genotypes available. Results The models achieved AUCs of 0.79–0.84 (mean 0.82) during cross-validation, 0.80 with the global test set and 0.78 with the southern African subset. The AUCs were significantly lower (0.56–0.57) for genotyping. Conclusions The models predicted virological response to HIV therapy without a genotype as accurately as previous models that included a genotype. They were accurate for cases from southern Africa and significantly more accurate than genotyping. These models will be accessible via the online treatment support tool HIV-TRePS and have the potential to help optimize antiretroviral therapy in resource-limited settings where genotyping is not generally available. PMID:24275116

  10. Micro RNA in Exosomes from HIV-Infected Macrophages.

    PubMed

    Roth, William W; Huang, Ming Bo; Addae Konadu, Kateena; Powell, Michael D; Bond, Vincent C

    2015-12-22

    Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

  11. Evaluation of a Cost Effective In-House Method for HIV-1 Drug Resistance Genotyping Using Plasma Samples

    PubMed Central

    Chaturbhuj, Devidas N.; Nirmalkar, Amit P.; Paranjape, Ramesh S.; Tripathy, Srikanth P.

    2014-01-01

    Objectives Validation of a cost effective in-house method for HIV-1 drug resistance genotyping using plasma samples. Design The validation includes the establishment of analytical performance characteristics such as accuracy, reproducibility, precision and sensitivity. Methods The accuracy was assessed by comparing 26 paired Virological Quality Assessment (VQA) proficiency testing panel sequences generated by in-house and ViroSeq Genotyping System 2.0 (Celera Diagnostics, US) as a gold standard. The reproducibility and precision were carried out on five samples with five replicates representing multiple HIV-1 subtypes (A, B, C) and resistance patterns. The amplification sensitivity was evaluated on HIV-1 positive plasma samples (n = 88) with known viral loads ranges from 1000–1.8 million RNA copies/ml. Results Comparison of the nucleotide sequences generated by ViroSeq and in-house method showed 99.41±0.46 and 99.68±0.35% mean nucleotide and amino acid identity respectively. Out of 135 Stanford HIVdb listed HIV-1 drug resistance mutations, partial discordance was observed at 15 positions and complete discordance was absent. The reproducibility and precision study showed high nucleotide sequence identities i.e. 99.88±0.10 and 99.82±0.20 respectively. The in-house method showed 100% analytical sensitivity on the samples with HIV-1 viral load >1000 RNA copies/ml. The cost of running the in-house method is only 50% of that for ViroSeq method (112$ vs 300$), thus making it cost effective. Conclusions The validated cost effective in-house method may be used to collect surveillance data on the emergence and transmission of HIV-1 drug resistance in resource limited countries. Moreover, the wide applications of a cost effective and validated in-house method for HIV-1 drug resistance testing will facilitate the decision making for the appropriate management of HIV infected patients. PMID:24533056

  12. HIV-1 RNA quantification in CRF02_AG HIV-1 infection: too easy to make mistakes.

    PubMed

    Tatarelli, Paola; Taramasso, Lucia; Di Biagio, Antonio; Sticchi, Laura; Nigro, Nicola; Barresi, Renata; Viscoli, Claudio; Bruzzone, Bianca

    2016-04-01

    The number of patients newly infected by HIV-1 non-B subtypes and circulating recombinant forms (CRFs) is increasing worldwide, including in the western countries. We report on a primary HIV-1 infection in a Caucasian patient. A routine quantitative assay (Nuclisens EasyQ HIV-1 2.0, BioMérieux SA) showed 6,700 HIV-1 RNA copies/ml. A combined antiretroviral therapy (cART) consistent with low baseline HIV-1 RNA was started. Few days later, the analysis performed with REGA HIV-1 Subtyping Tool - Version 3.0 attributed the HIV-1 sequence to the CRF02_AG recombinant form. Therefore, a second real-time PCR assay was performed, using the Versant HIV-1 RNA 1.0 Assay (kPCR) (Siemens HealthCare Diagnostics) which revealed a HIV-1 RNA of 230,000 copies/ml. Consequently, the ongoing cART was potentiated. This case suggests that the wide genetic variability of HIV-1 subtypes may affect the capability of the commonly used assays to detect and accurately quantify HIV-1 RNA in non-B subtypes and CRFs. In presence of CRFs different commercial HIV-1 RNA tests should be performed to find the most reliable for viral load quantification at the diagnosis, because it influences the choice of cART, and during the follow-up. Indeed, international guidelines for HIV-1 infection management suggest to monitor patient' HIV-RNA with the same assay over the course of treatment. As different commercial tests can be performed in the same laboratory with considerable difficulty, the laboratory should select an assay that is suitable not only for the more prevalent strain, but also for less frequent ones that, nevertheless, can occur. Then, knowing and investigating the spread of non-B strains has essential clinical and laboratory implications. PMID:27196556

  13. HCV genotype determination in monoinfected and HIV co-infected patients in Cuba.

    PubMed

    Rodríguez Lay, Licel de Los Angeles; Villalba, Maria Caridad Montalvo; Corredor, Marité Bello; Frómeta, Susel Sariego; Hernández, Jeny Marante; Carrera, Santiago Dueñas; Wong, Meilin Sánchez; Samada, Marcia; Núñez, Milay Bello; Alonso, Lidunka Valdes; da Silva Filho, Hermes Pedreira; Hübschen, Judith M; Reis, Mitermayer G

    2012-12-01

    With the aim to characterize the HCV genotype distribution in Cuba, sera were collected from two subgroups: HCV-monoinfected and HCV/HIV co-infected patients. A combination of reverse transcription-PCR using genotype-specific primers, restriction fragment length polymorphism and sequencing was used to determine the genotype of 84 samples. Seventy-nine (94%) showed single infections (10 [12%] were genotype 1a and 69 [82%] genotype 1b) and 5 (6%) samples corresponded to mixed infections (2 [2%] with genotypes 1a/3a and 1 sample [1%] each with 1b/3a, 1b/4a and 1a/1b/3a). HCV/HIV co-infected subjects had a higher frequency of mixed infections (p=0.08), infection with genotype 3a (p=0.18) and for the first time genotype 4a was found. There was no association of any demographic characteristics with any specific genotype although HCV/HIV co-infected patients showed a tendency to have mixed genotypes in those older than 45 years of age (p=0.11). Phylogenetic analysis showed that HCV isolates clustered with subtypes 1b (n=15, maximal genetic distance 2.51%) and 1a (n=2, maximal genetic distance 0.35%). This report presents the prevalence of HCV genotypes in monoinfected and HIV co-infected patients, mixed HCV infections in HCV/HIV co-infected men who have sex with men with high-risk sexual practices and for the first time identifies that the uncommon genotype 4a can be present in a patient co-infected with HIV.

  14. Genotypic resistance profiles of HIV-2-treated patients in West Africa

    PubMed Central

    Charpentier, Charlotte; Eholié, Serge; Anglaret, Xavier; Bertine, Mélanie; Rouzioux, Christine; Avettand-Fenoël, Véronique; Messou, Eugène; Minga, Albert; Damond, Florence; Plantier, Jean-Christophe; Dabis, François; Peytavin, Gilles; Brun-Vézinet, Françoise; Ekouevi, Didier K.

    2014-01-01

    Objective To assess the virological response, genotypic resistance profiles, and antiretroviral plasma concentrations in HIV-2 antiretroviral-treated (antiretroviral therapy, ART) patients in Côte d‘Ivoire. Methods A cross-sectional survey was conducted among HIV-2 patients receiving ART. Plasma HIV-2 viral load was performed using the Agence Nationale de Recherche sur le SIDA et les hépatites virales (ANRS) assay. Protease and reverse transcriptase sequencing was performed using in-house methods and antiretroviral plasma concentrations were assessed using ultra performance liquid chromatography combined with tandem mass spectrometry. Results One hundred and forty-five HIV-2-treated patients were enrolled with a median CD4+ cell count of 360 cells/µl (interquartile range, IQR = 215–528). Median duration of ART was 4 years (IQR = 2–7) and 74% of patients displayed viral load less than 50 copies/ml. Median plasma HIV-2 RNA among patients with viral load more than 50 copies/ml was 3016 copies/ml (IQR = 436–5156). Most patients (84%) received a lopinavir/ritonavir-based regimen. HIV-2 resistance mutations to nucleoside reverse transcriptase inhibitors and protease inhibitors were detected in 21 of 25 (84%) and 20 of 29 (69%) samples, respectively. The most prevalent nucleoside reverse transcriptase inhibitor resistance mutations were M184I/V (90%), Q151M (24%), and S215F/Y (24%). The most prevalent protease inhibitor resistance mutations were V47A (60%) and I54M (30%). Median CD4+ cell counts were 434 cells/µl (292–573) and 204 cells/µl (122–281) in patients with viral load less than 50 copies/ml and those exhibiting virological failure (P < 0.0001), respectively. The proportions of patients with adequate antiretroviral plasma concentrations were 81 and 93% in patients displaying virological failure and in those with viral load less than 50 copies/ml, respectively (P = 0.046), suggesting good treatment adherence. Conclusion We observed adequate drug

  15. Recognition of HIV TAR RNA by triazole linked neomycin dimers.

    PubMed

    Kumar, Sunil; Arya, Dev P

    2011-08-15

    A series of neomycin dimers have been synthesized using 'click chemistry' with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (trans activation response) RNA region, a 59 base pair stem loop structure located at 5'-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (T(m)) of the HIV TAR RNA up to 10°C. Ethidium bromide displacement titrations revealed nanomolar IC(50) between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC(50) in the micromolar range is observed.

  16. Recognition of HIV TAR RNA by triazole linked neomycin dimers

    PubMed Central

    Kumar, Sunil

    2013-01-01

    A series of neomycin dimers have been synthesized using “click chemistry” with varying linker functionality and length to target the TAR RNA region of HIV virus. TAR (Trans Activation Response) RNA region, a 59 base pair stem loop structure located at 5′-end of all nascent HIV-1 transcripts interacts with a key regulatory protein, Tat, and necessitates the replication of HIV-1 virus. Neomycin, an aminosugar, has been shown to exhibit more than one binding site with HIV TAR RNA. Multiple TAR binding sites of neomycin prompted us to design and synthesize a small library of neomycin dimers using click chemistry. The binding between neomycin dimers and HIV TAR RNA was characterized using spectroscopic techniques including FID (Fluorescent Intercalator Displacement) titration and UV-thermal denaturation. UV thermal denaturation studies demonstrate that neomycin dimer binding increase the melting temperature (Tm) of the HIV TAR RNA up to 10 °C. Ethidium bromide displacement titrations revealed nanomolar IC50 between neomycin dimers and HIV TAR RNA, whereas with neomycin, a much higher IC50 in the micromolar range is observed. PMID:21757341

  17. Clinical validation and applicability of different tipranavir/ritonavir genotypic scores in HIV-1 protease inhibitor-experienced patients.

    PubMed

    Saracino, Annalisa; Monno, Laura; Tartaglia, Alessandra; Tinelli, Carmine; Seminari, Elena; Maggiolo, Franco; Bonora, Stefano; Rusconi, Stefano; Micheli, Valeria; Lo Caputo, Sergio; Lazzaroni, Laura; Ferrara, Sergio; Ladisa, Nicoletta; Nasta, Paola; Parruti, Giustino; Bellagamba, Rita; Forbici, Federica; Angarano, Gioacchino

    2009-07-01

    Tipranavir, a non-peptidic protease inhibitor which shows in vitro efficacy against some HIV-1-resistant strains, can be used in salvage therapies for multi-experienced HIV patients due to its peculiar resistance profile including 21 mutations at 16 protease positions according to International AIDS Society (IAS). Other genotypic scores, however, which attribute a different weight to single amino-acid substitutions, have been recently proposed. To validate the clinical utility of four different genotypic scores for selecting tipranavir responders, the baseline resistance pattern of 176 HIV heavily experienced patients was correlated with virological success (HIV-RNA<50 copies/ml) after 24 weeks of a new treatment based on tipranavir/ritonavir. Virological suppression after 24 weeks was reached by 42.5% of patients. With univariate analysis, genotypic scores were all associated with outcome but showed a low accuracy with ROC analysis, with the weighted score (WS) by Scherer et al. demonstrating the best performance with an AUC of 68%. Only 52% of patients classified as susceptible (WS< or =3) responded to the new therapy. The following variables were significantly associated (p<0.05) to failure with multivariate analysis: WS, log peak of HIV-RNA, IAS mutations: L33F, I54AMV, Q58E, and non-IAS mutation: N37DES. On the contrary, the use of T20 in T20-naïve patients and the V82AFSI and F53LY non-IAS mutations were associated with virological success. The study suggests that even if the "weighted" scores are able to interpret correctly the antiretroviral resistance profile of multi-experienced patients, it is difficult to individuate a cut-off which can be easily applied to this population for discriminating responders.

  18. Minor Contribution of Chimeric Host-HIV Readthrough Transcripts to the Level of HIV Cell-Associated gag RNA.

    PubMed

    Pasternak, Alexander O; DeMaster, Laura K; Kootstra, Neeltje A; Reiss, Peter; O'Doherty, Una; Berkhout, Ben

    2015-11-11

    Cell-associated HIV unspliced RNA is an important marker of the viral reservoir. HIV gag RNA-specific assays are frequently used to monitor reservoir activation. Because HIV preferentially integrates into actively transcribed genes, some of the transcripts detected by these assays may not represent genuine HIV RNA but rather chimeric host-HIV readthrough transcripts. Here, we demonstrate that in HIV-infected patients on suppressive combination antiretroviral therapy, such host-derived transcripts do not significantly contribute to the HIV gag RNA level.

  19. Web Resources for HIV Type 1 Genotypic-Resistance Test Interpretation

    PubMed Central

    Liu, Tommy F.; Shafer, Robert W.

    2008-01-01

    Interpreting the results of plasma human immunodeficiency virus type 1 (HIV-1) genotypic drug-resistance tests is one of the most difficult tasks facing clinicians caring for HIV-1–infected patients. There are many drug-resistance mutations, and they arise in complex patterns that cause varying levels of drug resistance. In addition, HIV-1 exists in vivo as a virus population containing many genomic variants. Genotypic-resistance testing detects the drug-resistance mutations present in the most common plasma virus variants but may not detect drug-resistance mutations present in minor virus variants. Therefore, interpretation systems are necessary to determine the phenotypic and clinical significance of drug-resistance mutations found in a patient's plasma virus population. We describe the scientific principles of HIV-1 genotypic-resistance test interpretation and the most commonly used Web-based resources for clinicians ordering genotypic drug-resistance tests. PMID:16652319

  20. HIV infection among U.S. Army and Air Force military personnel: sociodemographic and genotyping analysis.

    PubMed

    Singer, Darrell E; Bautista, Christian T; O'Connell, Robert J; Sanders-Buell, Eric; Agan, Brian K; Kijak, Gustavo H; Hakre, Shilpa; Sanchez, Jose L; Sateren, Warren B; McCutchan, Francine E; Michael, Nelson L; Scott, Paul T

    2010-08-01

    Since 1985, the U.S. Department of Defense has periodically screened all military personnel for HIV allowing for the monitoring of the infection in this dynamic cohort population. A nested case-control study was performed to study sociodemographics, overseas assignment, and molecular analysis of HIV. Cases were newly identified HIV infections among U.S. Army and Air Force military personnel from 2000 to 2004. Controls were frequency matched to cases by gender and date of case first positive HIV screening test. Genotyping analysis was performed using high-throughput screening assays and partial genome sequencing. HIV was significantly associated with black race [odds ratio (OR) = 6.65], single marital status (OR = 4.45), and age (OR per year = 1.07). Ninety-seven percent were subtype B and 3% were non-B subtypes (A3, CRF01_AE, A/C recombinant, G, CRF02_AG). Among cases, overseas assignment in the period at risk prior to their first HIV-positive test was associated with non-B HIV subtype infection (OR = 8.44). Black and single military personnel remain disproportionately affected by HIV infection. Most non-B HIV subtypes were associated with overseas assignment. Given the increased frequency and length of assignments, and the expanding HIV genetic diversity observed in this population, there is a need for active HIV genotyping surveillance and a need to reinforce primary HIV prevention efforts.

  1. Evaluation of atazanavir Ctrough, atazanavir genotypic inhibitory quotient, and baseline HIV genotype as predictors of a 24-week virological response in highly drug-experienced, HIV-infected patients treated with unboosted atazanavir.

    PubMed

    Gianotti, Nicola; Seminari, Elena; Guffanti, Monica; Boeri, Enzo; Villani, Paola; Regazzi, Mario; Bigoloni, Alba; Schira, Giulia; Tiberi, Simon; Fusetti, Giuliana; Lazzarin, Adriano; Castagna, Antonella

    2005-04-01

    The objective of this study was to evaluate virological and pharmacological determinants of a 24-week virological response to unboosted atazanavir (ATV) in highly drug-experienced HIV-infected patients. Among patients enrolled in the ATV Expanded Access Program, those with HIV-RNA >1000 copies/mL, a genotype performed within three months from the baseline (BL), and who completed 24 weeks of treatment, were included. They received at least three antiretrovirals, including ATV 400 mg once daily without boosting. ATV plasma levels were evaluated after four weeks of treatment by high performance liquid chromatography (HPLC). ATV genotypic inhibitory quotient (GIQ) was calculated as the ratio between ATV Ctrough and the number of the BL ATV-related protease resistance mutations (among the following: 10I/V/F, 20R/M/I, 24I, 331/F/V, 36I/L/V, 46I/L, 48V, 54V/L, 63P, 71V/T/I, 73C/S/T/A, 82A/F/S/T, 84V, and 90M). Thirty-five subjects were included. At baseline, median (interquartile range) CD4+ T-lymphocytes, HIV-RNA, and ATV resistance mutations were 232.5 (106-303)/microL, 4.7 (4.2-5.1) log10 copies/mL, 2 (1-6), respectively. Thirteen (37.1%) subjects were off-therapy and 11 (31.4%) showed no PI mutation at baseline. Median steady-state ATV Ctrough was 230 ng/mL (87-520), for an ATV GIQ of 86.5 (25.5-165.5). Median HIV-RNA changes from baseline at weeks 4, 12 and 24 were -1.76 (from -0.44 to -2.12), -1.41 (from -0.41 to -2.81) and -1.44 (from -0.42 to -2.71) log10, respectively. The HIV-RNA changes were correlated to the number of ATV resistance mutations at each time point (P < 0.05), whereas no correlation was found between ATV Ctrough or ATV GIQ and HIV-RNA changes. In conclusion, the number of ATV resistance mutations is the only correlate to virological response through 24 weeks of treatment with unboosted atazanavir 400 mg once daily.

  2. N6-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression

    PubMed Central

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N6-methyladenosine (m6A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m6A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1–3) bind to m6A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1–3 proteins recognize m6A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4+ T-cells. We further mapped the YTHDF1–3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1–3 in cells had the opposite effects. Moreover, silencing the m6A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m6A erasers increased Gag expression. Our findings suggest an important role of m6A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. DOI: http://dx.doi.org/10.7554/eLife.15528.001 PMID:27371828

  3. N(6)-methyladenosine of HIV-1 RNA regulates viral infection and HIV-1 Gag protein expression.

    PubMed

    Tirumuru, Nagaraja; Zhao, Boxuan Simen; Lu, Wuxun; Lu, Zhike; He, Chuan; Wu, Li

    2016-01-01

    The internal N(6)-methyladenosine (m(6)A) methylation of eukaryotic nuclear RNA controls post-transcriptional gene expression, which is regulated by methyltransferases (writers), demethylases (erasers), and m(6)A-binding proteins (readers) in cells. The YTH domain family proteins (YTHDF1-3) bind to m(6)A-modified cellular RNAs and affect RNA metabolism and processing. Here, we show that YTHDF1-3 proteins recognize m(6)A-modified HIV-1 RNA and inhibit HIV-1 infection in cell lines and primary CD4(+) T-cells. We further mapped the YTHDF1-3 binding sites in HIV-1 RNA from infected cells. We found that the overexpression of YTHDF proteins in cells inhibited HIV-1 infection mainly by decreasing HIV-1 reverse transcription, while knockdown of YTHDF1-3 in cells had the opposite effects. Moreover, silencing the m(6)A writers decreased HIV-1 Gag protein expression in virus-producing cells, while silencing the m(6)A erasers increased Gag expression. Our findings suggest an important role of m(6)A modification of HIV-1 RNA in viral infection and HIV-1 protein synthesis. PMID:27371828

  4. The longitudinal and interactive effects of HIV status, stimulant use, and host genotype upon neurocognitive functioning.

    PubMed

    Levine, Andrew J; Reynolds, Sandra; Cox, Christopher; Miller, Eric N; Sinsheimer, Janet S; Becker, James T; Martin, Eileen; Sacktor, Ned

    2014-06-01

    Both human immunodeficiency virus (HIV)-1 infection and illicit stimulant use can adversely impact neurocognitive functioning, and these effects can be additive. However, significant variability exists such that as-of-yet unidentified exogenous and endogenous factors affect one's risk for neurocognitive impairment. Literature on both HIV and stimulant use indicates that host genetic variants in immunologic and dopamine-related genes are one such factor. In this study, the individual and interactive effects of HIV status, stimulant use, and genotype upon neurocognitive functioning were examined longitudinally over a 10-year period. Nine hundred fifty-two Caucasian HIV+ and HIV- cases from the Multicenter AIDS Cohort Study were included. All cases had at least two comprehensive neurocognitive evaluations between 1985 and 1995. Pre-highly active antiretroviral therapy (HAART) data were examined in order to avoid the confounding effect of variable drug regimens. Linear mixed models were used, with neurocognitive domain scores as the outcome variables. No four-way interactions were found, indicating that HIV and stimulant use do not interact over time to affect neurocognitive functioning as a function of genotype. Multiple three-way interactions were found that involved genotype and HIV status. All immunologically related genes found to interact with HIV status affected neurocognitive functioning in the expected direction; however, only C-C chemokine ligand 2 (CCL2) and CCL3 affected HIV+ individuals specifically. Dopamine-related genetic variants generally affected HIV-negative individuals only. Neurocognitive functioning among HIV+ individuals who also used stimulants was not significantly different from those who did not use stimulants. The findings support the role of immunologically related genetic differences in CCL2 and CCL3 in neurocognitive functioning among HIV+ individuals; however, their impact is minor. Being consistent with findings from another cohort

  5. Secondary structure of the HIV-2 leader RNA comprising the tRNA-primer binding site.

    PubMed Central

    Berkhout, B; Schoneveld, I

    1993-01-01

    The initiation of reverse transcription of a retroviral RNA genome occurs by a tRNA primer bound near the 5' end of the genomic RNA at a position called the primer-binding site (PBS). To understand the molecular basis for this RNA-RNA interaction, the secondary structure of the leader RNA of the human immunodeficiency virus type 2 (HIV-2) RNA was analyzed. In vitro synthesized HIV-2 RNA was probed with various structure-specific enzymes and chemicals. A computer program was then used to predict the secondary structure consistent with these data. In addition, the nucleotide sequences of different HIV-2 isolates were used to screen for the occurrence of covariation among putative base pairs. The primary sequences have diverged rapidly in some HIV-2 isolates, however, some strikingly conserved secondary structure elements were identified. Most nucleotides in the leader region are involved in base pairing. An exception is the PBS sequence, of which 15 out of 18 nucleotides are exposed in an internal loop. These findings suggest that the overall structure of the HIV-2 genome has evolved to facilitate an optimal interaction with its tRNA primer. Images PMID:8464701

  6. Interferon lambda 3 genotype predicts hepatitis C virus RNA levels in early acute infection among people who inject drugs: The InC3 Study

    PubMed Central

    Hajarizadeh, Behzad; Grady, Bart; Page, Kimberly; Kim, Arthur Y.; McGovern, Barbara H.; Cox, Andrea L.; Rice, Thomas M.; Sacks-Davis, Rachel; Bruneau, Julie; Morris, Meghan; Amin, Janaki; Schinkel, Janke; Applegate, Tanya; Maher, Lisa; Hellard, Margaret; Lloyd, Andrew R.; Prins, Maria; Geskus, Ronald B; Dore, Gregory J.; Grebely, Jason

    2014-01-01

    Background and Objectives Hepatitis C virus (HCV) RNA level in acute HCV infection is predictive of spontaneous clearance. This study assessed factors associated with HCV RNA levels during early acute infection among people who inject drugs with well-defined acute HCV infection. Study design Data were from International Collaboration of Incident HIV and Hepatitis C in Injecting Cohorts (InC3) Study, an international collaboration of nine prospective cohorts studying acute HCV infection. Individuals with available HCV RNA levels during early acute infection (first two months following infection) were included. The distribution of HCV RNA levels during early acute infection were compared by selected host and virological factors. Results A total of 195 individuals were included. Median HCV RNA levels were significantly higher among individuals with interferon lambda 3 (IFNL3, formerly called IL28B) CC genotype compared to those with TT/CT genotype (6.28 vs. 5.39 log IU/mL, respectively; P=0.01). IFNL3 CC genotype was also associated with top tertile HCV RNA levels (≥6.3 IU/mL; vs. TT/CT genotype; adjusted Odds Ratio: 4.28; 95%CI: 2.01, 9.10; P<0.01). Conclusions This study indicates that IFNL3 CC genotype predicts higher HCV RNA levels in early acute HCV infection. PMID:25256151

  7. Human Papillomavirus Prevalence and Genotype Distribution among HIV-Infected Women in Korea

    PubMed Central

    Park, Eun Kyoung; Cho, Heerim; Lee, Seung Geun; Lee, Sang Yeup; Kim, Ki Hyung; Lee, Chang Hun; Chung, Joo Seop; Kwak, Ihm Soo

    2014-01-01

    The epidemiology on human papillomavirus (HPV) among human immunodeficiency virus (HIV)-infected women in Korea is not well established. A retrospective study was conducted to determine the prevalence and genotype distribution of HPV infection among HIV-infected women in Korea. HPV DNA genotype and cervical cytology were examined in 60 HIV-positive women and 1,938 HIV-negative women. HPV genotypes were analyzed by using a HPV DNA chip. HIV-infected women had higher prevalence of high-risk HPV (hr-HPV) infection (30% vs 4.9%, adjusted odds ratio [AOR], 6.96; 95% confidence interval [CI], 3.63-13.34, P<0.001) and abnormal cervical cytology (18.3% vs 1.8%, AOR, 10.94; 95% CI, 5.18-23.1, P<0.001) compared with controls. The most common hr-HPV genotype detected in HIV-infected women was HPV 16 (10%), followed by 18 (6.7%) and 52 (5%). Prevalence of quadrivalent vaccine-preventable types (HPV 6, 11, 16, and 18) was 21.7% and 2.3% in HIV-positive women and HIV-negative women, respectively. Age was a significant risk factor for hr-HPV infection in HIV-infected women (P=0.039). The presence of hr-HPV was significantly associated with abnormal cervical cytology (P<0.001). These findings suggest that HPV testing for cervical cancer screening in HIV-infected women would be necessary, particularly among young age group. PMID:24431902

  8. CCR5 genotype and resistance to vertical transmission of HIV-1.

    PubMed

    Philpott, S; Burger, H; Charbonneau, T; Grimson, R; Vermund, S H; Visosky, A; Nachman, S; Kovacs, A; Tropper, P; Frey, H; Weiser, B

    1999-07-01

    A human gene has been identified that affects susceptibility to HIV-1 infection. The gene codes for CCR5, the coreceptor for macrophage-tropic strains of HIV-1. Individuals who are homozygous for a deleted, mutant form of the gene, delta32, display a high degree of natural resistance to sexual and parenteral transmission of HIV-1. To investigate whether delta32 plays a role in vertical transmission, we determined the CCR5 genotype of 552 children born to infected mothers in the United States and correlated the genotypes with HIV-1 infection status. Of these children, 13% were white, 30% Latino, and 56% African American, reflecting the ethnic makeup of infected women in the United States. The delta32 gene frequency varied among these groups, ranging from 0.08 in whites to 0.02 in both Latinos and African Americans. Approximately 27% of the children in each ethnic group were infected. Four children were identified as delta32 homozygotes, two uninfected whites (3.77%) and two uninfected Latinos (1.68%). None of the infected children displayed the delta32 homozygous genotype. Among Latinos and whites, the number of uninfected children who carried the homozygous delta32 mutation was significantly greater than that predicted by the Hardy-Weinberg equilibrium (p < .001 for Latinos, p = .044 for whites). This association was noted in Latino and white children whose mothers were either treated or untreated with zidovudine. These data document the occurrence of the homozygous delta32 genotype among children of HIV-1-infected mothers and suggest that this mutant genotype may confer protection from mother-to-child transmission of HIV-1. They also suggest that sexual, parenteral, and vertical transmission all involve processes that use CCR5 as a coreceptor for primary HIV-1 infection. Therefore, blocking the CCR5 receptor may provide an additional strategy to prevent HIV-1 vertical transmission.

  9. HIV and HCV coinfection: prevalence, associated factors and genotype characterization in the Midwest Region of Brazil.

    PubMed

    Freitas, Solange Zacalusni; Teles, Sheila Araújo; Lorenzo, Paulo Cesar; Puga, Marco Antonio Moreira; Tanaka, Tayana Serpa Ortiz; Thomaz, Danilo Yamamoto; Martins, Regina Maria Bringel; Druzian, Angelita Fernandes; Lindenberg, Andréa Siqueira Campos; Torres, Marina Sawada; Pereira, Sérgio A; Villar, Livia Melo; Lampe, Elisabete; Motta-Castro, Ana Rita Coimbra

    2014-01-01

    A cross-sectional study on prevalence, associated factors and genotype distribution of HCV infection was conducted among 848 HIV-infected patients recruited at reference centers in the Midwest Region of Brazil. The prevalence rate of HIV-HCV coinfection was 6.9% (95% CI: 5.2 to 8.6). In multivariable analysis, increasing age, use of illicit drugs (injection and non-injection), a history of blood transfusion before 1994, and the absence of a steady partnership were significant independent associated factors for HIV-HCV coinfection. The phylogenetic analysis based on the NS5B region revealed the presence of two major circulating genotypes of HCV: genotypes 1 (58.3%) and 3 (41.7%). The prevalence of HIV-HCV coinfection was lower than those reported in studies conducted with HIV-infected patients in different regions of Brazil, due to the fact that illicit drug use is not a frequent mode of HIV transmission in this region of Brazil. Serologic screening of HIV-patients for HCV before initiating antiretroviral treatment, a comprehensive identification of associated factors, and the implementation of effective harm reduction programs are highly recommended to provide useful information for treatment and to prevent HCV coinfection in these patients.

  10. HIV AND HCV COINFECTION: PREVALENCE, ASSOCIATED FACTORS AND GENOTYPE CHARACTERIZATION IN THE MIDWEST REGION OF BRAZIL

    PubMed Central

    Freitas, Solange Zacalusni; Teles, Sheila Araújo; Lorenzo, Paulo Cesar; Puga, Marco Antonio Moreira; Tanaka, Tayana Serpa Ortiz; Thomaz, Danilo Yamamoto; Martins, Regina Maria Bringel; Druzian, Angelita Fernandes; Lindenberg, Andréa Siqueira Campos; Torres, Marina Sawada; Pereira, Sérgio A.; Villar, Livia Melo; Lampe, Elisabete; Motta-Castro, Ana Rita Coimbra

    2014-01-01

    A cross-sectional study on prevalence, associated factors and genotype distribution of HCV infection was conducted among 848 HIV-infected patients recruited at reference centers in the Midwest Region of Brazil. The prevalence rate of HIV-HCV coinfection was 6.9% (95% CI: 5.2 to 8.6). In multivariable analysis, increasing age, use of illicit drugs (injection and non-injection), a history of blood transfusion before 1994, and the absence of a steady partnership were significant independent associated factors for HIV-HCV coinfection. The phylogenetic analysis based on the NS5B region revealed the presence of two major circulating genotypes of HCV: genotypes 1 (58.3%) and 3 (41.7%). The prevalence of HIV-HCV coinfection was lower than those reported in studies conducted with HIV-infected patients in different regions of Brazil, due to the fact that illicit drug use is not a frequent mode of HIV transmission in this region of Brazil. Serologic screening of HIV-patients for HCV before initiating antiretroviral treatment, a comprehensive identification of associated factors, and the implementation of effective harm reduction programs are highly recommended to provide useful information for treatment and to prevent HCV coinfection in these patients. PMID:25351547

  11. Genotype dependent QSAR for HIV-1 protease inhibition.

    PubMed

    Boutton, Carlo W; De Bondt, Hendrik L; De Jonge, Marc R

    2005-03-24

    The development of drug-resistant viruses limits the therapeutic success of anti-HIV therapies. Some of these genetic HIV-variants display complex mutational patterns in their pol gene that codes for protease and reverse transcriptase, the most investigated molecular targets for antiretroviral therapy. In this paper, we present a computational structure-based approach to predict the resistance of a HIV-1 protease strain to amprenavir by calculating the interaction energy of the drug with HIV-1 protease. By considering the interaction energy per residue, we can identify what residue mutations contribute to drug-resistance. This approach is presented here as a structure-based tool for the prediction of resistance of HIV-1 protease toward amprenavir, with a view to use the drug-protein interaction-energy pattern in a lead-optimization procedure for the discovery of new anti-HIV drugs. PMID:15771454

  12. Translational Control of the HIV Unspliced Genomic RNA

    PubMed Central

    Rojas-Araya, Bárbara; Ohlmann, Théophile; Soto-Rifo, Ricardo

    2015-01-01

    Post-transcriptional control in both HIV-1 and HIV-2 is a highly regulated process that commences in the nucleus of the host infected cell and finishes by the expression of viral proteins in the cytoplasm. Expression of the unspliced genomic RNA is particularly controlled at the level of RNA splicing, export, and translation. It appears increasingly obvious that all these steps are interconnected and they result in the building of a viral ribonucleoprotein complex (RNP) that must be efficiently translated in the cytosolic compartment. This review summarizes our knowledge about the genesis, localization, and expression of this viral RNP. PMID:26247956

  13. HIV-2 Integrase Polymorphisms and Longitudinal Genotypic Analysis of HIV-2 Infected Patients Failing a Raltegravir-Containing Regimen

    PubMed Central

    Cavaco-Silva, Joana; Abecasis, Ana; Miranda, Ana Cláudia; Poças, José; Narciso, Jorge; Águas, Maria João; Maltez, Fernando; Almeida, Isabel; Germano, Isabel; Diniz, António; Gonçalves, Maria de Fátima; Gomes, Perpétua; Cunha, Celso; Camacho, Ricardo Jorge

    2014-01-01

    To characterize the HIV-2 integrase gene polymorphisms and the pathways to resistance of HIV-2 patients failing a raltegravir-containing regimen, we studied 63 integrase strand transfer inhibitors (INSTI)-naïve patients, and 10 heavily pretreated patients exhibiting virological failure while receiving a salvage raltegravir-containing regimen. All patients were infected by HIV-2 group A. 61.4% of the integrase residues were conserved, including the catalytic motif residues. No INSTI-major resistance mutations were detected in the virus population from naïve patients, but two amino acids that are secondary resistance mutations to INSTIs in HIV-1 were observed. The 10 raltegravir-experienced patients exhibited resistance mutations via three main genetic pathways: N155H, Q148R, and eventually E92Q - T97A. The 155 pathway was preferentially used (7/10 patients). Other mutations associated to raltegravir resistance in HIV-1 were also observed in our HIV-2 population (V151I and D232N), along with several novel mutations previously unreported. Data retrieved from this study should help build a more robust HIV-2-specific algorithm for the genotypic interpretation of raltegravir resistance, and contribute to improve the clinical monitoring of HIV-2-infected patients. PMID:24681625

  14. The HIV Tat protein affects processing of ribosomal RNA precursor

    PubMed Central

    Ponti, Donatella; Troiano, Maria; Bellenchi, Gian Carlo; Battaglia, Piero A; Gigliani, Franca

    2008-01-01

    Background Inside the cell, the HIV Tat protein is mainly found in the nucleus and nucleolus. The nucleolus, the site of ribosome biogenesis, is a highly organized, non-membrane-bound sub-compartment where proteins with a high affinity for nucleolar components are found. While it is well known that Tat accumulates in the nucleolus via a specific nucleolar targeting sequence, its function in this compartment it still unknown. Results To clarify the significance of the Tat nucleolar localization, we induced the expression of the protein during oogenesis in Drosophila melanogaster strain transgenic for HIV-tat gene. Here we show that Tat localizes in the nucleoli of Drosophila oocyte nurse cells, where it specifically co-localizes with fibrillarin. Tat expression is accompanied by a significant decrease of cytoplasmic ribosomes, which is apparently related to an impairment of ribosomal rRNA precursor processing. Such an event is accounted for by the interaction of Tat with fibrillarin and U3 snoRNA, which are both required for pre-rRNA maturation. Conclusion Our data contribute to understanding the function of Tat in the nucleolus, where ribosomal RNA synthesis and cell cycle control take place. The impairment of nucleolar pre-rRNA maturation through the interaction of Tat with fibrillarin-U3snoRNA complex suggests a process by which the virus modulates host response, thus contributing to apoptosis and protein shut-off in HIV-uninfected cells. PMID:18559082

  15. Longitudinal analysis of HIV-1 coreceptor tropism by single and triplicate HIV-1 RNA and DNA sequencing in patients undergoing successful first-line antiretroviral therapy

    PubMed Central

    Meini, Genny; Rossetti, Barbara; Bianco, Claudia; Ceccherini-Silberstein, Francesca; Di Giambenedetto, Simona; Sighinolfi, Laura; Monno, Laura; Castagna, Antonella; Rozera, Gabriella; D'Arminio Monforte, Antonella; Zazzi, Maurizio; De Luca, Andrea; Moroni, M.; Angarano, G.; Antinori, A.; Armignacco, O.; d'Arminio Monforte, A.; Castelli, F.; Cauda, R.; Di Perri, G.; Galli, M.; Iardino, R.; Ippolito, G.; Lazzarin, A.; Perno, C. F.; von Schloesser, F.; Viale, P.; d'Arminio Monforte, A.; 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.; d'Arminio Monforte, 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.; Giacometti, A.; Costantini, A.; 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, G.; Alessandrini, A.; Piscopo, R.; Mazzarello, G.; Mastroianni, C.; Belvisi, V.; Bonfanti, P.; Caramma, I.; 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.; Cicconi, P.; Marchetti, G.; Mussini, C.; Puzzolante, C.; Gori, A.; Lapadula, G.; Abrescia, N.; Chirianni, A.; Guida, M. G.; Gargiulo, M.; Baldelli, F.; Francisci, D.; Parruti, G.; Ursini, T.; Magnani, G.; Ursitti, M. A.; Cauda, R.; Andreoni, M.; Antinori, A.; Vullo, V.; Cingolani, A.; d'Avino, A.; Ammassari, A.; Gallo, L.; Nicastri, E.; Acinapura, R.; Capozzi, M.; Libertone, R.; Tebano, G.; Cattelan, A.; Mura, M. S.; Madeddu, G.; Caramello, P.; Di Perri, G.; Orofino, G. C.; Bonora, S.; Sciandra, M.; Pellizzer, G.; Manfrin, V.

    2014-01-01

    Objectives Maraviroc has been shown to be effective in patients harbouring CCR5-tropic HIV-1. While this CCR5 antagonist has initially been used in salvage therapy, its excellent safety profile makes it ideal for antiretroviral treatment simplification strategies in patients with suppressed plasma viraemia. The aim of this study was to compare HIV-1 tropism as detected in baseline plasma RNA and peripheral blood mononuclear cell (PBMC) DNA prior to first-line therapy and to analyse tropism evolution while on successful treatment. Methods HIV-1 tropism was determined using triplicate genotypic testing combined with geno2pheno[coreceptor] analysis at a 10% false positive rate in 42 patients. Paired pre-treatment plasma RNA and PBMC DNA and two subsequent PBMC DNA samples (the first obtained after reaching undetectable plasma HIV-1 RNA and the second after at least 2 years of suppression of plasma viraemia) were evaluated. Results Coreceptor tropism was completely concordant in paired pre-treatment RNA and DNA, with 26.2% of HIV-1 sequences predicted to be non-CCR5-tropic. During follow-up, coreceptor tropism switches were detected in 4 (9.5%) patients without any preferential direction. Although false positive rate discrepancies within triplicates were common, the rate of discordance of coreceptor tropism assignment among triplicate results in this mostly CCR5-tropic dataset was only 2.1%, questioning the added value of triplicate testing compared with single testing. Conclusions HIV-1 coreceptor tropism changes during virologically successful first-line treatment are infrequent. HIV-1 DNA analysis may thus support the choice of a CCR5 antagonist in treatment switch strategies; however, maraviroc treatment outcome data are required to confirm this option. PMID:24155059

  16. Thermodynamic profiling of HIV RREIIB RNA-zinc finger interactions.

    PubMed

    Mishra, Subrata H; Spring, Alexander M; Germann, Markus W

    2009-10-23

    The interactions between the HIV Rev-responsive element (RRE) RNA and the HIV regulatory protein Rev, are crucial for the HIV life-cycle. Earlier, we showed that single C(2)H(2) zinc fingers (znfs) have the same binding site as the Rev peptide and exhibit nanomolar affinity. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation of the binding energetics via changes in temperature, pH, buffers, and salt concentrations, as well as znf and RNA mutations, by isothermal titration calorimetry. Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized by NMR to a histidine on the znf beta-sheet. The DeltaC(p) of znf-RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent-accessible surface area models. An alternative model, based on the extent of hydrogen bond changes as a result of differences in ligand-induced water displacement at the binding site, provided reasonable explanation of the trends in DeltaC(p), as well as DeltaH and DeltaS. Our studies show that incorporation of favorable interactions at the solvent-excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface.

  17. Thermodynamic profiling of HIV RREIIB RNA – zinc finger interactions

    PubMed Central

    Mishra, Subrata H.; Spring, Alexander M.; Germann, Markus W.

    2009-01-01

    The interactions between the HIV Rev responsive element (RRE) RNA and the HIV regulatory protein Rev, is crucial for the HIV life cycle. We have previously shown that single C2H2 zinc fingers, have the same binding site as the Rev peptide and exhibit nanomolar affinities. In this study, the specific role of amino acid side chains and molecular processes involved with complex formation were investigated by perturbation to the binding energetics via changes in temperature, pH, buffers, salt concentrations as well as zinc finger (znf) and RNA mutations, by isothermal titration calorimetry (ITC). Interestingly, despite the large cationic charge on the znfs, the number of interactions with the RNA phosphate backbone was lower than intuitively expected. The presence of binding induced protonation was established by ITC and localized to a histidine on the znf β-sheet, by NMR. The ΔCp of znf-RNA binding was observed to be substantially negative and could not be accounted for by conventional solvent accessible surface area models. An alternative model (50), based on the extent of hydrogen bond changes as a result of differences in ligand induced water displacement at the binding site, provided reasonable explanation of the trends in ΔCp, as well as ΔH and ΔS. Our studies show that incorporation of favorable interactions at the solvent excluded binding interface can be used to alleviate the unfavorable enthalpic penalties of displacing water molecules from the hydrated RNA surface. PMID:19646998

  18. On the Selective Packaging of Genomic RNA by HIV-1.

    PubMed

    Comas-Garcia, Mauricio; Davis, Sean R; Rein, Alan

    2016-01-01

    Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag-gRNA complex will nucleate particle assembly more efficiently than other Gag-RNA complexes. New data shows that among cellular mRNAs, those with long 3'-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3'-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.

  19. Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

    PubMed Central

    2011-01-01

    Background HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was

  20. Examining small molecule: HIV RNA interactions using arrayed imaging reflectometry

    NASA Astrophysics Data System (ADS)

    Chaimayo, Wanaruk; Miller, Benjamin L.

    2014-03-01

    Human Immunodeficiency Virus (HIV) has been the subject of intense research for more than three decades as it causes an uncurable disease: Acquired Immunodeficiency Syndrome, AIDS. In the pursuit of a medical treatment, RNAtargeted small molecules are emerging as promising targets. In order to understand the binding kinetics of small molecules and HIV RNA, association (ka) and dissociation (kd) kinetic constants must be obtained, ideally for a large number of sequences to assess selectivity. We have developed Aqueous Array Imaged Reflectometry (Aq-AIR) to address this challenge. Using a simple light interference phenomenon, Aq-AIR provides real-time high-throughput multiplex capabilities to detect binding of targets to surface-immobilized probes in a label-free microarray format. The second generation of Aq-AIR consisting of high-sensitivity CCD camera and 12-μL flow cell was fabricated. The system performance was assessed by real-time detection of MBNL1-(CUG)10 and neomycin B - HIV RNA bindings. The results establish this second-generation Aq-AIR to be able to examine small molecules binding to RNA sequences specific to HIV.

  1. Antiviral Stratagems Against HIV-1 Using RNA Interference (RNAi) Technology

    PubMed Central

    Vlachakis, Dimitrios; Tsiliki, Georgia; Pavlopoulou, Athanasia; Roubelakis, Maria G.; Champeris Tsaniras, Spyridon; Kossida, Sophia

    2013-01-01

    The versatility of human immunodeficiency virus (HIV)-1 and its evolutionary potential to elude antiretroviral agents by mutating may be its most invincible weapon. Viruses, including HIV, in order to adapt and survive in their environment evolve at extremely fast rates. Given that conventional approaches which have been applied against HIV have failed, novel and more promising approaches must be employed. Recent studies advocate RNA interference (RNAi) as a promising therapeutic tool against HIV. In this regard, targeting multiple HIV sites in the context of a combinatorial RNAi-based approach may efficiently stop viral propagation at an early stage. Moreover, large high-throughput RNAi screens are widely used in the fields of drug development and reverse genetics. Computer-based algorithms, bioinformatics, and biostatistical approaches have been employed in traditional medicinal chemistry discovery protocols for low molecular weight compounds. However, the diversity and complexity of RNAi screens cannot be efficiently addressed by these outdated approaches. Herein, a series of novel workflows for both wet- and dry-lab strategies are presented in an effort to provide an updated review of state-of-the-art RNAi technologies, which may enable adequate progress in the fight against the HIV-1 virus. PMID:23761954

  2. Estimation of HIV-1 DNA Level Interfering with Reliability of HIV-1 RNA Quantification Performed on Dried Blood Spots Collected from Successfully Treated Patients.

    PubMed

    Zida, Sylvie; Tuaillon, Edouard; Barro, Makoura; Kwimatouo Lekpa Franchard, Arnaud; Kagoné, Thérèse; Nacro, Boubacar; Ouedraogo, Abdoul Salam; Bolloré, Karine; Sanosyan, Armen; Plantier, Jean-Christophe; Meda, Nicolas; Sangaré, Lassana; Rouzioux, Christine; Rouet, François; Kania, Dramane

    2016-06-01

    The impact of HIV-1 DNA coamplification during HIV-1 RNA quantification on dried blood spots (DBS) was explored. False-positive HIV RNA detection (22/62, 35%) was associated with high HIV-1 DNA levels. Specificity of HIV-1 RNA assays on DBS should be evaluated following manufacturer protocols on samples with HIV-1 DNA levels of ≥1,000 copies/10(6) peripheral blood mononuclear cells. PMID:27008874

  3. Replicative phenotyping adds value to genotypic resistance testing in heavily pre-treated HIV-infected individuals - the Swiss HIV Cohort Study

    PubMed Central

    2011-01-01

    Background Replicative phenotypic HIV resistance testing (rPRT) uses recombinant infectious virus to measure viral replication in the presence of antiretroviral drugs. Due to its high sensitivity of detection of viral minorities and its dissecting power for complex viral resistance patterns and mixed virus populations rPRT might help to improve HIV resistance diagnostics, particularly for patients with multiple drug failures. The aim was to investigate whether the addition of rPRT to genotypic resistance testing (GRT) compared to GRT alone is beneficial for obtaining a virological response in heavily pre-treated HIV-infected patients. Methods Patients with resistance tests between 2002 and 2006 were followed within the Swiss HIV Cohort Study (SHCS). We assessed patients' virological success after their antiretroviral therapy was switched following resistance testing. Multilevel logistic regression models with SHCS centre as a random effect were used to investigate the association between the type of resistance test and virological response (HIV-1 RNA <50 copies/mL or ≥1.5log reduction). Results Of 1158 individuals with resistance tests 221 with GRT+rPRT and 937 with GRT were eligible for analysis. Overall virological response rates were 85.1% for GRT+rPRT and 81.4% for GRT. In the subgroup of patients with >2 previous failures, the odds ratio (OR) for virological response of GRT+rPRT compared to GRT was 1.45 (95% CI 1.00-2.09). Multivariate analyses indicate a significant improvement with GRT+rPRT compared to GRT alone (OR 1.68, 95% CI 1.31-2.15). Conclusions In heavily pre-treated patients rPRT-based resistance information adds benefit, contributing to a higher rate of treatment success. PMID:21255386

  4. Field study of dried blood spot specimens for HIV-1 drug resistance genotyping.

    PubMed

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

    2014-08-01

    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

  5. 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

  6. HIV genotypes and primary drug resistance among HIV seropositive blood donors in Brazil: role of infected blood donors as sentinel populations for molecular surveillance of HIV

    PubMed Central

    Alencar, CS; Sabino, EC; Carvalho, SMF; Leao, S; Carneiro- Proietti, AB; Capuani, L; Oliveira, CL; Carrick, D; Birch, RJ; Gonçalez, TT; Keating, S; Swanson, P; Hackett, J; Busch, MP

    2013-01-01

    Background There are few surveillance studies analyzing genotypes or primary (transmitted) drug resistance in HIV-infected blood donors in Brazil. The aim of this study was to characterize patterns of HIV genotypes and primary resistance among HIV seropositive donors identified at 4 geographically dispersed blood centers in Brazil. Methods All HIV-infected donors who returned for counseling at the 4 REDS-II Hemocenters in Brazil from January 2007–March 2011 were invited to participate in a case-control study involving a questionnaire on risk factors. Viral sequencing was also offered to positive cases to assign genotypes and to detect and characterize primary resistance to RT and protease inhibitors according to WHO guidelines. Results Of the 341 HIV seropositive donors who consented to participate in the risk-factor and genetics study, pol sequences were obtained for 331 (97%). Clade B was predominant (76%) followed by F (15%) and C (5%). Primary resistance was present in 36 (12.2%; 95% confidence interval [CI] 8.2,15.5) of the 303 individuals not exposed to antiretroviral therapy (ART), varying from 8.2% (95%CI 2,7,13.6) in Recife to 19.4% in São Paulo (95%CI 9.5,29.2); there were no significant correlations with other demographics or risk factors. Conclusion Although subtype B remains the most prevalent genotype in all 4 areas, increasing rates of subtype C in Sao Paulo and F in Recife were documented relative to earlier reports. Transmitted drug resistance was relatively frequent, particularly in the city of Sao Paulo which showed an increase compared to previous HIV seropositive donor data from 10 years ago. PMID:23507660

  7. Prediction of HIV drug resistance from genotype with encoded three-dimensional protein structure

    PubMed Central

    2014-01-01

    Background Drug resistance has become a severe challenge for treatment of HIV infections. Mutations accumulate in the HIV genome and make certain drugs ineffective. Prediction of resistance from genotype data is a valuable guide in choice of drugs for effective therapy. Results In order to improve the computational prediction of resistance from genotype data we have developed a unified encoding of the protein sequence and three-dimensional protein structure of the drug target for classification and regression analysis. The method was tested on genotype-resistance data for mutants of HIV protease and reverse transcriptase. Our graph based sequence-structure approach gives high accuracy with a new sparse dictionary classification method, as well as support vector machine and artificial neural networks classifiers. Cross-validated regression analysis with the sparse dictionary gave excellent correlation between predicted and observed resistance. Conclusion The approach of encoding the protein structure and sequence as a 210-dimensional vector, based on Delaunay triangulation, has promise as an accurate method for predicting resistance from sequence for drugs inhibiting HIV protease and reverse transcriptase. PMID:25081370

  8. Novel Biochemical Tools for Probing HIV RNA Structure.

    PubMed

    Rausch, Jason W; Sztuba-Solinska, Joanna; Lusvarghi, Sabrina; Le Grice, Stuart F J

    2016-01-01

    Functional analysis of viral RNA requires knowledge of secondary structure arrangements and tertiary base interactions. Thus, high-throughput and comprehensive methods for assessing RNA structure are highly desirable. Selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) has proven highly useful for modeling the secondary structures of HIV and other retroviral RNAs in recent years. This technology is not without its limitations however, as SHAPE data can be severely compromised when the RNA under study is structurally heterogeneous. In addition, the method reveals little information regarding the three-dimensional (3D) organization of an RNA. This chapter outlines four detailed SHAPE-related methodologies that circumvent these limitations. "Ensemble" and "in-gel" variations of SHAPE permit structural analysis of individual conformers within structurally heterogeneous mixtures of RNA, while probing strategies that utilize "through-space" cleavage reagents such as methidiumpropyl-EDTA (MPE) and peptides appended with an ATCUN (amino terminal copper/nickel binding motif) can provide insight into 3D organization. Combinational application of these techniques provides a formidable arsenal for exploring the structures of HIV RNAs and associated nucleoprotein complexes.

  9. On the Selective Packaging of Genomic RNA by HIV-1

    PubMed Central

    Comas-Garcia, Mauricio; Davis, Sean R.; Rein, Alan

    2016-01-01

    Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag–gRNA complex will nucleate particle assembly more efficiently than other Gag–RNA complexes. New data shows that among cellular mRNAs, those with long 3′-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3′-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag. PMID:27626441

  10. On the Selective Packaging of Genomic RNA by HIV-1.

    PubMed

    Comas-Garcia, Mauricio; Davis, Sean R; Rein, Alan

    2016-01-01

    Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its cis-acting packaging signal, the mechanism of this selection is not understood. The affinity of Gag (the viral structural protein) for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag-gRNA complex will nucleate particle assembly more efficiently than other Gag-RNA complexes. New data shows that among cellular mRNAs, those with long 3'-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3'-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag. PMID:27626441

  11. Quantitation of HIV-1 RNA in breast milk by real time PCR.

    PubMed

    Becquart, Pierre; Foulongne, Vincent; Willumsen, Juana; Rouzioux, Christine; Segondy, Michel; Van de Perre, Philippe

    2006-04-01

    HIV-1 RNA in breast milk is a strong predictor of HIV-1 transmission through breastfeeding. In the present report, breast milk samples from HIV-1 uninfected donors were spiked with dilution of quantified culture supernatant from HIV-1(NDK) infected PBMC. Two RNA extraction techniques based on silica extraction, Nuclisens (BioMerieux) and Triazol (Qiagen), two techniques based on guanidine thiocynanate/chloroforme extraction, TRIzol (Life Technologie) and Amplicor HIV-1 Monitor (Roche Diagnostic Systems), and one technique based on electrostatic adsorption on iron oxide micro beads (Promega) were compared. HIV-1 RNA was quantitated by real time PCR (LTR gene) and Amplicor HIV-1 Monitor. Combining magnetic micro beads extraction and real time PCR quantitation allowed to correctly quantify breast milk HIV-1 RNA, with a difference between the expected and measured HIV-1 RNA levels always lower than 0.3 log copies/ml. The same combination was confirmed on 25 breast milk samples from HIV-1 infected women collected in Kwazulu-Natal, South Africa, by comparing measurements with those obtained by the Amplicor HIV-1 Monitor (r(2)=0.88). Nucleic acid extraction by magnetic micro beads followed by real time PCR is a reliable, sensitive, rapid and simple procedure to quantify HIV-1 RNA in breast milk and allows for PCR inhibitors found frequently in these samples.

  12. Apolipoprotein E4 genotype does not increase risk of HIV-associated neurocognitive disorders.

    PubMed

    Morgan, E E; Woods, S P; Letendre, S L; Franklin, D R; Bloss, C; Goate, A; Heaton, R K; Collier, A C; Marra, C M; Gelman, B B; McArthur, J C; Morgello, S; Simpson, D M; McCutchan, J A; Ellis, R J; Abramson, I; Gamst, A; Fennema-Notestine, C; Smith, D M; Grant, I; Vaida, F; Clifford, D B

    2013-04-01

    This is a cross-sectional, observational study to evaluate the hypothesis that HIV-seropositive (HIV+) apolipoprotein E4 (APOE4) carriers are at increased risk for HIV-associated neurocognitive disorders (HAND) compared to APOE4 noncarriers with HIV in the CNS HIV Antiretroviral Therapy Effects Research (CHARTER) Group sample. APOE genotype was determined in 466 CHARTER participants with varying disease stages and histories of antiretroviral treatment who did not have severe psychiatric or medical comorbid conditions that preclude diagnosis of HAND. HAND diagnoses were based on results of comprehensive neurobehavioral evaluation and use of current neuroAIDS diagnostic criteria. HAND status consists of two levels: neuropsychologically normal status (i.e., no HAND) and any HAND diagnosis (i.e., asymptomatic neurocognitive impairment, minor neurocognitive disorder, HIV-associated dementia). Logistic regression analyses revealed no association between APOE4 carrier status and HAND, and there were no interactions between APOE4 carrier status and ethnicity, age, substance use disorders, duration of infection, or nadir CD4. Results did not differ when analysis was restricted to symptomatic HAND, and no APOE4 gene dose-dependent relationship to HAND emerged. APOE4 status was not associated with concurrent HAND in this large, well-characterized sample. This does not preclude emergence of an association between APOE4 status and HAND as this population ages. Prospective, longitudinal studies are needed to examine APOE4 as a risk factor for neurocognitive decline, incident HAND at older ages, and potential associations with cerebrospinal fluid amyloid.

  13. Mycobacterium tuberculosis Beijing Genotype Is Associated with HIV Infection in Mozambique

    PubMed Central

    Viegas, Sofia O.; Machado, Adelina; Groenheit, Ramona; Ghebremichael, Solomon; Pennhag, Alexandra; Gudo, Paula S.; Cuna, Zaina; Langa, Egídio; Miotto, Paolo; Cirillo, Daniela M.; Rastogi, Nalin; Warren, Rob M.; van Helden, Paul D.; Koivula, Tuija; Källenius, Gunilla

    2013-01-01

    The Beijing genotype is a lineage of Mycobacterium tuberculosis that is distributed worldwide and responsible for large epidemics, associated with multidrug-resistance. However, its distribution in Africa is less understood due to the lack of data. Our aim was to investigate the prevalence and possible transmission of Beijing strains in Mozambique by a multivariate analysis of genotypic, geographic and demographic data. A total of 543 M. tuberculosis isolates from Mozambique were spoligotyped. Of these, 33 were of the Beijing lineage. The genetic relationship between the Beijing isolates were studied by identification of genomic deletions within some Regions of Difference (RD), Restriction Fragment Length Polymorphism (RFLP) and Mycobacterial Interspersed Repetivie Unit – variable number tandem repeat (MIRU-VNTR). Beijing strains from South Africa, representing different sublineages were included as reference strains. The association between Beijing genotype, Human Immunodeficiency Virus (HIV) serology and baseline demographic data was investigated. HIV positive serostatus was significantly (p=0.023) more common in patients with Beijing strains than in patients with non-Beijing strains in a multivariable analysis adjusted for age, sex and province (14 (10.9%) of the 129 HIV positive patients had Beijing strains while 6/141 (4.3%) of HIV negative patients had Beijing strains). The majority of Beijing strains were found in the Southern region of Mozambique, particularly in Maputo City (17%). Only one Beijing strain was drug resistant (multi-drug resistant). By combined use of RD and spoligotyping, three genetic sublineages could be tentatively identified where a distinct group of four isolates had deletion of RD150, a signature of the “sublineage 7” recently emerging in South Africa. The same group was very similar to South African “sublineage 7” by RFLP and MIRU-VNTR, suggesting that this sublineage could have been recently introduced in Mozambique from

  14. Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA

    PubMed Central

    Harwig, Alex; Jongejan, Aldo; van Kampen, Antoine H. C.; Berkhout, Ben; Das, Atze T.

    2016-01-01

    Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3′ side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome. PMID:26984525

  15. Tat-dependent production of an HIV-1 TAR-encoded miRNA-like small RNA.

    PubMed

    Harwig, Alex; Jongejan, Aldo; van Kampen, Antoine H C; Berkhout, Ben; Das, Atze T

    2016-05-19

    Evidence is accumulating that retroviruses can produce microRNAs (miRNAs). To prevent cleavage of their RNA genome, retroviruses have to use an alternative RNA source as miRNA precursor. The transacting responsive (TAR) hairpin structure in HIV-1 RNA has been suggested as source for miRNAs, but how these small RNAs are produced without impeding virus replication remained unclear. We used deep sequencing analysis of AGO2-bound HIV-1 RNAs to demonstrate that the 3' side of the TAR hairpin is processed into a miRNA-like small RNA. This ∼21 nt RNA product is able to repress the expression of mRNAs bearing a complementary target sequence. Analysis of the small RNAs produced by wild-type and mutant HIV-1 variants revealed that non-processive transcription from the HIV-1 LTR promoter results in the production of short TAR RNAs that serve as precursor. These TAR RNAs are cleaved by Dicer and processing is stimulated by the viral Tat protein. This biogenesis pathway differs from the canonical miRNA pathway and allows HIV-1 to produce the TAR-encoded miRNA-like molecule without cleavage of the RNA genome. PMID:26984525

  16. Sustained virological response to peginterferon therapy in patients infected with HCV (genotypes 2 and 3), with or without HIV

    PubMed Central

    2014-01-01

    Background HIV infection leads to a faster progression of liver disease in subjects infected with HCV, as compared with HCV mono-infected patients. Previous reports suggest that sustained virological response (SVR) rates are lower in HIV/HCV coinfection than in HCV monoinfection. We aimed to compare SVR rates of these two populations. Methods We retrospectively analyzed clinical, biochemical and virological data of HCV and HIV/HCV infected patients with HCV genotypes 2 and 3 who started anti-HCV treatment between March 2004 and November 2012, at a single large center. Intention-to-treat (ITT) and per-protocol (PP) analysis were performed. Univariate and multivariate logistic regression analyses were performed to assess predictors of SVR. Results 461 patients were analyzed: 307 (66.6%) males, 76 (16.5%) infected with HIV. Several differences at baseline between HCV monoinfected and HIV/HCV coinfected patients were observed. HCV monoinfected group was characterized by higher prevalence of genotype 2 (53% vs 5.3%), higher baseline HCV viral load (50% vs 35%), shorter mean duration of treatment (19 vs 41 weeks), more frequent use of peginterferon alfa-2a (84.5% vs 69.7%), lower prevalence of cirrhosis (6% vs 31.6%). Globally, SVR was achieved by 353 (76.6%) patients and 321 (83.8%) in the PP analysis. No statistically relevant differences were found in SVR rates between the two groups, either in ITT [78.2% (n = 301/385) vs 68.4% (n = 52/76), p =0.066, respectively] than in PP analysis [83.6% (n = 276/330) vs 84.9% (n = 45/53), p = 0.8]. ITT analysis At univariate and multivariate analysis, baseline HCV-RNA >500.000 IU/ml [OR 0.4 (0.24-0.66), p = 0.0004], use of peginterferon alfa-2b [OR 0.5 (0.27-0.93) p = 0.033], platelets count <130.000/mm3 [OR 0.45 (0.2-0.99), p = 0.045], interruption of peginterferon therapy [OR 0.2 (0.1-0.4), p<0.0001], interruption of ribavirin treatment [OR 0.34 (0.17-0.69), p = 0.0026] were related with lower rate of SVR. PP analysis Only HCV-RNA

  17. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array

    NASA Astrophysics Data System (ADS)

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-beom; Choi, Heon-Jin

    2016-07-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes.

  18. Sensitive and Selective Detection of HIV-1 RRE RNA Using Vertical Silicon Nanowire Electrode Array.

    PubMed

    Lee, Jaehyung; Hong, Min-Ho; Han, Sanghun; Na, Jukwan; Kim, Ilsoo; Kwon, Yong-Joon; Lim, Yong-Beom; Choi, Heon-Jin

    2016-12-01

    In this study, HIV-1 Rev response element (RRE) RNA was detected via an Au-coated vertical silicon nanowire electrode array (VSNEA). The VSNEA was fabricated by combining bottom-up and top-down approaches and then immobilized by artificial peptides for the recognition of HIV-1 RRE. Differential pulse voltammetry (DPV) analysis was used to measure the electrochemical response of the peptide-immobilized VSNEA to the concentration and types of HIV-1 RRE RNA. DPV peaks showed linearity to the concentration of RNA with a detection limit down to 1.513 fM. It also showed the clear different peaks to the mutated HIV-1 RRE RNA. The high sensitivity and selectivity of VSNEA for the detection of HIV-1 RRE RNA may be attributed to the high surface-to-volume ratio and total overlap diffusion mode of ions of the one-dimensional nanowire electrodes.

  19. Prevalences, genotypes, and risk factors for HIV transmission in South America.

    PubMed

    Montano, Silvia M; Sanchez, Jose L; Laguna-Torres, Alberto; Cuchi, Paloma; Avila, Maria M; Weissenbacher, Mercedes; Serra, Margarita; Viñoles, Jose; Russi, Jose C; Aguayo, Nicolas; Galeano, Adolfo H; Gianella, Alberto; Andrade, Ronald; Arredondo, Anabella; Ramirez, Eugenio; Acosta, Maria E; Alava, Aracely; Montoya, Orlando; Guevara, Angel; Manrique, Hugo; Sanchez, Jorge L; Lama, Javier R; de la Hoz, Fernando; Sanchez, Gloria I; Ayala, Claudia; Pacheco, Maria E; Carrion, Gladys; Chauca, Gloria; Perez, Juan J; Negrete, Monica; Russell, Kevin L; Bautista, Christian T; Olson, James G; Watts, Douglas M; Birx, Deborah L; Carr, Jean K

    2005-09-01

    HIV cross-sectional studies were conducted among high-risk populations in 9 countries of South America. Enzyme-linked immunosorbent assay screening and Western blot confirmatory testing were performed, and env heteroduplex mobility assay genotyping and DNA sequencing were performed on a subset of HIV-positive subjects. HIV prevalences were highest among men who have sex with men (MSM; 2.0%-27.8%) and were found to be associated with multiple partners, noninjection drug use (non-IDU), and sexually transmitted infections (STIs). By comparison, much lower prevalences were noted among female commercial sex workers (FCSWs; 0%-6.3%) and were associated mainly with a prior IDU and STI history. Env subtype B predominated among MSM throughout the region (more than 90% of strains), whereas env subtype F predominated among FCSWs in Argentina and male commercial sex workers in Uruguay (more than 50% of strains). A renewed effort in controlling STIs, especially among MSM groups, could significantly lessen the impact of the HIV epidemic in South America.

  20. [Titration and genotyping of hepatitis C virus RNA in chronic hepatitis C patients treated with interferon].

    PubMed

    Konishi, M

    1994-02-01

    In order to evaluate the relation of the response to IFN therapy with viral factors, we quantified and typed HCV-RNA in sera and liver specimens from hepatitis C patients treated with IFN by using the single tube polymerase chain reaction. Sustained serum aminotransferase (sALT) normalization was resulted in 11 (19.6%) of the 51 patients. At 6 months after IFN therapy, serum HCV-RNA was negative in 13 of 44 patients. The pretreatment serum HCV-RNA titers tended to be higher in no responders than in complete responders and partial responders. HCV-RNA concentration in the liver correlated with serum HCV-RNA quantity. HCV infections with type I, II, III, IV, II + III, III + IV or II + III + IV were detected in 0, 24, 9, 2, 9, 2 or 3 patients, respectively. Mixed infection with more than 2 genotypes of HCV was found commonly than previously reported. IFN therapy induced complete response in 6 (66.7%) of the 9 patients with type III infection, and this response rate was apparently higher than that in the patients infected with type II or mixed type of HCV. Serum HCV-RNA titers were significantly higher in the patients with type III infection than in the patients with type II or mixed type infection. These findings implied that the determinations of pretreatment serum HCV-RNA titers and HCV-genotypes could be useful to predict sALT improvement and HCV eradication by IFN therapy.

  1. A Rapid, Self-confirming Assay for HIV: Simultaneous Detection of Anti-HIV Antibodies and Viral RNA

    PubMed Central

    Chen, Zongyuan; Zhu, Hui; Malamud, Daniel; Barber, Cheryl; Ongagna, Yhombi Yvon Serge; Yasmin, Rubina; Modak, Sayli; Janal, Malvin N.; Abrams, William R.; Montagna, Richard A.

    2016-01-01

    Objective We developed a microfluidic system to simultaneously detect host anti-HIV antibodies and viral RNA in the same specimen in order to satisfy two important diagnostic criteria, especially within resource-limited settings. First, the system can detect acute HIV infection and allow immediate confirmation of a seropositive screening result by detection of HIV RNA. It also addresses the well-known "seroconversion window" during early HIV infection when antibodies are not yet detectable and viral loads are at their highest. Methods We first developed and optimized two separate manual assays for the detection of host anti-HIV antibodies and viral RNA and then converted them to the microfluidic system. We optimized a commercially available serologic assay to run within the microfluidic device while we incorporated the isothermal LAMP assay to detect the presence of viral RNA. The microfluidic device and instrumentation were developed to simultaneously perform both assays without any user intervention. Results The finalized system consists of a disposable injection molded and film-laminated microfluidic CARD disposable device and a portable, software controlled instrument, which together can automatically perform all steps of both assays without any user intervention after the initial loading of samples and reagents. The microfluidic CARD cartridge has multiple microchannels, valves, pumps and reservoirs, which perform the immunoassay, isolates viral RNA for detection by magnetic bead based purification, and Reverse Transcriptase loop-mediated isothermal amplification (RT-LAMP). The microfluidic system was able to detect host anti-HIV antibodies and viral RNA in either a blood or saliva sample. Conclusion The ability to detect antibodies and simultaneously confirm a seropositive HIV-RNA result provides healthcare workers with a complete and accurate appraisal of a patient's infection status in the earliest stages of the disease and represents an important tool for

  2. CCR5Δ32 Genotypes in a German HIV-1 Seroconverter Cohort and Report of HIV-1 Infection in a CCR5Δ32 Homozygous Individual

    PubMed Central

    Oh, Djin-Ye; Jessen, Heiko; Kücherer, Claudia; Neumann, Konrad; Oh, Nari; Poggensee, Gabriele; Bartmeyer, Barbara; Jessen, Arne; Pruss, Axel

    2008-01-01

    Background Homozygosity (Δ32/Δ32) for the 32 bp deletion in the chemokine receptor 5 (CCR5) gene is associated with strong resistance against HIV infection. Heterozygosity is associated with protection of HIV-1 disease progression. Methodology/Principal Findings We genotyped a population of 737 HIV-positive adults and 463 healthy controls for the CCR5Δ32 deletion and found heterozygous frequencies of 16.2% (HIV-negative) and 17.5% (HIV-positive) among Caucasian individuals. Analysis of CCR5Δ32 influence on disease progression showed notably lower viral setpoints and a longer time to a CD4 count of <200 µl−1 in seroconverters heterozygous for the deletion. Furthermore, we identified one HIV-positive man homozygous for the Δ32 deletion. Conclusions/Significance The protective effect of CCR5 Δ32 heterozygosity is confimed in a large cohort of German seroconverters. The HIV-infected CCR5 Δ32 homozygous individual, however, displays extremely rapid disease progression. This is the 12th case of HIV-infection in this genotype described worldwide. PMID:18648518

  3. 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

  4. 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

  5. APOL1 Genotype and Glomerular and Tubular Kidney Injury in Women With HIV

    PubMed Central

    Jotwani, Vasantha; Shlipak, Michael G.; Scherzer, Rebecca; Parekh, Rulan S.; Kao, W.H. Linda; Bennett, Michael; Cohen, Mardge H.; Nowicki, Marek; Sharma, Anjali; Young, Mary; Tien, Phyllis C.; Parikh, Chirag R.; Estrella, Michelle M.

    2015-01-01

    Background APOL1 genotype is associated with advanced kidney disease in African-Americans, but the pathogenic mechanisms are unclear. Here, associations of APOL1 genotype with urine biomarkers of glomerular and tubular injury, and with kidney function decline, were evaluated. Study Design Observational study. Setting & Participants 431 HIV-infected African-American women enrolled in Women's Interagency HIV Study (WIHS). Predictor APOL1 genotype. Outcomes Albumin-creatinine ratio (ACR), four tubular injury biomarkers (interleukin 18 [IL-18], kidney injury molecule 1 [KIM-1], neutrophil gelatinase-associated lipocalin [NGAL], and α1-microglobulin [α1m]), and kidney function estimated using the CKD-EPI cystatin C equation. Measurements Participants were genotyped for APOL1 single-nucleotide polymorphisms rs73885319 (G1 allele) and rs71785313 (G2 allele). Urine biomarker levels were measured using stored samples from 1999-2000. Cystatin C was measured using serum collected at baseline and 4- and 8-year follow-up. Results At baseline, ACR levels were higher among 47 women with 2 APOL1 risk alleles versus 384 women with 0/1 risk allele (median, 24 vs. 11 mg/g; p < 0.001). Compared to women with 0/1 risk allele, women with 2 risk alleles had 104% higher ACR (95% CI, 29-223 mg/g) and 2-fold greater risk of ACR > 30 mg/g (95% CI, 1.17-3.44) after multivariable adjustment. APOL1 genotype showed little association with urine IL-18:Cr, KIM-1:Cr, and NGAL:Cr (estimates of -5% [95% CI, -24% to 18%], -20% [95% CI, -36% to 1%], and 10% [95% CI, -26% to 64%], respectively), or detectable urine α1m (prevalence ratio, 1.13; 95% CI, 0.65-1.97) in adjusted analyses. Compared to women with 0/1 allele, women with 2 risk alleles had faster eGFR decline, by 1.2 (95% CI, -2.2 to -0.2) ml/min/1.73 m2 per year, and had 1.7- and 3.4-fold greater rates of incident chronic kidney disease (95% CI, 1.1-2.5) and 10% annual eGFR decline (95% CI, 1.7-6.7), respectively, with minimal attenuation

  6. 48 week outcomes of maraviroc-containing regimens following the genotypic or Trofile assay in HIV-1 failing subjects: the OSCAR Study.

    PubMed

    Nozza, Silvia; Pignataro, Angela Rosa; Galli, Laura; Svicher, Valentina; Alteri, Claudia; Boeri, Enzo; Ripa, Marco; Castagna, Antonella; Sampaolo, Michela; Clementi, Massimo; Perno, Carlo Federico; Lazzarin, Adriano

    2016-09-01

    This study assessed the 48-week efficacy of an antiretroviral therapy including maraviroc following the assessment of co-receptor tropism by use of Geno2Pheno algorithm or the Trofile phenotypic assay in failing treatment-experienced HIV-1 patients. This was a multicenter, randomized, open-label, non-inferiority trial. Treatment-experienced subjects with HIV-RNA ≥500 copies/mL were randomized (1:1) to undergo co-receptor tropism testing by the Geno- 2Pheno algorithm (with a false positive rate >10%) or the Trofile assay before starting a new antiretroviral treatment which included maraviroc. The primary endpoint was the 48 week proportion of patients with treatment success (TS). Intention-to-treat analyses are also reported. One hundred and fifty-five experienced patients were analysed: 77 patients in the Trofile arm and 78 in the Genotype arm. The 48-week proportion of TS was 87% in the Trofile arm and 89% in the Genotype arm (difference: 1.5%, 95%CI: -8.9% to 11.8%) suggesting non-inferiority. In the Trofile arm, 10 patients had treatment failure: 5 viral rebound, 5 discontinuations. In the Genotype arm, 9 patients had treatment failure: 7 viral rebound, 2 lost to follow-up. CD4+ significantly increased from baseline to week 48 in both arms. 48-week treatment success was similar for maraviroc-including therapy prescribed following the Trofile phenotypic assay or Geno2Pheno algorithm. PMID:27602417

  7. HIV-1 RNA genome dimerizes on the plasma membrane in the presence of Gag protein.

    PubMed

    Chen, Jianbo; Rahman, Sheikh Abdul; Nikolaitchik, Olga A; Grunwald, David; Sardo, Luca; Burdick, Ryan C; Plisov, Sergey; Liang, Edward; Tai, Sheldon; Pathak, Vinay K; Hu, Wei-Shau

    2016-01-12

    Retroviruses package a dimeric genome comprising two copies of the viral RNA. Each RNA contains all of the genetic information for viral replication. Packaging a dimeric genome allows the recovery of genetic information from damaged RNA genomes during DNA synthesis and promotes frequent recombination to increase diversity in the viral population. Therefore, the strategy of packaging dimeric RNA affects viral replication and viral evolution. Although its biological importance is appreciated, very little is known about the genome dimerization process. HIV-1 RNA genomes dimerize before packaging into virions, and RNA interacts with the viral structural protein Gag in the cytoplasm. Thus, it is often hypothesized that RNAs dimerize in the cytoplasm and the RNA-Gag complex is transported to the plasma membrane for virus assembly. In this report, we tagged HIV-1 RNAs with fluorescent proteins, via interactions of RNA-binding proteins and motifs in the RNA genomes, and studied their behavior at the plasma membrane by using total internal reflection fluorescence microscopy. We showed that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane. Dynamic interactions occur among HIV-1 RNAs, and stabilization of the RNA dimer requires Gag protein. Dimerization often occurs at an early stage of the virus assembly process. Furthermore, the dimerization process is probably mediated by the interactions of two RNA-Gag complexes, rather than two RNAs. These findings advance the current understanding of HIV-1 assembly and reveal important insights into viral replication mechanisms.

  8. Heterogeneous structures formed by conserved RNA sequences within the HIV reverse transcription initiation site

    PubMed Central

    Coey, Aaron; Larsen, Kevin; Puglisi, Joseph D.; Viani Puglisi, Elisabetta

    2016-01-01

    Reverse transcription is a key process in the early steps of HIV infection. This process initiates within a specific complex formed by the 5′ UTR of the HIV genomic RNA (vRNA) and a host primer tRNALys3. Using nuclear magnetic resonance (NMR) spectroscopy and single-molecule fluorescence spectroscopy, we detect two distinct conformers adopted by the tRNA/vRNA initiation complex. We directly show that an interaction between the conserved 8-nucleotide viral RNA primer activation signal (PAS) and the primer tRNA occurs in one of these conformers. This intermolecular PAS interaction likely induces strain on a vRNA intramolecular helix, which must be broken for reverse transcription to initiate. We propose a mechanism by which this vRNA/tRNA conformer relieves the kinetic block formed by the vRNA intramolecular helix to initiate reverse transcription. PMID:27613581

  9. The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid.

    PubMed

    York, Ashley; Kutluay, Sebla B; Errando, Manel; Bieniasz, Paul D

    2016-08-01

    The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions. PMID:27541140

  10. The RNA Binding Specificity of Human APOBEC3 Proteins Resembles That of HIV-1 Nucleocapsid

    PubMed Central

    Errando, Manel; Bieniasz, Paul D.

    2016-01-01

    The APOBEC3 (A3) cytidine deaminases are antiretroviral proteins, whose targets include human immunodeficiency virus type-1 (HIV-1). Their incorporation into viral particles is critical for antiviral activity and is driven by interactions with the RNA molecules that are packaged into virions. However, it is unclear whether A3 proteins preferentially target RNA molecules that are destined to be packaged and if so, how. Using cross-linking immunoprecipitation sequencing (CLIP-seq), we determined the RNA binding preferences of the A3F, A3G and A3H proteins. We found that A3 proteins bind preferentially to RNA segments with particular properties, both in cells and in virions. Specifically, A3 proteins target RNA sequences that are G-rich and/or A-rich and are not scanned by ribosomes during translation. Comparative analyses of HIV-1 Gag, nucleocapsid (NC) and A3 RNA binding to HIV-1 RNA in cells and virions revealed the striking finding that A3 proteins partially mimic the RNA binding specificity of the HIV-1 NC protein. These findings suggest a model for A3 incorporation into HIV-1 virions in which an NC-like RNA binding specificity is determined by nucleotide composition rather than sequence. This model reconciles the promiscuity of A3 RNA binding that has been observed in previous studies with a presumed advantage that would accompany selective binding to RNAs that are destined to be packaged into virions. PMID:27541140

  11. Cell-specific RNA aptamer against human CCR5 specifically targets HIV-1 susceptible cells and inhibits HIV-1 infectivity.

    PubMed

    Zhou, Jiehua; Satheesan, Sangeetha; Li, Haitang; Weinberg, Marc S; Morris, Kevin V; Burnett, John C; Rossi, John J

    2015-03-19

    The C-C chemokine receptor type 5 (CCR5) is a receptor expressed by T cells and macrophages that serves as a coreceptor 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 small interfering RNA [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 inhibitory concentration 50%. 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.

  12. Hierarchical Modeling and Differential Expression Analysis for RNA-seq Experiments with Inbred and Hybrid Genotypes

    PubMed Central

    Lithio, Andrew; Nettleton, Dan

    2016-01-01

    The performance of inbred and hybrid genotypes is of interest in plant breeding and genetics. High-throughput sequencing of RNA (RNA-seq) has proven to be a useful tool in the study of the molecular genetic responses of inbreds and hybrids to environmental stresses. Commonly used experimental designs and sequencing methods lead to complex data structures that require careful attention in data analysis. We demonstrate an analysis of RNA-seq data from a split-plot design involving drought stress applied to two inbred genotypes and two hybrids formed by crosses between the inbreds. Our generalized linear modeling strategy incorporates random effects for whole-plot experimental units and uses negative binomial distributions to allow for overdispersion in count responses for split-plot experimental units. Variations in gene length and base content, as well as differences in sequencing intensity across experimental units, are also accounted for. Hierarchical modeling with thoughtful parameterization and prior specification allows for borrowing of information across genes to improve estimation of dispersion parameters, genotype effects, treatment effects, and interaction effects of primary interest. PMID:27110090

  13. Differences in HIV type 1 RNA plasma load profile of closely related cocirculating Ethiopian subtype C strains: C and C'.

    PubMed

    Ayele, Workenesh; Mekonnen, Yared; Messele, Tsehaynesh; Mengistu, Yohannes; Tsegaye, Aster; Bakker, Margreet; Berkhout, Ben; Dorigo-Zetsma, Wendelien; Wolday, Dawit; Goudsmit, Jaap; Coutinho, Roel; de Baar, Michel; Paxton, William A; Pollakis, Georgios

    2010-07-01

    Two HIV-1 subtype C subclusters have been identified in Ethiopia (C and C') with little knowledge regarding their biological or clinical differences. We longitudinally monitored HIV-1 viral loads and CD4(+) T cell counts for 130 subtype C-infected individuals from Ethiopia over 5 years. The genetic subclusters C and C' were determined and comparisons were made between the groups. None of the study individuals received antiretroviral therapy. Subcluster C' was found to be the more prevalent (72.3%) genotype circulating. Individuals infected with subcluster C' harbored higher viral loads in comparison to subcluster C-infected individuals when the CD4(+) T cell counts were high (500-900 cells/mm(3)), whereas at low CD4(+) T cell counts (0-150 cells/mm(3)) individuals infected with subcluster C viruses showed higher viral loads. We identified a greater number of deaths among individuals infected with subcluster C viruses in comparison to C'. Our results indicate that infection with subcluster C viruses leads to a more rapid onset of disease, despite the initial lower HIV-1 RNA plasma loads. Additionally, the higher viral loads seen for HIV-1 subcluster C' infections at higher CD4(+) T cell counts can help explain the higher prevalence of this subtype in Ethiopia.

  14. Selection and Characterization of Small Molecules That Bind the HIV-1 Frameshift Site RNA

    PubMed Central

    Marcheschi, Ryan J.; Mouzakis, Kathryn D.; Butcher, Samuel E.

    2009-01-01

    HIV-1 requires a −1 translational frameshift to properly synthesize the viral enzymes required for replication. The frameshift mechanism is dependent upon two RNA elements, a seven-nucleotide slippery sequence (UUUUUUA) and a downstream RNA structure. Frameshifting occurs with a frequency of ~5%, and increasing or decreasing this frequency may result in a decrease in viral replication. Here, we report the results of a high-throughput screen designed to find small molecules that bind to the HIV-1 frameshift site RNA. Out of 34,500 compounds screened, 202 were identified as positive hits. We show that one of these compounds, doxorubicin, binds the HIV-1 RNA with low micromolar affinity (Kd = 2.8 μM). This binding was confirmed and localized to the RNA using NMR. Further analysis revealed that this compound increased the RNA stability by approximately 5 °C and decreased translational frameshifting by 28% (±14%), as measured in vitro. PMID:19673541

  15. Epidemiology of HPV Genotypes among HIV Positive Women in Kenya: A Systematic Review and Meta-Analysis

    PubMed Central

    Menon, Sonia; Wusiman, Aibibula; Boily, Marie Claude; Kariisa, Mbabazi; Mabeya, Hillary; Luchters, Stanley; Forland, Frode; Rossi, Rodolfo; Callens, Steven; vanden Broeck, Davy

    2016-01-01

    Background There is a scarcity of data on the distribution of human papillomavirus (HPV) genotypes in the HIV positive population and in invasive cervical cancer (ICC) in Kenya. This may be different from genotypes found in abnormal cytology. Yet, with the advent of preventive HPV vaccines that target HPV 16 and 18, and the nonavalent vaccine targeting 90% of all ICC cases, such HPV genotype distribution data are indispensable for predicting the impact of vaccination and HPV screening on prevention. Even with a successful vaccination program, vaccinated women will still require screening to detect those who will develop ICC from other High risk (HR) HPV genotypes not prevented by current vaccines. The aim of this review is to report on the prevalence of pHR/HR HPV types and multiple pHR/HR HPV genotypes in Kenya among HIV positive women with normal, abnormal cytology and ICC. Methods PUBMED, EMBASE, SCOPUS, and PROQUEST were searched for articles on HPV infection up to August 2nd 2016. Search terms were HIV, HPV, Cervical Cancer, Incidence or Prevalence, and Kenya. Results The 13 studies included yielded a total of 2116 HIV-infected women, of which 89 had ICC. The overall prevalence of pHR/HR HPV genotypes among HIV-infected women was 64% (95%CI: 50%-77%). There was a borderline significant difference in the prevalence of pHR/HR HPV genotypes between Female Sex workers (FSW) compared to non-FSW in women with both normal and abnormal cytology. Multiple pHR/HR HPV genotypes were highly prominent in both normal cytology/HSIL and ICC. The most prevalent HR HPV genotypes in women with abnormal cytology were HPV 16 with 26%, (95%CI: 23.0%-30.0%) followed by HPV 35 and 52, with 21% (95%CI: 18%-25%) and 18% (95%CI: 15%-21%), respectively. In women with ICC, the most prevalent HPV genotypes were HPV 16 (37%; 95%CI: 28%-47%) and HPV 18 (24%; 95%CI: 16%-33%). Conclusion HPV 16/18 gains prominence as the severity of cervical disease increases, with HPV 16/18 accounting for 61

  16. 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

  17. Performance of genotypic tools for prediction of tropism in HIV-1 subtype C V3 loop sequences.

    PubMed

    Gupta, Soham; Neogi, Ujjwal; Srinivasa, Hiresave; Shet, Anita

    2015-01-01

    Currently, there is no consensus on the genotypic tools to be used for tropism analysis in HIV-1 subtype C strains. Thus, the aim of the study was to evaluate the performance of the different V3 loop-based genotypic algorithms available. We compiled a dataset of 645 HIV-1 subtype C V3 loop sequences of known coreceptor phenotypes (531 R5-tropic/non-syncytium-inducing and 114 X4-tropic/R5X4-tropic/syncytium-inducing sequences) from the Los Alamos database (http://www.hiv.lanl.gov/) and previously published literature. Coreceptor usage was predicted based on this dataset using different software-based machine-learning algorithms as well as simple classical rules. All the sophisticated machine-learning methods showed a good concordance of above 85%. Geno2Pheno (false-positive rate cutoff of 5-15%) and CoRSeqV3-C were found to have a high predicting capability in determining both HIV-1 subtype C X4-tropic and R5-tropic strains. The current sophisticated genotypic tropism tools based on V3 loop perform well for tropism prediction in HIV-1 subtype C strains and can be used in clinical settings. PMID:25573618

  18. Titration of human immunodeficiency virus type 1 (HIV-1) and quantitative analysis of virus expression in vitro using liquid RNA-RNA hybridization.

    PubMed

    Volsky, D J; Pellegrino, M G; Li, G; Logan, K A; Aswell, J E; Lawrence, N P; Decker, S R

    1990-06-01

    An assay is described for titration of human immunodeficiency virus type 1 (HIV-1) and for quantitative analysis of virus expression in vitro. The assay utilizes a liquid RNA-RNA hybridization method coupled with reversible target capture (RTC) on oligo(dT) derivatized magnetic particles. The assay provides a rapid, specific, and sensitive method for quantitation of HIV-1 RNA molecules present either in cells or in viral particles from cell-free culture media. Chronically infected monocytoid U1.1 cells were found to carry 52 pg HIV-1 RNA per 200,000 cells (160 HIV-1 RNA molecules per cell). In contrast, acutely infected CEM and H9 cells carried 3010 and 4370 pg HIV-1 RNA per 200,000 cells (9040 and 13,110 HIV-1 RNA molecules per cell, respectively). No hybridization was observed with uninfected cells or cells infected with HIV-2, HTLV-I, HTLV-II, or EBV. Use of liquid HIV-1 RNA hybridization in association with HIV-1 protein detection methods permits more complete characterization of HIV-1 expression in host cells than either method alone, and also provides a method for standardizing preparations of virus.

  19. Editing of HIV-1 RNA by the double-stranded RNA deaminase ADAR1 stimulates viral infection

    PubMed Central

    Doria, Margherita; Neri, Francesca; Gallo, Angela; Farace, Maria Giulia; Michienzi, Alessandro

    2009-01-01

    Adenosine deaminases that act on dsRNA (ADARs) are enzymes that target double-stranded regions of RNA converting adenosines into inosines (A-to-I editing) thus contributing to genome complexity and fine regulation of gene expression. It has been described that a member of the ADAR family, ADAR1, can target viruses and affect their replication process. Here we report evidence showing that ADAR1 stimulates human immuno deficiency virus type 1 (HIV-1) replication by using both editing-dependent and editing-independent mechanisms. We show that over-expression of ADAR1 in HIV-1 producer cells increases viral protein accumulation in an editing-independent manner. Moreover, HIV-1 virions generated in the presence of over-expressed ADAR1 but not an editing-inactive ADAR1 mutant are released more efficiently and display enhanced infectivity, as demonstrated by challenge assays performed with T cell lines and primary CD4+ T lymphocytes. Finally, we report that ADAR1 associates with HIV-1 RNAs and edits adenosines in the 5′ untranslated region (UTR) and the Rev and Tat coding sequence. Overall these results suggest that HIV-1 has evolved mechanisms to take advantage of specific RNA editing activity of the host cell and disclose a stimulatory function of ADAR1 in the spread of HIV-1. PMID:19651874

  20. Full Viral Suppression, Low-Level Viremia, and Quantifiable Plasma HIV-RNA at the End of Pregnancy in HIV-Infected Women on Antiretroviral Treatment

    PubMed Central

    Baroncelli, Silvia; Pirillo, Maria F.; Tamburrini, Enrica; Guaraldi, Giovanni; Pinnetti, Carmela; Antoni, Anna Degli; Galluzzo, Clementina M.; Stentarelli, Chiara; Amici, Roberta

    2015-01-01

    Abstract There is limited information on full viral suppression and low-level HIV-RNA viremia in HIV-infected women at the end of pregnancy. We investigated HIV-RNA levels close to delivery in women on antiretroviral treatment in order to define rates of complete suppression, low-level viremia, and quantifiable HIV-RNA, exploring as potential determinants some clinical and viroimmunological variables. Plasma samples from a national study in Italy, collected between 2003 and 2012, were used. According to plasma HIV-RNA levels, three groups were defined: full suppression (target not detected), low-level viremia (target detected but <37 copies/ml), and quantifiable HIV-RNA (≥37 copies/ml). Multivariable logistic regression was used to define determinants of full viral suppression and of quantifiable HIV-RNA. Among 107 women evaluated at a median gestational age of 35 weeks, 90 (84.1%) had HIV-RNA <37 copies/ml. Most of them (59/90, 65.6%) had full suppression, with the remaining (31/90, 34.4%) showing low-level viremia (median: 11.9 copies/ml; IQR 7.4–16.3). Among the 17 women with quantifiable viral load, median HIV-RNA was 109 copies/ml (IQR 46–251), with only one case showing resistance (mutation M184V; rate: 9.1%). In multivariable analyses, women with higher baseline HIV-RNA levels and with hepatitis C virus (HCV) coinfection were significantly more likely to have quantifiable HIV-RNA in late pregnancy. Full viral suppression was significantly more likely with nonnucleoside reverse transcriptase inhibitor (NNRTI)-based regimens and significantly less likely with higher HIV-RNA in early pregnancy. No cases of HIV transmission occurred. In conclusion, HIV-infected pregnant women showed a high rate of viral suppression and a low resistance rate before delivery. In most cases no target HIV-RNA was detected in plasma, suggesting a low risk of subsequent virological rebound and development of resistance. Women with high levels of HIV-RNA in early pregnancy and

  1. The Evolving Genotypic Profile of HIV-1 Mutations Related to Antiretroviral Treatment in the North Region of Brazil.

    PubMed

    Lopes, Carmen Andréa F; Soares, Marcelo A; Falci, Diego R; Sprinz, Eduardo

    2015-01-01

    HIV related mutations can be associated with decreased susceptibility to antiretrovirals and treatment failures. There is scarce information about HIV mutations in persons failing HIV treatment in North of Brazil. Our aim was to evaluate evolution of HIV subtypes and mutations patterns related to antiretroviral therapy in this region. We investigated HIV resistance profile in adults failing antiretroviral regimen in Northern Brazil from January, 2004, through December, 2013. Genotype data was evaluated through Stanford University algorithm. There were 377 genotypes from different individuals to evaluate. Resistance mutations were similar to worldwide reports and related to antiretroviral exposure. Most prevalent mutations in the reverse transcriptase gene were M184V (80.1%) and K130N (40.6%). Thymidine associated mutations were more frequent in multiexperienced patients. Most common protease mutations were M46I, V82A, I54V, L90M, I84V, M46L, and L76V. Subtype B was the most prevalent (90.7%). There were differences between subtypes B and non-B mutations. We documented for the first time subtypes and patterns of HIV associated mutations in Northern Brazil. A1 subtype was identified for the first time in this area. Depending on drug regimen and how experienced the patient is, an empirical switch of a failing antiretroviral treatment could be a reasonable option.

  2. The Evolving Genotypic Profile of HIV-1 Mutations Related to Antiretroviral Treatment in the North Region of Brazil.

    PubMed

    Lopes, Carmen Andréa F; Soares, Marcelo A; Falci, Diego R; Sprinz, Eduardo

    2015-01-01

    HIV related mutations can be associated with decreased susceptibility to antiretrovirals and treatment failures. There is scarce information about HIV mutations in persons failing HIV treatment in North of Brazil. Our aim was to evaluate evolution of HIV subtypes and mutations patterns related to antiretroviral therapy in this region. We investigated HIV resistance profile in adults failing antiretroviral regimen in Northern Brazil from January, 2004, through December, 2013. Genotype data was evaluated through Stanford University algorithm. There were 377 genotypes from different individuals to evaluate. Resistance mutations were similar to worldwide reports and related to antiretroviral exposure. Most prevalent mutations in the reverse transcriptase gene were M184V (80.1%) and K130N (40.6%). Thymidine associated mutations were more frequent in multiexperienced patients. Most common protease mutations were M46I, V82A, I54V, L90M, I84V, M46L, and L76V. Subtype B was the most prevalent (90.7%). There were differences between subtypes B and non-B mutations. We documented for the first time subtypes and patterns of HIV associated mutations in Northern Brazil. A1 subtype was identified for the first time in this area. Depending on drug regimen and how experienced the patient is, an empirical switch of a failing antiretroviral treatment could be a reasonable option. PMID:26543866

  3. Identification of RNA helicases in human immunodeficiency virus 1 (HIV-1) replication - a targeted small interfering RNA library screen using pseudotyped and WT HIV-1.

    PubMed

    Williams, Claire A; Abbink, Truus E M; Jeang, Kuan-Teh; Lever, Andrew M L

    2015-06-01

    Central to the development of new treatments for human immunodeficiency virus 1 (HIV-1) is a more thorough understanding of the viral life cycle and the cellular cofactors upon which this depends. Targeting cellular proteins and their interaction with HIV-1 has the potential to reduce the problem of emerging viral resistance to drugs as mutational escape is more difficult. We performed a short interfering RNA (siRNA) library screen targeting 59 cellular RNA helicases, assessing the effect on both viral capsid protein production and infectious virion formation. Five RNA helicases were identified which, when knocked down, reproducibly decreased infectious particle production: DDX5, DDX10, DDX17, DDX28 and DDX52. Two of these proteins (DDX5 and DDX17) have known roles in HIV-1 replication. A further helicase (DDX10) was a positive hit from a previous genome-wide siRNA screen; however, DDX28 and DDX52 have not previously been implicated as essential cofactors for HIV-1.

  4. Low Efficacy of Pegylated Interferon plus Ribavirin plus Nitazoxanide for HCV Genotype 4 and HIV Coinfection

    PubMed Central

    Macías, Juan; López-Cortés, Luis F.; Téllez, Francisco; Recio, Eva; Ojeda-Burgos, Guillermo; Ríos, MªJosé; Rivero-Juárez, Antonio; Delgado, Marcial; Jeremías, Rivas-; Pineda, Juan A.

    2015-01-01

    Background Nitazoxanide (NTZ) plus pegylated interferon and ribavirin (Peg-IFN/RBV) improved the sustained virological response (SVR) achieved with Peg-IFN/RBV in hepatitis C virus genotype 4 (HCV-4)-monoinfected patients. There are no data currently on the efficacy of Peg-IFN/RBV plus NTZ for human immunodeficiency virus (HIV)/HCV-4 coinfection. Therefore, the objectives of this clinical trial were to assess the efficacy and to evaluate the safety of Peg-IFN/RBV plus NTZ in HIV/HCV-4-coinfected patients. Patients and Methods This was an open-label, single arm, multicenter phase II pilot clinical trial (NCT01529073) enrolling HIV-infected individuals with HCV-4 chronic infection, naïve to HCV therapy. Patients were treated with NTZ 500 mg bid for 4 weeks, followed by NTZ 500 mg bid plus Peg-IFN alpha-2b 1.5 μg/kg/week plus weight-adjusted RBV during 48 weeks. Analyses were done by intention-to-treat (ITT, missing = failure). A historical cohort of HIV/HCV-4-infected patients treated with Peg-IFN alpha-2b and RBV at the same area was used as control. Results Two (9.5%) of 21 patients included in the trial compared with 5 (21.7%) of 23 patients included in the historical cohort achieved SVR (SVR risk difference, -12.2%; 95% confidence interval, -33.2% to 8.8%; p = 0.416). Virological failure was due to lack of response in 13 (62%) individuals recruited in the trial. Two (9.5%) patients included in the trial and two (9.5%) individuals from the historical cohort discontinued permanently due to adverse events. Conclusions No increase in SVR was observed among HIV/HCV-4-coinfected patients receiving Peg-IFN/RBV plus NTZ compared with a historical cohort treated with Peg-IFN/RBV. Interruptions due to adverse events of Peg-IFN/RBV plus NTZ were similar to those of dual therapy. Trial Registration ClinicalTrials.gov NCT01529073 PMID:26640956

  5. HIV type 1 genetic diversity and genotypic drug susceptibility in the Republic of Moldova.

    PubMed

    Pandrea, I; Descamps, D; Collin, G; Robertson, D L; Damond, F; Dimitrienco, V; Gheorghita, S; Pecec, M; Simon, F; Brun-Vézinet, F; Apetrei, C

    2001-09-01

    HIV-1 genetic diversity and, for the first time, genotypic drug susceptibility was investigated for strains circulating in the Republic of Moldova (of the former Soviet Union). Eighty-three samples from adults recently infected by intravenous drug use (IDU) (n = 60), heterosexual contact (n = 8), and from blood donors (n = 15) that tested positive from 1997 to 1998, and originating from different regions of Moldova were serotyped. By group-specific and subtype-specific peptide ELISA, patients were infected by serotype A (n = 65), serotype B (n = 1), or were nontypable (n = 17). Heteroduplex mobility assay (HMA) confirmed 11 subtype A and the one subtype B infection. Analyses of pol and env sequences for six of the IDUs confirmed that they were infected with subtype A strain. These strains clustered tightly with subtype A strains isolated from the former Soviet Union in phylogenetic analysis. No mutations associated with drug resistance were detected. The Republic of Moldova is culturally more closely related to Romania (where subtype F dominates the epidemic), but depends economically on Russia (where subtype A is established among IDUs). Thus, our results suggest that the spread of HIV in this region is driven by drug networks rather than being due to cultural similarities.

  6. 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

  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. Parameters Influencing Baseline HIV-1 Genotypic Tropism Testing Related to Clinical Outcome in Patients on Maraviroc

    PubMed Central

    Sierra, Saleta; Dybowski, J. Nikolai; Pironti, Alejandro; Heider, Dominik; Güney, Lisa; Thielen, Alex; Reuter, Stefan; Esser, Stefan; Fätkenheuer, Gerd; Lengauer, Thomas; Hoffmann, Daniel; Pfister, Herbert; Jensen, Björn; Kaiser, Rolf

    2015-01-01

    Objectives We analysed the impact of different parameters on genotypic tropism testing related to clinical outcome prediction in 108 patients on maraviroc (MVC) treatment. Methods 87 RNA and 60 DNA samples were used. The viral tropism was predicted using the geno2pheno[coreceptor] and T-CUP tools with FPR cut-offs ranging from 1%-20%. Additionally, 27 RNA and 28 DNA samples were analysed in triplicate, 43 samples with the ESTA assay and 45 with next-generation sequencing. The influence of the genotypic susceptibility score (GSS) and 16 MVC-resistance mutations on clinical outcome was also studied. Results Concordance between single-amplification testing compared to ESTA and to NGS was in the order of 80%. Concordance with NGS was higher at lower FPR cut-offs. Detection of baseline R5 viruses in RNA and DNA samples by all methods significantly correlated with treatment success, even with FPR cut-offs of 3.75%-7.5%. Triple amplification did not improve the prediction value but reduced the number of patients eligible for MVC. No influence of the GSS or MVC-resistance mutations but adherence to treatment, on the clinical outcome was detected. Conclusions Proviral DNA is valid to select candidates for MVC treatment. FPR cut-offs of 5%-7.5% and single amplification from RNA or DNA would assure a safe administration of MVC without excluding many patients who could benefit from this drug. In addition, the new prediction system T-CUP produced reliable results. PMID:25970632

  9. Comparative efficacy, pharmacokinetic, pharmacodynamic activity, and interferon stimulated gene expression of different interferon formulations in HIV/HCV genotype-1 infected patients.

    PubMed

    Osinusi, Anu; Bon, Dimitra; Nelson, Amy; Lee, Yu-Jin; Poonia, Seerat; Shivakumar, Bhavana; Cai, Shu Yi; Wood, Brad; Haagmans, Bart; Lempicki, Richard; Herrmann, Eva; Sneller, Michael; Polis, Michael; Masur, Henry; Kottilil, Shyam

    2014-02-01

    The effect of different formulations of interferon on therapeutic response in patients coinfected with HIV and HCV is unclear. In this study, the safety, tolerability, viral kinetics (VK) modeling and host responses among HIV/HCV coinfected patients treated with pegylated-IFN or albinterferon alfa-2b (AlbIFN) with weight-based ribavirin were compared. Three trials treated 57 HIV/HCV coinfected genotype-1 patients with PegIFN alfa-2b (1.5 µg/kg/week) (n = 30), PegIFN alfa-2a (180 µg/week) (n = 10), and AlbIFN (900 µg/q2week) (n = 17) in combination with weight-based ribavirin (RBV). HCV RNA, safety labs, and interferon stimulated gene expression (ISG) was evaluated. Adverse events were documented at all study visits. HCV viral kinetics using a full pharmacokinetic/pharmacodynamic model was also evaluated. Baseline patient characteristics were similar across the three studies. All three formulations exhibited comparable safety and tolerability profiles and efficacy. VK/PK/PD parameters for all three studies as measured by mean efficiency and rate of infected cell loss were similar between the three groups. Host responses (ISG expression and immune activation markers) were similar among the three groups. All three regimens induced significant ISG at week 4 (P < 0.05) and ISG expression strongly correlated with therapeutic response (r = 0.65; P < 0.01). In summary, a comprehensive analysis of responses to three different interferon formulations in HIV/HCV coinfected patients demonstrated similar effects. Notably, interferon-based therapy results in a blunted host response followed by modest antiviral effect in HIV/HCV coinfected patients. This suggests that future treatment options that do not rely on host immune responses such as direct antiviral agents would be particularly beneficial in these difficult to treat patients. PMID:24166150

  10. Adenosine Deaminase Acting on RNA-1 (ADAR1) Inhibits HIV-1 Replication in Human Alveolar Macrophages

    PubMed Central

    Levy, David N.; Li, Yonghua; Kumar, Rajnish; Burke, Sean A.; Dawson, Rodney; Hioe, Catarina E.; Borkowsky, William; Rom, William N.; Hoshino, Yoshihiko

    2014-01-01

    While exploring the effects of aerosol IFN-γ treatment in HIV-1/tuberculosis co-infected patients, we observed A to G mutations in HIV-1 envelope sequences derived from bronchoalveolar lavage (BAL) of aerosol IFN-γ-treated patients and induction of adenosine deaminase acting on RNA 1 (ADAR1) in the BAL cells. IFN-γ induced ADAR1 expression in monocyte-derived macrophages (MDM) but not T cells. ADAR1 siRNA knockdown induced HIV-1 expression in BAL cells of four HIV-1 infected patients on antiretroviral therapy. Similar results were obtained in MDM that were HIV-1 infected in vitro. Over-expression of ADAR1 in transformed macrophages inhibited HIV-1 viral replication but not viral transcription measured by nuclear run-on, suggesting that ADAR1 acts post-transcriptionally. The A to G hyper-mutation pattern observed in ADAR1 over-expressing cells in vitro was similar to that found in the lungs of HIV-1 infected patients treated with aerosol IFN-γ suggesting the model accurately represented alveolar macrophages. Together, these results indicate that ADAR1 restricts HIV-1 replication post-transcriptionally in macrophages harboring HIV-1 provirus. ADAR1 may therefore contribute to viral latency in macrophages. PMID:25272020

  11. The structure of the genotype-phenotype map strongly constrains the evolution of non-coding RNA.

    PubMed

    Dingle, Kamaludin; Schaper, Steffen; Louis, Ard A

    2015-12-01

    The prevalence of neutral mutations implies that biological systems typically have many more genotypes than phenotypes. But, can the way that genotypes are distributed over phenotypes determine evolutionary outcomes? Answering such questions is difficult, in part because the number of genotypes can be hyper-astronomically large. By solving the genotype-phenotype (GP) map for RNA secondary structure (SS) for systems up to length L = 126 nucleotides (where the set of all possible RNA strands would weigh more than the mass of the visible universe), we show that the GP map strongly constrains the evolution of non-coding RNA (ncRNA). Simple random sampling over genotypes predicts the distribution of properties such as the mutational robustness or the number of stems per SS found in naturally occurring ncRNA with surprising accuracy. Because we ignore natural selection, this strikingly close correspondence with the mapping suggests that structures allowing for functionality are easily discovered, despite the enormous size of the genetic spaces. The mapping is extremely biased: the majority of genotypes map to an exponentially small portion of the morphospace of all biophysically possible structures. Such strong constraints provide a non-adaptive explanation for the convergent evolution of structures such as the hammerhead ribozyme. These results present a particularly clear example of bias in the arrival of variation strongly shaping evolutionary outcomes and may be relevant to Mayr's distinction between proximate and ultimate causes in evolutionary biology. PMID:26640651

  12. Analysis of sequences of hepatitis C virus NS5A genotype 1 in HIV-coinfected patients with a null response to nitazoxanide or peg-interferon plus ribavirin.

    PubMed

    Sede, M; Laufer, N; Ojeda, D; Gun, A; Cahn, P; Quarleri, J

    2013-09-01

    Even though new drugs have been approved for treatment of hepatitis C virus (HCV) infection, the risk of drug-drug interactions and concern about overlapping toxicities has hindered the development of studies in HIV/HCV-coinfected individuals. Traditional treatment with pegylated interferon plus ribavirin (peg-IFN + RBV) is very expensive and has a low rate of sustained virological response in coinfected patients, especially if they are infected with HCV genotype 1. Nitazoxanide (NTZ) is a drug that is being evaluated for the treatment of chronic HCV infection, both in HCV-monoinfected and HIV/HCV-coinfected patients. Understanding the NTZ resistance mechanism could allow the development of resistance to be minimized and would expand the treatment options, mainly in special populations such as HIV/HCV-coinfected patients. Similarly to IFN, NTZ increases the activity of the cellular protein kinase activated by double-stranded RNA (PKR), a key kinase in the innate antiviral response. In order to elucidate whether sequence heterogeneity in the PKR-binding domain of HCV NS5A genotype 1 could influence the antiviral activity of either NTZ monotherapy or peg-IFN + RBV, baseline and end-of-therapy plasma samples from two groups of eleven non-responder HIV/HCV-coinfected patients that had received NTZ or peg-IFN + RBV were studied. Most of the HCV NS5A sequences examined at the end of therapy did not change from the baseline, even after 30 days course of antiviral therapy. An extensive comparison of HCV NS5A genotype 1 and 4 sequences from the database with reported IFN therapy outcome was performed in order to infer their phylogenetic relationships. The HCV genotype 1 NS5A nucleotide sequences from therapy-non-responder patients were intermingled amongst those from the database, irrespective of their IFN-therapy outcome. When comparing NS5A-PKRBD amino acid sequences, significant differences were observed in genotype 4, but not in genotype 1 (p < 0.0001 and p

  13. Unusual Enterocytozoon bieneusi genotypes and Cryptosporidium hominis subtypes in HIV-infected patients on highly active antiretroviral therapy.

    PubMed

    Akinbo, Frederick O; Okaka, Christopher E; Omoregie, Richard; Adamu, Haileeyesus; Xiao, Lihua

    2013-07-01

    Human immunodeficiency virus (HIV)-infected persons are commonly infected with Cryptosporidium species and Enterocytozoon bieneusi in both developed and developing countries, particularly patients with CD4+ cell counts below 200 cells/μL; 285 HIV-infected patients on highly active antiretroviral therapy (HAART) were enrolled in this study, and both stool and blood specimens were collected from participants. The stool specimens were analyzed and typed for E. bieneusi and Cryptosporidium spp. by polymerase chain reaction (PCR) and DNA sequencing. CD4 count was analyzed using flow cytometry. E. bieneusi and Cryptosporidium were detected in 18 (6.3%) and 4 (1.4%) patients, respectively. The E. bieneusi detected mostly belonged to a new genotype group that, thus far, has only been found in a few humans: genotype Nig4 in 2 patients and two new genotypes related to Nig4 in 12 patients. The Cryptosporidium detected included C. hominis (two patients), C. parvum (one patient), and C. felis (one patient), with the two C. hominis infections belonging to an unusual subtype family. Additional studies are required to determine whether some E. bieneusi genotypes and C. hominis subtypes are more prevalent in HIV patients on HAART. PMID:23629938

  14. High Genotypic Discordance of Concurrent Mycobacterium tuberculosis Isolates from Sputum and Blood of HIV-Infected Individuals

    PubMed Central

    Ssengooba, Willy; Cobelens, Frank G.; Nakiyingi, Lydia; Mboowa, Gerald; Armstrong, Derek T.; Manabe, Yukari C.; Joloba, Moses L.; de Jong, Bouke C.

    2015-01-01

    Background Among HIV-infected individuals with CD4 less than 200 cells/mm3, tuberculosis often has an atypical presentation, is more likely to be disseminated and is diagnostically challenging. We sought to understand the genotypic discordance of concurrent sputum and blood M. tuberculosis (MTB) isolates from HIV-infected individuals. Methods From a prospective diagnostic accuracy study with 182 HIV-infected culture-positive TB adults, isolates were obtained from 51 of 66 participants who were MTB culture-positive by both sputum and blood. Isolates were subjected to susceptibility testing to 1st line drugs, spoligotyping and 24 locus- MIRU-VNTR. Results The median age of the participants was 31 (IQR; 27–38) years and 51% were male. The median CD4 count was 29 (IQR; 10–84) cells/mm3 with 20% taking ART; 8.0% were previously treated for TB, and 63% were AFB smear-negative. The isolates belonged to two of the main global MTB-lineages; East-African-Indian (L3) 17 (16.7%) and Euro-American (L4) 85 (83.3%). We identified 26 (51.0%) participants with discordant MTB-genotypes between sputum and blood, including two patients with evidence of mixed infection in either compartment. Having discordant MTB-genotypes was not predicted by the MTB-lineage in either blood or sputum, CD4 cell count, or any other clinical characteristic. Conclusions There is a high genotypic discordance among M. tuberculosis concurrently isolated from sputum and blood of HIV-infected individuals. These findings suggest that infection with more than one strain of M. tuberculosis occurs in at least half of patients with advanced HIV infection. PMID:26176604

  15. RNA-Seq reveals genotype-specific molecular responses to water deficit in eucalyptus

    PubMed Central

    2011-01-01

    Background In a context of climate change, phenotypic plasticity provides long-lived species, such as trees, with the means to adapt to environmental variations occurring within a single generation. In eucalyptus plantations, water availability is a key factor limiting productivity. However, the molecular mechanisms underlying the adaptation of eucalyptus to water shortage remain unclear. In this study, we compared the molecular responses of two commercial eucalyptus hybrids during the dry season. Both hybrids differ in productivity when grown under water deficit. Results Pyrosequencing of RNA extracted from shoot apices provided extensive transcriptome coverage - a catalog of 129,993 unigenes (49,748 contigs and 80,245 singletons) was generated from 398 million base pairs, or 1.14 million reads. The pyrosequencing data enriched considerably existing Eucalyptus EST collections, adding 36,985 unigenes not previously represented. Digital analysis of read abundance in 14,460 contigs identified 1,280 that were differentially expressed between the two genotypes, 155 contigs showing differential expression between treatments (irrigated vs. non irrigated conditions during the dry season), and 274 contigs with significant genotype-by-treatment interaction. The more productive genotype displayed a larger set of genes responding to water stress. Moreover, stress signal transduction seemed to involve different pathways in the two genotypes, suggesting that water shortage induces distinct cellular stress cascades. Similarly, the response of functional proteins also varied widely between genotypes: the most productive genotype decreased expression of genes related to photosystem, transport and secondary metabolism, whereas genes related to primary metabolism and cell organisation were over-expressed. Conclusions For the most productive genotype, the ability to express a broader set of genes in response to water availability appears to be a key characteristic in the maintenance

  16. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release

    SciTech Connect

    López, Claudia S.; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L.; Kabat, David; Barklis, Eric

    2014-08-15

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. - Highlights: • At the protein level, full-length HIV-1 Env alters Gag protein expression. • HIV-1 Env RNA expression reduces Gag levels and virus release. • Env RNA effects on Gag are dependent on the RRE. • RRE-containing Env RNAs compete with vRNAs for nuclear export.

  17. Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies

    PubMed Central

    Carlson, Lars-Anders; Bai, Yun; Keane, Sarah C; Doudna, Jennifer A; Hurley, James H

    2016-01-01

    HIV-1 Gag selects and packages a dimeric, unspliced viral RNA in the context of a large excess of cytosolic human RNAs. As Gag assembles on the plasma membrane, the HIV-1 genome is enriched relative to cellular RNAs by an unknown mechanism. We used a minimal system consisting of purified RNAs, recombinant HIV-1 Gag and giant unilamellar vesicles to recapitulate the selective packaging of the 5’ untranslated region of the HIV-1 genome in the presence of excess competitor RNA. Mutations in the CA-CTD domain of Gag which subtly affect the self-assembly of Gag abrogated RNA selectivity. We further found that tRNA suppresses Gag membrane binding less when Gag has bound viral RNA. The ability of HIV-1 Gag to selectively package its RNA genome and its self-assembly on membranes are thus interdependent on one another. DOI: http://dx.doi.org/10.7554/eLife.14663.001 PMID:27343348

  18. Collaborative study for the calibration of HCV RNA, HBV DNA and HIV RNA reference preparations against the relative international standards.

    PubMed

    Pisani, Giulio; Marino, Francesco; Cristiano, Karen; Bisso, Guillermo Mario; Mele, Caludio; Luciani, Francesca; Wirz, Maria; Gentili, Giuliano

    2007-01-01

    We organised a collaborative study to calibrate three new ISS reference preparations (ISS: Istituto Superiore di Sanità), one for HCV RNA, one for HIV RNA and one for HBV DNA, to be used for nucleic acid amplification techniques (NAT) in blood testing. Serial dilution of the ISS reference preparations and the respective international standards were tested in different days by each participating laboratory using two commercial NAT assays. Data were collected by the ISS for statistical analysis. Based on the mean potency of the HCV RNA and HIV RNA preparations, calculated from the results provided by the 12 participating laboratories, a definitive concentrations of 5700 IU/mL and 4000 IU/mL, respectively, were assigned to the reference materials. On the contrary, it was not possible to obtain a consensus titre for the HBV DNA reference material. These new Italian reference preparations (HCV RNA ISS/1005 and HIV RNA ISS/1005) calibrated against the respective international standards are available free of charge to any laboratory upon request.

  19. Computational models can predict response to HIV therapy without a genotype and may reduce treatment failure in different resource-limited settings

    PubMed Central

    Revell, A. D.; Wang, D.; Wood, R.; Morrow, C.; Tempelman, H.; Hamers, R. L.; Alvarez-Uria, G.; Streinu-Cercel, A.; Ene, L.; Wensing, A. M. J.; DeWolf, F.; Nelson, M.; Montaner, J. S.; Lane, H. C.; Larder, B. A.

    2013-01-01

    Objectives Genotypic HIV drug-resistance testing is typically 60%–65% predictive of response to combination antiretroviral therapy (ART) and is valuable for guiding treatment changes. Genotyping is unavailable in many resource-limited settings (RLSs). We aimed to develop models that can predict response to ART without a genotype and evaluated their potential as a treatment support tool in RLSs. Methods Random forest models were trained to predict the probability of response to ART (≤400 copies HIV RNA/mL) using the following data from 14 891 treatment change episodes (TCEs) after virological failure, from well-resourced countries: viral load and CD4 count prior to treatment change, treatment history, drugs in the new regimen, time to follow-up and follow-up viral load. Models were assessed by cross-validation during development, with an independent set of 800 cases from well-resourced countries, plus 231 cases from Southern Africa, 206 from India and 375 from Romania. The area under the receiver operating characteristic curve (AUC) was the main outcome measure. Results The models achieved an AUC of 0.74–0.81 during cross-validation and 0.76–0.77 with the 800 test TCEs. They achieved AUCs of 0.58–0.65 (Southern Africa), 0.63 (India) and 0.70 (Romania). Models were more accurate for data from the well-resourced countries than for cases from Southern Africa and India (P < 0.001), but not Romania. The models identified alternative, available drug regimens predicted to result in virological response for 94% of virological failures in Southern Africa, 99% of those in India and 93% of those in Romania. Conclusions We developed computational models that predict virological response to ART without a genotype with comparable accuracy to genotyping with rule-based interpretation. These models have the potential to help optimize antiretroviral therapy for patients in RLSs where genotyping is not generally available. PMID:23485767

  20. Genital Tract HIV RNA Levels and Their Associations with Human Papillomavirus Infection and Risk of Cervical Pre-Cancer

    PubMed Central

    GHARTEY, Jeny; KOVACS, Andrea; BURK, Robert D.; MASSAD, L. Stewart; MINKOFF, Howard; XIE, Xianhong; D’SOUZA, Gypsyamber; XUE, Xiaonan; WATTS, D. Heather; LEVINE, Alexandra M.; EINSTEIN, Mark H.; COLIE, Christine; ANASTOS, Kathryn; ELTOUM, Isam-Eldin; HEROLD, Betsy C.; PALEFSKY, Joel M.; STRICKLER, Howard D.

    2014-01-01

    Objective Plasma HIV RNA levels have been associated with risk of human papillomavirus (HPV) and cervical neoplasia in HIV-seropositive women. However, little is known regarding local genital tract HIV RNA levels and their relation with cervical HPV and neoplasia. Design/Methods In an HIV-seropositive women’s cohort with semi-annual follow-up, we conducted a nested case-control study of genital tract HIV RNA levels and their relation with incident high-grade squamous intraepithelial lesions sub-classified as severe (severe HSIL), as provided for under the Bethesda 2001 classification system. Specifically, 66 incident severe HSIL were matched to 130 controls by age, CD4+ count, HAART use, and other factors. We also studied HPV prevalence, incident detection, and persistence in a random sample of 250 subjects. Results Risk of severe HSIL was associated with genital tract HIV RNA levels (odds ratio comparing HIV RNA ≥ the median among women with detectable levels versus undetectable [ORVL] 2.96; 95% CI: 0.99–8.84; Ptrend=0.03). However, this association became non-significant (Ptrend=0.51) following adjustment for plasma HIV RNA levels. There was also no association between genital tract HIV RNA levels and the prevalence of any HPV or oncogenic HPV. However, the incident detection of any HPV (Ptrend=0.02) and persistence of oncogenic HPV (Ptrend=0.04) were associated with genital tract HIV RNA levels, after controlling plasma HIV RNA levels. Conclusion These prospective data suggest that genital tract HIV RNA levels are not a significant independent risk factor for cervical pre-cancer in HIV-seropositive women, but leave open the possibility that they may modestly influence HPV infection, an early stage of cervical tumoriogenesis. PMID:24694931

  1. In vivo SELEX of single-stranded domains in the HIV-1 leader RNA.

    PubMed

    van Bel, Nikki; Das, Atze T; Berkhout, Ben

    2014-02-01

    The 5' untranslated leader region of the human immunodeficiency virus type 1 (HIV-1) RNA genome is a strongly conserved sequence that encodes several regulatory motifs important for viral replication. Most of these motifs are exposed as hairpin structures, including the dimerization initiation signal (DIS), the major splice donor site (SD), and the packaging signal (Ψ), which are connected by short single-stranded regions. Mutational analysis revealed many functions of these hairpins, but only a few studies have focused on the single-stranded purine-rich sequences. Using the in vivo SELEX (systematic evolution of ligands by exponential enrichment) approach, we probed the sequence space in these regions that is compatible with efficient HIV-1 replication and analyzed the impact on the RNA secondary structure of the leader RNA. Our results show a strong sequence requirement for the DIS hairpin flanking regions. We postulate that these sequences are important for the binding of specific protein factors that support leader RNA-mediated functions. The sequence between the SD and Ψ hairpins seems to have a less prominent role, despite the strong conservation of the stretch of 5 A residues in natural isolates. We hypothesize that this may reflect the subtle evolutionary pressure on HIV-1 to acquire an A-rich RNA genome. In silico analyses indicate that sequences are avoided in all 3 single-stranded domains that affect the local or overall leader RNA folding. IMPORTANCE Many regulatory RNA sequences are clustered in the untranslated leader domain of the HIV-1 RNA genome. Several RNA hairpin structures in this domain have been proposed to fulfill specific roles, e.g., mediating RNA dimer formation to facilitate HIV-1 recombination. We now focus on the importance of a few well-conserved single-stranded sequences that connect these hairpins. We created libraries of HIV-1 variants in which these segments were randomized and selected the best-replicating variants. For two

  2. Global changes in the RNA binding specificity of HIV-1 Gag regulate virion genesis

    PubMed Central

    Kutluay, Sebla B.; Zang, Trinity; Blanco-Melo, Daniel; Powell, Chelsea; Jannain, David; Errando, Manel; Bieniasz, Paul D.

    2014-01-01

    Summary The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes. PMID:25416948

  3. Global changes in the RNA binding specificity of HIV-1 gag regulate virion genesis.

    PubMed

    Kutluay, Sebla B; Zang, Trinity; Blanco-Melo, Daniel; Powell, Chelsea; Jannain, David; Errando, Manel; Bieniasz, Paul D

    2014-11-20

    The HIV-1 Gag protein orchestrates all steps of virion genesis, including membrane targeting and RNA recruitment into virions. Using crosslinking-immunoprecipitation (CLIP) sequencing, we uncover several dramatic changes in the RNA-binding properties of Gag that occur during virion genesis, coincident with membrane binding, multimerization, and proteolytic maturation. Prior to assembly, and after virion assembly and maturation, the nucleocapsid domain of Gag preferentially binds to psi and Rev Response elements in the viral genome, and GU-rich mRNA sequences. However, during virion genesis, this specificity transiently changes in a manner that facilitates genome packaging; nucleocapsid binds to many sites on the HIV-1 genome and to mRNA sequences with a HIV-1-like, A-rich nucleotide composition. Additionally, we find that the matrix domain of Gag binds almost exclusively to specific tRNAs in the cytosol, and this association regulates Gag binding to cellular membranes.

  4. Prevalence of HIV, Hepatitis B and C Infections and an Assessment of HCV-Genotypes and Two IL28B SNPs among People Who Inject Drugs in Three Regions of Nepal.

    PubMed

    Kinkel, Hans-Tilmann; Karmacharya, Dibesh; Shakya, Jivan; Manandhar, Sulochana; Panthi, Santosh; Karmacharya, Prajwola; Sitaula, Deepika; Thapaliya, Reenu; K C, Prawachan; Rai, Apurva; Dixit, Sameer

    2015-01-01

    As part of a comprehensive health care programme for people who use drugs in Nepal, HIV and viral hepatitis B and C status--including risk factors, HCV-genotypes and co-infections--as well as two IL28B Single-nucleotide polymorphisms (SNPs) were assessed for a random sample of 401 people who inject drugs in three regions of Nepal: mid-western Terrai (Nepalgunj), the eastern region (Dharan, Biratnagar) and the central region (Kathmandu, Lalitpur and Chitwan). Individuals were included who showed at least a minimum of health care seeking behaviour. This latter criterion was defined by being registered with any organisation offering health services. The average age of the participants was 30.5 yrs, and the average length of intravenous drug use was 8.5 yrs. The prevalence rates of HBsAg, anti-HIV antibodies and HCV-RNA were 3.5%, 13.8% and 41.9%, respectively. Spontaneous HCV clearance was evident in 16% of all of those who tested positive for anti-HCV antibodies. Independent risk factors for HCV-RNA positivity were age, gender, geographical region, duration of injecting drug use, history of imprisonment and HIV co-infection. In the age group ≤24 yrs, the rate of spontaneous HCV clearance was 43.5%. Overall, 59.8% of HCV infections were caused by HCV genotype 3 and 40.2% by HCV genotype 1. No other HCV genotypes were identified in this study. The IL28B SNP rs12979860 and rs8099917 were identified in 122 patients, and 75.4% of all participants had both favourable genotypes rs12979860 C/C and rs8099917 T/T.

  5. Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA*

    PubMed Central

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-01-01

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

  6. The use of dried blood spot specimens for HIV-1 drug resistance genotyping in young children initiating antiretroviral therapy

    PubMed Central

    Salimo, Anna T.; Ledwaba, Johanna; Coovadia, Ashraf; Abrams, Elaine J.; Technau, Karl-Günter; Kuhn, Louise; Morris, Lynn; Hunt, Gillian M.

    2015-01-01

    Paired plasma and dried blood spots (DBS) from 232 South African HIV-infected children initiating antiretroviral therapy (ART) were genotyped for drug resistance mutations, most of who had prior exposure to ART for prevention-of-mother-to-child-transmission. Non-nucleoside reverse transcriptase inhibitor mutations were most commonly detected in both specimen types, particularly Y181C/I and K103N/S. Resistance interpretation concordance was achieved in 97% of pairs with 7 children having mutations detected in DBS only. These results validate the preferential use of DBS specimens for HIVDR genotyping in this patient group. PMID:26192603

  7. LETTER TO THE EDITOR Performance of the ViroSeq® HIV-1 Genotyping System v2.0 in Central Africa.

    PubMed

    Mouafo, Linda Chapdeleine Mekue; Péré, Hélène; Ndjoyi-Mbiguino, Angélique; Koyalta, Donato; Longo, Jean De Dieu; Mbopi-Kéou, François-Xavier; Kane, Coumba Toure; Bélec, Laurent

    2015-01-01

    Resistance genotypes in pol gene of HIV-1 were obtained by the ViroSeq(®) HIV-1 Genotyping System v2.0 (Celera Diagnostics, Alameda, CA, USA) in 138 of 145 (95%) antiretroviral treatment-experienced adults in virological failure living in Central Africa (Cameroon, Central African Republic, Chad, Gabon). HIV-1 group M exhibited broad genetic diversity. Performance of the 7 ViroSeq(®) sequencing primers showed high failure rate, from 3% to 76% (D: 76%; F: 17%; A and H: 15%; G and B: 4%; C: 3%). These findings emphasize the need of updating the ViroSeq(®) HIV-1 genotyping system for non-B subtypes HIV-1.

  8. HPV Genotyping from the high risk mRNA Aptima assay- a direct approach using DNA from Aptima sample tubes.

    PubMed

    Lillsunde Larsson, G; Kaliff, M; Bergengren, L; Karlsson, M G; Helenius, G

    2016-09-01

    The underlying cause of cervical cancer is an infection with the human papilloma virus (HPV) and HPV testing can be used for cervical cancer screening. The Aptima HPV assay from Hologic is an mRNA HPV test used to identify clinically relevant infections but the method does not discriminate between the different high risk genotypes. The aim of the current study was to evaluate if analyzed Aptima sample transfer tubes could be used as a source for HPV genotyping, using sample DNA. Study samples (n=108); were HPV-tested with mRNA Aptima assay and in parallel DNA was extracted and genotyped with Anyplex II HPV28. Analyzed mRNA Aptima tubes were thereafter used as source for a second DNA extraction and genotyping. Using mRNA Aptima result as reference, 90% of the samples (35/39) were high risk positive with the Anyplex II HPV28. Cohen's kappa 0.78 (95% CI: 0.66-0.90), sensitivity 0.90 (95% CI: 0.76-0.97) and specificity 0.90 (95% CI: 0.80-0.96). Two discordant samples carried low-risk genotypes (HPV 82 and HPV 44) and two were negative. DNA-genotyping results, in parallel to and after mRNA testing, were compared and differed significantly (McNemar test: P=0.021) possibly due to sample extraction volume difference. Cohen's kappa 0.81 (95% CI: 0.70-0.92), sensitivity 0.85 (95% CI: 0.74-0.93) and specificity 0.98 (95% CI: 0.88-1.00). In conclusion, analyzed mRNA Aptima sample tubes could be used as a source for DNA HPV genotyping. The sample volume used for extraction needs to be further explored.

  9. HIV-1 and the microRNA-Guided Silencing Pathway: An Intricate and Multifaceted Encounter

    PubMed Central

    Provost, Patrick; Barat, Corinne; Plante, Isabelle; Tremblay, Michel J.

    2010-01-01

    Summary MicroRNAs (miRNAs) are ~21 to 24-nucleotide RNAs that mediate repression of messenger RNA (mRNA) translation through recognition of specific miRNA binding sites usually located in the 3′ nontranslated region. Designed to simulate miRNAs, small interfering RNAs represent a powerful genetic approach to potently inhibit gene expression by mediating cleavage of the intended mRNA target. This strategy has been applied successfully to suppress replication of several viruses, including human immunodeficiency virus type 1 (HIV-1). However, recent evidences indicate that viral RNAs may themselves be processed, to some extent, by the endogenous miRNA biosynthetic machinery in mammalian cells, extending previous observations in plants. The resulting viral miRNAs may exert regulatory effects towards host and/or viral genes that may influence viral replication and modulate the course of infection. Viral miRNA generation and/or action may be limited by counteraction through inhibitory viral RNAs and/or proteins. This review article will focus on the relationship between HIV-1 and miRNA-guided RNA silencing, and discuss the different aspects of their interaction. As we learn more about the mechanism and importance of small RNA-based antiviral systems, a more intricate picture of the interaction between HIV-1 and a proven antiviral defense mechanism in lower eukaryotes is emerging. PMID:16889864

  10. Estimating HIV-1 Fitness Characteristics from Cross-Sectional Genotype Data

    PubMed Central

    Gopalakrishnan, Sathej; Montazeri, Hesam; Menz, Stephan; Beerenwinkel, Niko; Huisinga, Wilhelm

    2014-01-01

    Despite the success of highly active antiretroviral therapy (HAART) in the management of human immunodeficiency virus (HIV)-1 infection, virological failure due to drug resistance development remains a major challenge. Resistant mutants display reduced drug susceptibilities, but in the absence of drug, they generally have a lower fitness than the wild type, owing to a mutation-incurred cost. The interaction between these fitness costs and drug resistance dictates the appearance of mutants and influences viral suppression and therapeutic success. Assessing in vivo viral fitness is a challenging task and yet one that has significant clinical relevance. Here, we present a new computational modelling approach for estimating viral fitness that relies on common sparse cross-sectional clinical data by combining statistical approaches to learn drug-specific mutational pathways and resistance factors with viral dynamics models to represent the host-virus interaction and actions of drug mechanistically. We estimate in vivo fitness characteristics of mutant genotypes for two antiretroviral drugs, the reverse transcriptase inhibitor zidovudine (ZDV) and the protease inhibitor indinavir (IDV). Well-known features of HIV-1 fitness landscapes are recovered, both in the absence and presence of drugs. We quantify the complex interplay between fitness costs and resistance by computing selective advantages for different mutants. Our approach extends naturally to multiple drugs and we illustrate this by simulating a dual therapy with ZDV and IDV to assess therapy failure. The combined statistical and dynamical modelling approach may help in dissecting the effects of fitness costs and resistance with the ultimate aim of assisting the choice of salvage therapies after treatment failure. PMID:25375675

  11. Long noncoding RNA NRON contributes to HIV-1 latency by specifically inducing tat protein degradation

    PubMed Central

    Li, Jun; Chen, Cancan; Ma, Xiancai; Geng, Guannan; Liu, Bingfeng; Zhang, Yijun; Zhang, Shaoyang; Zhong, Fudi; Liu, Chao; Yin, Yue; Cai, Weiping; Zhang, Hui

    2016-01-01

    Long noncoding RNAs (lncRNAs) play multiple key regulatory roles in various cellular pathways. However, their functions in HIV-1 latent infection remain largely unknown. Here we show that a lncRNA named NRON, which is highly expressed in resting CD4+ T lymphocytes, could be involved in HIV-1 latency by specifically inducing Tat protein degradation. Our results suggest that NRON lncRNA potently suppresses the viral transcription by decreasing the cellular abundance of viral transactivator protein Tat. NRON directly links Tat to the ubiquitin/proteasome components including CUL4B and PSMD11, thus facilitating Tat degradation. Depletion of NRON, especially in combination with a histone deacetylase (HDAC) inhibitor, significantly reactivates the viral production from the HIV-1-latently infected primary CD4+ T lymphocytes. Our data indicate that lncRNAs play a role in HIV-1 latency and their manipulation could be a novel approach for developing latency-reversing agents. PMID:27291871

  12. 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

  13. Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA

    PubMed Central

    Aga, Evgenia; Cillo, Anthony R.; Yates, Aarika L.; Besson, Guillaume; Fyne, Elizabeth; Koontz, Dianna L.; Jennings, Cheryl; Zheng, Lu; Mellors, John W.

    2016-01-01

    Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1 pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMCs) from 5 viremic patients and 20 patients receiving effective ART. Total and resting CD4+ T cells were isolated from a subset of patients and tested for comparison with PBMCs. The estimated standard deviations including interassay variability and intra-assay variability of the assays were modest, i.e., 0.15 and 0.10 log10 copies/106 PBMCs, respectively, for CA HIV-1 DNA and 0.40 and 0.19 log10 copies/106 PBMCs for CA HIV-1 RNA. Testing of longitudinally obtained PBMC samples showed little variation for either viremic patients (median fold differences of 0.80 and 0.88 for CA HIV-1 DNA and RNA, respectively) or virologically suppressed patients (median fold differences of 1.14 and 0.97, respectively). CA HIV-1 DNA and RNA levels were strongly correlated (r = 0.77 to 1; P = 0.0001 to 0.037) for assays performed using PBMCs from different sources (phlebotomy versus leukapheresis) or using total or resting CD4+ T cells purified by either bead selection or flow cytometric sorting. Their sensitivity, reproducibility, and broad applicability to small numbers of mononuclear cells make these assays useful for observational and interventional studies that examine longitudinal changes in the numbers of HIV-1-infected cells and their levels of transcription. PMID:26763968

  14. Nuclear Retention of Multiply Spliced HIV-1 RNA in Resting CD4+ T Cells

    PubMed Central

    Lassen, Kara G; Ramyar, Kasra X; Bailey, Justin R; Zhou, Yan; Siliciano, Robert F

    2006-01-01

    HIV-1 latency in resting CD4+ T cells represents a major barrier to virus eradication in patients on highly active antiretroviral therapy (HAART). We describe here a novel post-transcriptional block in HIV-1 gene expression in resting CD4+ T cells from patients on HAART. This block involves the aberrant localization of multiply spliced (MS) HIV-1 RNAs encoding the critical positive regulators Tat and Rev. Although these RNAs had no previously described export defect, we show that they exhibit strict nuclear localization in resting CD4+ T cells from patients on HAART. Overexpression of the transcriptional activator Tat from non-HIV vectors allowed virus production in these cells. Thus, the nuclear retention of MS HIV-1 RNA interrupts a positive feedback loop and contributes to the non-productive nature of infection of resting CD4+ T cells. To define the mechanism of nuclear retention, proteomic analysis was used to identify proteins that bind MS HIV-1 RNA. Polypyrimidine tract binding protein (PTB) was identified as an HIV-1 RNA-binding protein differentially expressed in resting and activated CD4+ T cells. Overexpression of PTB in resting CD4+ T cells from patients on HAART allowed cytoplasmic accumulation of HIV-1 RNAs. PTB overexpression also induced virus production by resting CD4+ T cells. Virus culture experiments showed that overexpression of PTB in resting CD4+ T cells from patients on HAART allowed release of replication-competent virus, while preserving a resting cellular phenotype. Whether through effects on RNA export or another mechanism, the ability of PTB to reverse latency without inducing cellular activation is a result with therapeutic implications. PMID:16839202

  15. Interaction of drugs of abuse and microRNA with HIV: a brief review

    PubMed Central

    Pilakka-Kanthikeel, Sudheesh; Nair, Madhavan P. N.

    2015-01-01

    MicroRNAs (miRNAs), the post-transcriptional regulators of gene expression, play key roles in modulating many cellular processes. The changes in the expression profiles of several specific miRNAs affect the interactions between miRNA and their targets in various illnesses, including addiction, HIV, cancer etc. The presence of anti-HIV-1 microRNAs (which regulate the level of infectivity of HIV-1) have been validated in the cells which are the primary targets of HIV infection. Drugs of abuse impair the intracellular innate anti-HIV mechanism(s) in monocytes, contributing to cell susceptibility to HIV infection. Emerging evidence has implicated miRNAs are differentially expressed in response to chronic morphine treatment. Activation of mu opioid receptors (MOR) by morphine is shown to down regulate the expression of anti-HIV miRNAs. In this review, we summarize the results which demonstrate that several drugs of abuse related miRNAs have roles in the mechanisms that define addiction, and how they interact with HIV. PMID:26483757

  16. MicroRNA binding to the HIV-1 Gag protein inhibits Gag assembly and virus production

    PubMed Central

    Chen, Antony K.; Sengupta, Prabuddha; Waki, Kayoko; Van Engelenburg, Schuyler B.; Ochiya, Takahiro; Ablan, Sherimay D.; Freed, Eric O.; Lippincott-Schwartz, Jennifer

    2014-01-01

    MicroRNAs (miRNAs) are small, 18–22 nt long, noncoding RNAs that act as potent negative gene regulators in a variety of physiological and pathological processes. To repress gene expression, miRNAs are packaged into RNA-induced silencing complexes (RISCs) that target mRNAs for degradation and/or translational repression in a sequence-specific manner. Recently, miRNAs have been shown to also interact with proteins outside RISCs, impacting cellular processes through mechanisms not involving gene silencing. Here, we define a previously unappreciated activity of miRNAs in inhibiting RNA–protein interactions that in the context of HIV-1 biology blocks HIV virus budding and reduces virus infectivity. This occurs by miRNA binding to the nucleocapsid domain of the Gag protein, the main structural component of HIV-1 virions. The resulting miRNA–Gag complexes interfere with viral–RNA-mediated Gag assembly and viral budding at the plasma membrane, with imperfectly assembled Gag complexes endocytosed and delivered to lysosomes. The blockade of virus production by miRNA is reversed by adding the miRNA’s target mRNA and stimulated by depleting Argonaute-2, suggesting that when miRNAs are not mediating gene silencing, they can block HIV-1 production through disruption of Gag assembly on membranes. Overall, our findings have significant implications for understanding how cells modulate HIV-1 infection by miRNA expression and raise the possibility that miRNAs can function to disrupt RNA-mediated protein assembly processes in other cellular contexts. PMID:24938790

  17. The in vitro loose dimer structure and rearrangements of the HIV-2 leader RNA

    PubMed Central

    Purzycka, Katarzyna J.; Pachulska-Wieczorek, Katarzyna; Adamiak, Ryszard W.

    2011-01-01

    RNA dimerization is an essential step in the retroviral life cycle. Dimerization and encapsidation signals, closely linked in HIV-2, are located in the leader RNA region. The SL1 motif and nucleocapsid protein are considered important for both processes. In this study, we show the structure of the HIV-2 leader RNA (+1–560) captured as a loose dimer. Potential structural rearrangements within the leader RNA were studied. In the loose dimer form, the HIV-2 leader RNA strand exists in vitro as a single global fold. Two kissing loop interfaces within the loose dimer were identified: SL1/SL1 and TAR/TAR. Evidence for these findings is provided by RNA probing using SHAPE, chemical reagents, enzymes, non-denaturing PAGE mobility assays, antisense oligonucleotides hybridization and analysis of an RNA mutant. Both TAR and SL1 as isolated domains are bound by recombinant NCp8 protein with high affinity, contrary to the hairpins downstream of SL1. Foot-printing of the SL1/NCp8 complex indicates that the major binding site maps to the SL1 upper stem. Taken together, these data suggest a model in which TAR hairpin III, the segment of SL1 proximal to the loop and the PAL palindromic sequence play specific roles in the initiation of dimerization. PMID:21622659

  18. PCR-derived ssDNA probes for fluorescent in situ hybridization to HIV-1 RNA.

    PubMed

    Knuchel, M C; Graf, B; Schlaepfer, E; Kuster, H; Fischer, M; Weber, R; Cone, R W

    2000-02-01

    We developed a simple and rapid technique to synthesize single-stranded DNA (ssDNA) probes for fluorescent in situ hybridization (ISH) to human immunodeficiency virus 1 (HIV-1) RNA. The target HIV-1 regions were amplified by the polymerase chain reaction (PCR) and were simultaneously labeled with dUTP. This product served as template for an optimized asymmetric PCR (one-primer PCR) that incorporated digoxigenin (dig)-labeled dUTP. The input DNA was subsequently digested by uracil DNA glycosylase, leaving intact, single-stranded, digoxigenin-labeled DNA probe. A cocktail of ssDNA probes representing 55% of the HIV-1 genome was hybridized to HIV-1-infected 8E5 T-cells and uninfected H9 T-cells. For comparison, parallel hybridizations were done with a plasmid-derived RNA probe mix covering 85% of the genome and a PCR-derived RNA probe mix covering 63% of the genome. All three probe types produced bright signals, but the best signal-to-noise ratios and the highest sensitivities were obtained with the ssDNA probe. In addition, the ssDNA probe syntheses generated large amounts of probe (0.5 to 1 microg ssDNA probe per synthesis) and were easier to perform than the RNA probe syntheses. These results suggest that ssDNA probes may be preferable to RNA probes for fluorescent ISH. (J Histochem Cytochem 48:285-293, 2000)

  19. In-Depth Analysis of the Interaction of HIV-1 with Cellular microRNA Biogenesis and Effector Mechanisms

    PubMed Central

    Whisnant, Adam W.; Bogerd, Hal P.; Flores, Omar; Ho, Phong; Powers, Jason G.; Sharova, Natalia; Stevenson, Mario; Chen, Chin-Ho; Cullen, Bryan R.

    2013-01-01

    ABSTRACT The question of how HIV-1 interfaces with cellular microRNA (miRNA) biogenesis and effector mechanisms has been highly controversial. Here, we first used deep sequencing of small RNAs present in two different infected cell lines (TZM-bl and C8166) and two types of primary human cells (CD4+ peripheral blood mononuclear cells [PBMCs] and macrophages) to unequivocally demonstrate that HIV-1 does not encode any viral miRNAs. Perhaps surprisingly, we also observed that infection of T cells by HIV-1 has only a modest effect on the expression of cellular miRNAs at early times after infection. Comprehensive analysis of miRNA binding to the HIV-1 genome using the photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) technique revealed several binding sites for cellular miRNAs, a subset of which were shown to be capable of mediating miRNA-mediated repression of gene expression. However, the main finding from this analysis is that HIV-1 transcripts are largely refractory to miRNA binding, most probably due to extensive viral RNA secondary structure. Together, these data demonstrate that HIV-1 neither encodes viral miRNAs nor strongly influences cellular miRNA expression, at least early after infection, and imply that HIV-1 transcripts have evolved to avoid inhibition by preexisting cellular miRNAs by adopting extensive RNA secondary structures that occlude most potential miRNA binding sites. PMID:23592263

  20. Evaluation of genotypic prediction of HIV-1 tropism using population sequencing of replicates.

    PubMed

    Ferreira, Joao Leandro de Paula; Coelho, Luana Portes Ozorio; Rodrigues, Rosangela; Cabral, Gabriela Bastos; Cavalcanti, Jaqueline de Souza; Guimaraes, Paula Morena de Souza; Brigido, Luis Fernando de Macedo

    2012-02-01

    Determination of human immunodeficiency virus tropism has contributed to the understanding of the pathogenesis of HIV and is necessary prior to the use of CCR5 antagonists. Replicate V3 sequences may generate different sequences and improve viral tropism prediction. The diversity of HIV was evaluated to access its influence on prediction. Plasma RNA was retro-transcribed and amplified using a one-step protocol, followed by nested PCR and sequencing using an ABI3130XL. Eighty-one patients, 74% male and 26% female, with a median age of 44 years had either a single sequence (n=50) or 2-4 replicates (n=31) evaluated. Most patients (92%) had used multiple anti-retroviral regimens. Tropism prediction was performed using the Geno2pheno clonal option. The number of ambiguous nucleotides, the deduced non-synonymous amino acids at V3 and the genetic distance were quantified. Using a 20% false positive rate (FPR) cut-off, 41/81 (50.6%) was predicted as X4. TCD4 was lower, 226 cells/mm(3) (IQR 82-378), in patients infected with X4; TCD4 for R5 was 324 cells/mm(3) (IQR 200-538, p<0.05). The number of ambiguous nucleotides correlated with a lower FPR value (p<0.0027). Although different sequences may be generated, the number of replicates was not associated to a lower FPR or X4 assignment, and may allow a better prediction of this biological characteristic. Ambiguous nucleotides correlate inversely to a lower FPR.

  1. Influence of IL28B Polymorphisms on Response to a Lower-Than-Standard Dose peg-IFN-α 2a for Genotype 3 Chronic Hepatitis C in HIV-Coinfected Patients

    PubMed Central

    López-Cortés, Luis F.; Ruiz-Valderas, Rosa; Jimenez-Jimenez, Luis; González-Escribano, María F.; Torres-Cornejo, Almudena; Mata, Rosario; Rivero, Antonio; Pineda, Juan A.; Marquez-Solero, Manuel; Viciana, Pompeyo

    2012-01-01

    Background Data on which to base definitive recommendations on the doses and duration of therapy for genotype 3 HCV/HIV-coinfected patients are scarce. We evaluated the efficacy of a lower peginterferon-α 2a dose and a shorter duration of therapy than the current standard of care in genotype 3 HCV/HIV-coinfected patients. Methods and Findings Pilot, open-label, single arm clinical trial which involved 58 Caucasian HCV/HIV-coinfected patients who received weekly 135 µg peginterferon-α 2a plus ribavirin 400 mg twice daily during 20 weeks after attaining undetectable viremia. The relationships between baseline patient-related variables, including IL28B genotype, plasma HCV-RNA, ribavirin dose/kg, peginterferon-α 2a and ribavirin levels with virological responses were analyzed. Only 4 patients showed lack of response and 5 patients dropped out due to adverse events related to the study medication. Overall, sustained virologic response (SVR) rates were 58.3% by intention-to-treat and 71.4% by per protocol analysis, respectively. Among patients with rapid virologic response (RVR), SVR and relapses rates were 92.6% and 7.4%, respectively. No relationships were observed between viral responses and ribavirin dose/kg, peginterferon-α 2a concentrations, ribavirin levels or rs129679860 genotype. Conclusions Weekly 135 µg pegIFN-α 2a could be as effective as the standard 180 µg dose, with a very low incidence of severe adverse events. A 24-week treatment duration appears to be appropriate in patients achieving RVR, but extending treatment up to just 20 weeks beyond negativization of viremia is associated with a high relapse rate in those patients not achieving RVR. There was no influence of IL28B genotype on the virological responses. Trial Registration: ClinicalTrials.gov NCT00553930 PMID:22235243

  2. Cell-type Specific Aptamer and Aptamer-siRNA Conjugates for Targeted HIV-1 Therapy

    PubMed Central

    Zhou, Jiehua; Rossi, John

    2014-01-01

    Human immunodeficiency virus (HIV) is a virus that causes acquired immunodeficiency syndrome (AIDS), a chronic and incurable disease of the human immune system. As the standard of care for the patients with HIV-1, current highly active antiretroviral treatment (HAART) has been therapeutically effective in the majority of patients; however, it is not curative, and HAART is intolerable due to server side effects. Therefore, nucleic acid-based therapeutics, such as antisense oligonucleotide, ribozyme, mRNA, RNAi-based therapeutics, aptamer, etc., have been actively developed as alternative or adjuvant agents for those chemical antiviral drugs in order to surmount those drawbacks. The combinatorial use of various antiviral nucleic acids could be more efficacious in blocking viral replication and preventing the emergence of resistant variants. In this regard, RNA interference (RNAi) can function as a gene-specific therapeutic option for controlling HIV-1 replication. Another type of therapeutic nucleic acid – aptamers – shows promise as a new and potent class of anti-HIV agent and can additionally function as a cell-type-specific delivery vehicle for targeted RNAi. The combined use of small interfering RNA (siRNAs) and aptamers could effectively block viral replication and prevent the emergence of resistant variants. The present review offers a brief overview of the use of cell-type specific aptamer and aptamer-siRNA conjugates development in our group for the treatment of HIV-1. Their potentials for targeted delivering RNAi therapeutics (e.g. siRNA) and suppressing HIV-1 replication in vitro and in humanized animal model will be highlighted here. PMID:25118114

  3. HIV infection and hepatitis C virus genotype 1a are associated with phylogenetic clustering among people with recently acquired hepatitis C virus infection.

    PubMed

    Bartlett, Sofia R; Jacka, Brendan; Bull, Rowena A; Luciani, Fabio; Matthews, Gail V; Lamoury, Francois M J; Hellard, Margaret E; Hajarizadeh, Behzad; Teutsch, Suzy; White, Bethany; Maher, Lisa; Dore, Gregory J; Lloyd, Andrew R; Grebely, Jason; Applegate, Tanya L

    2016-01-01

    The aim of this study was to identify factors associated with phylogenetic clustering among people with recently acquired hepatitis C virus (HCV) infection. Participants with available sample at time of HCV detection were selected from three studies; the Australian Trial in Acute Hepatitis C, the Hepatitis C Incidence and Transmission Study - Prison and Community. HCV RNA was extracted and Core to E2 region of HCV sequenced. Clusters were identified from maximum likelihood trees with 1000 bootstrap replicates using 90% bootstrap and 5% genetic distance threshold. Among 225 participants with available Core-E2 sequence (ATAHC, n=113; HITS-p, n=90; and HITS-c, n=22), HCV genotype prevalence was: G1a: 38% (n=86), G1b: 5% (n=12), G2a: 1% (n=2), G2b: 5% (n=11), G3a: 48% (n=109), G6a: 1% (n=2) and G6l 1% (n=3). Of participants included in phylogenetic trees, 22% of participants were in a pair/cluster (G1a-35%, 30/85, mean maximum genetic distance=0.031; G3a-11%, 12/106, mean maximum genetic distance=0.021; other genotypes-21%, 6/28, mean maximum genetic distance=0.023). Among HCV/HIV co-infected participants, 50% (18/36) were in a pair/cluster, compared to 16% (30/183) with HCV mono-infection (P=<0.001). Factors independently associated with phylogenetic clustering were HIV co-infection [vs. HCV mono-infection; adjusted odds ratio (AOR) 4.24; 95%CI 1.91, 9.39], and HCV G1a infection (vs. other HCV genotypes; AOR 3.33, 95%CI 0.14, 0.61).HCV treatment and prevention strategies, including enhanced antiviral therapy, should be optimised. The impact of targeting of HCV treatment as prevention to populations with higher phylogenetic clustering, such as those with HIV co-infection, could be explored through mathematical modelling.

  4. Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries

    PubMed Central

    Chow, Wei Zhen; Bon, Abdul Hamid; Keating, Sheila; Anderios, Fread; Halim, Hazwan Abdul; Takebe, Yutaka; Kamarulzaman, Adeeba; Busch, Michael P.; Tee, Kok Keng

    2016-01-01

    Transfusion-transmissible infections including HIV-1 continue to pose major risks for unsafe blood transfusions due to both window phase infections and divergent viruses that may not be detected by donor screening assays. Given the recent emergence of several HIV-1 circulating recombinant forms (CRFs) in high-risk populations in the Southeast Asia region, we investigated the genetic diversity of HIV-1 among the blood donors in Kuala Lumpur, Malaysia. A total of 211 HIV-positive plasma samples detected among 730,188 donations to the National Blood Centre between 2013 and 2014 were provided (90.5% male, median age: 27.0 years old). Recent or long-term infection status at the time of donation was determined using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA). HIV-1 gag-pol genes were amplified and sequenced from residual plasma for 149 cases followed by genotype determination using phylogenetic and recombination analyses. Transmitted antiretroviral resistance mutations were not observed among the blood donors, among which 22.7% were classified as recent or incident infections. Major circulating HIV-1 genotypes determined by neighbour-joining phylogenetic inference included CRF01_AE at 40.9% (61/149), CRF33_01B at 21.5% (32/149), and subtype B at 10.1% (15/149). Newly-described CRFs including CRF54_01B circulated at 4.0%, CRF74_01B at 2.0%, and CRF53_01B and CRF48_01B at 0.7% each. Interestingly, unique HIV-1 genotypes including African subtype G (8.7%), CRF45_cpx (1.3%), CRF02_AG (0.7%) and CRF07_BC (0.7%) from China were detected for the first time in the country. A cluster of subtype G sequences formed a distinct founder sub-lineage within the African strains. In addition, 8.7% (13/149) of HIV-infected donors had unique recombinant forms (URFs) including CRF01_AE/B' (4.7%), B'/C (2.7%) and B'/G (1.3%) recombinants. Detailed analysis identified similar recombinant structures with shared parental strains among the B'/C and B'/G URFs, some of which

  5. Extensive Genetic Diversity of HIV-1 in Incident and Prevalent Infections among Malaysian Blood Donors: Multiple Introductions of HIV-1 Genotypes from Highly Prevalent Countries.

    PubMed

    Chow, Wei Zhen; Bon, Abdul Hamid; Keating, Sheila; Anderios, Fread; Halim, Hazwan Abdul; Takebe, Yutaka; Kamarulzaman, Adeeba; Busch, Michael P; Tee, Kok Keng

    2016-01-01

    Transfusion-transmissible infections including HIV-1 continue to pose major risks for unsafe blood transfusions due to both window phase infections and divergent viruses that may not be detected by donor screening assays. Given the recent emergence of several HIV-1 circulating recombinant forms (CRFs) in high-risk populations in the Southeast Asia region, we investigated the genetic diversity of HIV-1 among the blood donors in Kuala Lumpur, Malaysia. A total of 211 HIV-positive plasma samples detected among 730,188 donations to the National Blood Centre between 2013 and 2014 were provided (90.5% male, median age: 27.0 years old). Recent or long-term infection status at the time of donation was determined using a limiting antigen avidity enzyme immunoassay (LAg-Avidity EIA). HIV-1 gag-pol genes were amplified and sequenced from residual plasma for 149 cases followed by genotype determination using phylogenetic and recombination analyses. Transmitted antiretroviral resistance mutations were not observed among the blood donors, among which 22.7% were classified as recent or incident infections. Major circulating HIV-1 genotypes determined by neighbour-joining phylogenetic inference included CRF01_AE at 40.9% (61/149), CRF33_01B at 21.5% (32/149), and subtype B at 10.1% (15/149). Newly-described CRFs including CRF54_01B circulated at 4.0%, CRF74_01B at 2.0%, and CRF53_01B and CRF48_01B at 0.7% each. Interestingly, unique HIV-1 genotypes including African subtype G (8.7%), CRF45_cpx (1.3%), CRF02_AG (0.7%) and CRF07_BC (0.7%) from China were detected for the first time in the country. A cluster of subtype G sequences formed a distinct founder sub-lineage within the African strains. In addition, 8.7% (13/149) of HIV-infected donors had unique recombinant forms (URFs) including CRF01_AE/B' (4.7%), B'/C (2.7%) and B'/G (1.3%) recombinants. Detailed analysis identified similar recombinant structures with shared parental strains among the B'/C and B'/G URFs, some of which

  6. VS411 Reduced Immune Activation and HIV-1 RNA Levels in 28 Days: Randomized Proof-of-Concept Study for AntiViral-HyperActivation Limiting Therapeutics

    PubMed Central

    Lori, Franco; De Forni, Davide; Katabira, Elly; Baev, Denis; Maserati, Renato; Calarota, Sandra A.; Cahn, Pedro; Testori, Marco; Rakhmanova, Aza; Stevens, Michael R.

    2012-01-01

    Background A new class of antiretrovirals, AntiViral-HyperActivation Limiting Therapeutics (AV-HALTs), has been proposed as a disease-modifying therapy to both reduce Human Immunodeficiency Virus Type 1 (HIV-1) RNA levels and the excessive immune activation now recognized as the major driver of not only the continual loss of CD4+ T cells and progression to Acquired Immunodeficiency Syndrome (AIDS), but also of the emergence of both AIDS-defining and non-AIDS events that negatively impact upon morbidity and mortality despite successful (ie, fully suppressive) therapy. VS411, the first-in-class AV-HALT, combined low-dose, slow-release didanosine with low-dose hydroxycarbamide to accomplish both objectives with a favorable toxicity profile during short-term administration. Five dose combinations were administered as VS411 to test the AV-HALT Proof-of-Concept in HIV-1-infected subjects. Methods Multinational, double-blind, 28-day Phase 2a dose-ranging Proof-of-Concept study of antiviral activity, immunological parameters, safety, and genotypic resistance in 58 evaluable antiretroviral-naïve HIV-1-infected adults. Randomization and allocation to study arms were carried out by a central computer system. Results were analyzed by ANOVA, Kruskal-Wallis, ANCOVA, and two-tailed paired t tests. Results VS411 was well-tolerated, produced significant reductions of HIV-1 RNA levels, increased CD4+ T cell counts, and led to significant, rapid, unprecedented reductions of immune activation markers after 28 days despite incomplete viral suppression and without inhibiting HIV-1-specific immune responses. The didanosine 200 mg/HC 900 mg once-daily formulation demonstrated the greatest antiviral efficacy (HIV-1 RNA: −1.47 log10 copies/mL; CD4+ T cell count: +135 cells/mm3) and fewest adverse events. Conclusions VS411 successfully established the Proof-of-Concept that AV-HALTs can combine antiviral efficacy with rapid, potentially beneficial reductions in the excessive immune system

  7. Controlling HIV-1: Non-Coding RNA Gene Therapy Approaches to a Functional Cure

    PubMed Central

    Ahlenstiel, Chantelle L.; Suzuki, Kazuo; Marks, Katherine; Symonds, Geoff P.; Kelleher, Anthony D.

    2015-01-01

    The current treatment strategy for HIV-1 involves prolonged and intensive combined antiretroviral therapy (cART), which successfully suppresses plasma viremia. It has transformed HIV-1 infection into a chronic disease. However, despite the success of cART, a latent form of HIV-1 infection persists as integrated provirus in resting memory CD4+ T cells. Virus can reactivate from this reservoir upon cessation of treatment, and hence HIV requires lifelong therapy. The reservoir represents a major barrier to eradication. Understanding molecular mechanisms regulating HIV-1 transcription and latency are crucial to develop alternate treatment strategies, which impact upon the reservoir and provide a path toward a “functional cure” in which there is no detectable viremia in the absence of cART. Numerous reports have suggested ncRNAs are involved in regulating viral transcription and latency. This review will discuss the latest developments in ncRNAs, specifically short interfering (si)RNA and short hairpin (sh)RNA, targeting molecular mechanisms of HIV-1 transcription, which may represent potential future therapeutics. It will also briefly address animal models for testing potential therapeutics and current gene therapy clinical trials. PMID:26441979

  8. Genotypic heterogeneity based on 18S-rRNA gene sequences among Acanthamoeba isolates from clinical samples in Italy.

    PubMed

    Di Cave, David; D' Alfonso, Rossella; Dussey Comlavi, Kodjo A; D' Orazi, Carlo; Monno, Rosa; Berrilli, Federica

    2014-11-01

    Acanthamoeba keratitis (AK) is an ocular disease caused by members of a genus of free-living amoebae and it is associated predominantly with contact lens (CL) use. This study reports 55 cases of AK diagnosed in Italy. Genotype identification was carried out by PCR assay followed by sequence analysis of the 18S rRNA gene using the genus specific primers JDP1 and JDP2. Genotype assignment was based on phenetic analysis of the ASA.S1 subset of the small-subunit rRNA gene sequences. The material has been collected at the Polyclinic Tor Vergata of Rome for a total of 19 isolates and at the Polyclinic Hospital of Bari (36 isolates). Thirty-three out of the 55 genetically characterized isolates were assigned to the genotype T4. Ten isolates were identified as belonging to the genotype T15 thus confirming the first association between the genotype T15 and human amoebic keratitis previously described from the same area. We underline the occurrence of the genotype T3 and T11 identified for the first time in the country.

  9. The Life-Cycle of the HIV-1 Gag–RNA Complex

    PubMed Central

    Mailler, Elodie; Bernacchi, Serena; Marquet, Roland; Paillart, Jean-Christophe; Vivet-Boudou, Valérie; Smyth, Redmond P.

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA) by the viral Pr55Gag precursor polyprotein, and the processes leading to its incorporation into viral particles. PMID:27626439

  10. The Life-Cycle of the HIV-1 Gag-RNA Complex.

    PubMed

    Mailler, Elodie; Bernacchi, Serena; Marquet, Roland; Paillart, Jean-Christophe; Vivet-Boudou, Valérie; Smyth, Redmond P

    2016-01-01

    Human immunodeficiency virus type 1 (HIV-1) replication is a highly regulated process requiring the recruitment of viral and cellular components to the plasma membrane for assembly into infectious particles. This review highlights the recent process of understanding the selection of the genomic RNA (gRNA) by the viral Pr55(Gag) precursor polyprotein, and the processes leading to its incorporation into viral particles. PMID:27626439

  11. Ultrasensitive in-house reverse transcription-competitive PCR for quantitation of HIV-1 RNA in plasma.

    PubMed

    Venturi, G; Ferruzzi, R; Romano, L; Catucci, M; Valensin, P E; Zazzi, M

    2000-06-01

    An ultrasensitive version of an 'in-house' reverse transcription-competitive polymerase chain reaction assay described previously for quantitation of human immunodeficiency virus type 1 (HIV-1) RNA in plasma was developed. The increase in sensitivity from 400 to 50 HIV-1 RNA copies/ml was achieved by pelleting virus particles from 1.8 ml plasma by centrifugation prior to RNA extraction, modifying competitor DNA structure and amounts, and redesigning primers. Quantitation of HIV-1 RNA in 130 samples tested previously by the standard assay showed that the two procedures yield comparable results (mean absolute difference, 0.26+/-0.20 log) and that the ultrasensitive version detects HIV-1 RNA below the threshold of sensitivity of the standard method. The ultrasensitive 'in-house assay' and the reference QUANTIPLEX HIV-1 RNA 3.0 had the same sensitivity and gave equivalent results (mean absolute difference, 0.19+/-0.11 log), as shown by parallel blinded testing of 47 plasma samples. Titration experiments with reconstructed plasma samples allowed the determination of a dynamic range of 50-500000 HIV-1 RNA copies/ml for the 'in-house' system. The interassay coefficient of variation for samples nominally containing 200, 4000 and 80000 HIV-1 RNA copies/ml were 33.4, 22.9 and 38.2%, respectively. The performance, turnaround time, and cost-effectiveness of this system make it suitable for medium-scale clinical application.

  12. Changes in the Plasticity of HIV-1 Nef RNA during the Evolution of the North American Epidemic

    PubMed Central

    Manzourolajdad, Amirhossein; Gonzalez, Mileidy; Spouge, John L.

    2016-01-01

    Because of a high mutation rate, HIV exists as a viral swarm of many sequence variants evolving under various selective pressures from the human immune system. Although the Nef gene codes for the most immunogenic of HIV accessory proteins, which alone makes it of great interest to HIV research, it also encodes an RNA structure, whose contribution to HIV virulence has been largely unexplored. Nef RNA helps HIV escape RNA interference (RNAi) through nucleotide changes and alternative folding. This study examines Historic and Modern Datasets of patient HIV-1 Nef sequences during the evolution of the North American epidemic for local changes in RNA plasticity. By definition, RNA plasticity refers to an RNA molecule’s ability to take alternative folds (i.e., alternative conformations). Our most important finding is that an evolutionarily conserved region of the HIV-1 Nef gene, which we denote by R2, recently underwent a statistically significant increase in its RNA plasticity. Thus, our results indicate that Modern Nef R2 typically accommodates an alternative fold more readily than Historic Nef R2. Moreover, the increase in RNA plasticity resides mostly in synonymous nucleotide changes, which cannot be a response to selective pressures on the Nef protein. R2 may therefore be of interest in the development of antiviral RNAi therapies. PMID:27685447

  13. Novel RNA Duplex Locks HIV-1 in a Latent State via Chromatin-mediated Transcriptional Silencing

    PubMed Central

    Ahlenstiel, Chantelle; Mendez, Catalina; Lim, Steven T H; Marks, Katherine; Turville, Stuart; Cooper, David A; Kelleher, Anthony D; Suzuki, Kazuo

    2015-01-01

    Transcriptional gene silencing (TGS) of mammalian genes can be induced by short interfering RNA (siRNA) targeting promoter regions. We previously reported potent TGS of HIV-1 by siRNA (PromA), which targets tandem NF-κB motifs within the viral 5′LTR. In this study, we screened a siRNA panel with the aim of identifying novel 5′LTR targets, to provide multiplexing potential with enhanced viral silencing and application toward developing alternate therapeutic strategies. Systematic examination identified a novel siRNA target, si143, confirmed to induce TGS as the silencing mechanism. TGS was prolonged with virus suppression >12 days, despite a limited ability to induce post- TGS. Epigenetic changes associated with silencing were suggested by partial reversal by histone deacetylase inhibitors and confirmed by chromatin immunoprecipitation analyses, which showed induction of H3K27me3 and H3K9me3, reduction in H3K9Ac, and recruitment of argonaute-1, all characteristic marks of heterochromatin and TGS. Together, these epigenetic changes mimic those associated with HIV-1 latency. Further, robust resistance to reactivation was observed in the J-Lat 9.2 cell latency model, when transduced with shPromA and/or sh143. These data support si/shRNA-mediated TGS approaches to HIV-1 and provide alternate targets to pursue a functional cure, whereby the viral reservoir is locked in latency following antiretroviral therapy cessation. PMID:26506039

  14. Acquisition of HIV-1 resistance in T lymphocytes using an ACA-specific E. coli mRNA interferase.

    PubMed

    Chono, Hideto; Matsumoto, Kazuya; Tsuda, Hiroshi; Saito, Naoki; Lee, Karim; Kim, Sujeong; Shibata, Hiroaki; Ageyama, Naohide; Terao, Keiji; Yasutomi, Yasuhiro; Mineno, Junichi; Kim, Sunyoung; Inouye, Masayori; Kato, Ikunoshin

    2011-01-01

    Transcriptional activation of gene expression directed by the long terminal repeat (LTR) of HIV-1 requires both the transactivation response element (TAR) and Tat protein. HIV-1 mutants lacking a functional tat gene are not able to proliferate. Here we take a genetic approach to suppress HIV-1 replication based on Tat-dependent production of MazF, an ACA-specific endoribonuclease (mRNA interferase) from Escherichia coli. When induced, MazF is known to cause Bak- and NBK-dependent apoptotic cell death in mammalian cells. We first constructed a retroviral vector, in which the mazF (ACA-less) gene was inserted under the control of the HIV-1 LTR, which was then transduced into CD4+ T-lymphoid CEM-SS cells in such a way that, upon HIV-1 infection, the mazF gene is induced to destroy the infecting HIV-1 mRNA, preventing HIV-1 replication. Indeed, when the transduced cells were infected with HIV-1 IIIB, the viral replication was effectively inhibited, as HIV-1 IIIB p24 could not be detected in the culture medium. Consistently, not only cell growth but also the CD4 level was not affected by the infection. These results suggest that the HIV-1-LTR-regulated mazF gene was effectively induced upon HIV-1 IIIB infection, which is sufficient enough to destroy the viral mRNA from the infected HIV-1 IIIB to completely block viral proliferation in the cells, but not to affect normal cell growth. These results indicate that the T cells transduced with the HIV-1-LTR-regulated mazF gene acquire HIV-1 resistance, providing an intriguing potential for the use of the HIV-1-LTR-regulated mazF gene in anti-HIV gene therapy.

  15. Isolation of human immunodeficiency virus type 1 (HIV-1) RNA from feces by a simple method and difference between HIV-1 subpopulations in feces and serum.

    PubMed Central

    van der Hoek, L; Boom, R; Goudsmit, J; Snijders, F; Sol, C J

    1995-01-01

    A simple method for the isolation and subsequent detection of human immunodeficiency virus type 1 (HIV-1) RNA from feces is described. Viral RNA was isolated by the method developed by Boom et al. (R. Boom, C.J.A. Sol, M.M.M. Salimans, C.L. Jansen, P.M.E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990), which was adapted for feces. HIV-1 RNA was detected by reverse transcription (RT) followed by a nested PCR encompassing the V3 region. Reconstruction experiments revealed that the efficiencies of the extraction technique and the subsequent RT-PCR were not considerably affected by the varied composition of feces. The method was applied on fecal specimens from 18 HIV-1-infected individuals, among which were samples that had been stored for 9 years. It appeared that HIV-1 RNA was detectable in the feces of 12 persons (67%). Viral RNA was present in the feces of persons who fulfilled the criteria for CDC class II and CDC class III HIV infection as well as in patients who were diagnosed with AIDS (CDC class IV). Direct sequencing of amplimers obtained from paired fecal and serum specimens showed that differences in sequence heterogeneity existed. In one patient a remarkable difference in the HIV-1 sequences between isolates from feces and serum was observed. In conclusion, HIV-1 RNA is frequently present in the feces of HIV-1-infected individuals, and in some cases the HIV-1 subpopulation in feces differs from the HIV-1 subpopulation in serum. PMID:7751361

  16. HIV-1 Suppressive Sequences Are Modulated by Rev Transport of Unspliced RNA and Are Required for Efficient HIV-1 Production

    PubMed Central

    Noguchi, Kousei; Ishibashi, Keisuke; Miyokawa, Kaori; Hokari, Manami; Kanno, Tomoyuki; Hirano, Tomoya; Yamamoto, Norio; Takaku, Hiroshi

    2012-01-01

    The unspliced human immunodeficiency virus type 1 (HIV-1) RNAs are translated as Gag and Gag-Pol polyproteins or packaged as genomes into viral particles. Efficient translation is necessary before the transition to produce infective virions. The viral protein Rev exports all intron-containing viral RNAs; however, it also appears to enhance translation. Cellular microRNAs target cellular and viral mRNAs to silence their translation and enrich them at discrete cytoplasmic loci that overlap with the putative interim site of Gag and the genome. Here, we analyzed how Rev-mediated transport and the splicing status of the mRNA influenced the silencing status imposed by microRNA. Through identification and mutational analysis of the silencing sites in the HIV-1 genome, we elucidated the effect of silencing on virus production. Renilla luciferase mRNA, which contains a let-7 targeting site in its 3′ untranslated region, was mediated when it was transported by Rev and not spliced, but it was either not mediated when it was spliced even in a partial way or it was Rev-independent. The silencing sites in the pol and env-nef regions of the HIV-1 genome, which were repressed in T cells and other cell lines, were Drosha-dependent and could also be modulated by Rev in an unspliced state. Mutant viruses that contained genomic mutations that reflect alterations to show more derepressive effects in the 3′ untranslated region of the Renilla luciferase gene replicated more slowly than wild-type virus. These findings yield insights into the HIV-1 silencing sites that might allow the genome to avoid translational machinery and that might be utilized in coordinating virus production during initial virus replication. However, the function of Rev to modulate the silencing sites of unspliced RNAs would be advantageous for the efficient translation that is required to support protein production prior to viral packaging and particle production. PMID:23251516

  17. RNA and Nucleocapsid Are Dispensable for Mature HIV-1 Capsid Assembly

    PubMed Central

    Mattei, Simone; Flemming, Annica; Anders-Össwein, Maria; Kräusslich, Hans-Georg

    2015-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) is released from infected cells in an immature, noninfectious form in which the structural polyprotein Gag is arranged in a hexameric lattice, forming an incomplete spherical shell. Maturation to the infectious form is mediated by the viral protease, which cleaves Gag at five sites, releasing the CA (capsid) protein, which forms a conical capsid encasing the condensed RNA genome. The pathway of this structural rearrangement is currently not understood, and it is unclear how cone assembly is initiated. RNA represents an integral structural component of retroviruses, and the viral nucleoprotein core has previously been proposed to nucleate mature capsid assembly. We addressed this hypothesis by replacing the RNA-binding NC (nucleocapsid) domain of HIV-1 Gag and the adjacent spacer peptide 2 (SP2) by a leucine zipper (LZ) protein-protein interaction domain [Gag(LZ)] in the viral context. We found that Gag(LZ)-carrying virus [HIV(LZ)] was efficiently released and viral polyproteins were proteolytically processed, though with reduced efficiency. Cryo-electron tomography revealed that the particles lacked a condensed nucleoprotein and contained an increased proportion of aberrant core morphologies caused either by the absence of RNA or by altered Gag processing. Nevertheless, a significant proportion of HIV(LZ) particles contained mature capsids with the wild-type morphology. These results clearly demonstrate that the nucleoprotein complex is dispensable as a nucleator for mature HIV-1 capsid assembly in the viral context. IMPORTANCE Formation of a closed conical capsid encasing the viral RNA genome is essential for HIV-1 infectivity. It is currently unclear what viral components initiate and regulate the formation of the capsid during virus morphogenesis, but it has been proposed that the ribonucleoprotein complex plays a role. To test this, we prepared virus-like particles lacking the viral nucleocapsid protein and

  18. Imaging HIV-1 RNA dimerization in cells by multicolor super-resolution and fluctuation microscopies

    PubMed Central

    Ferrer, Mireia; Clerté, Caroline; Chamontin, Célia; Basyuk, Eugenia; Lainé, Sébastien; Hottin, Jérome; Bertrand, Edouard; Margeat, Emmanuel; Mougel, Marylène

    2016-01-01

    Dimerization is a unique and vital characteristic of retroviral genomes. It is commonly accepted that genomic RNA (gRNA) must be dimeric at the plasma membrane of the infected cells to be packaged during virus assembly. However, where, when and how HIV-1 gRNA find each other and dimerize in the cell are long-standing questions that cannot be answered using conventional approaches. Here, we combine two state-of-the-art, multicolor RNA labeling strategies with two single-molecule microscopy technologies to address these questions. We used 3D-super-resolution structured illumination microscopy to analyze and quantify the spatial gRNA association throughout the cell and monitored the dynamics of RNA-RNA complexes in living-cells by cross-correlation fluctuation analysis. These sensitive and complementary approaches, combined with trans-complementation experiments, reveal for the first time the presence of interacting gRNA in the cytosol, a challenging observation due to the low frequency of these events and their dilution among the bulk of other RNAs, and allow the determination of the subcellular orchestration of the HIV-1 dimerization process. PMID:27280976

  19. Correlation between changes in plasma HIV RNA levels and in plasma infectivity in response to antiretroviral therapy.

    PubMed

    Andreoni, M; Sarmati, L; Ercoli, L; Nicastri, E; Giannini, G; Galluzzo, C; Pirillo, M F; Vella, S

    1997-05-01

    We evaluated the degree of correlation between the variation of different HIV-1 viral load measures in response to antiretroviral therapy. A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) for plasma HIV-RNA, and HIV plasma infectivity titration, were performed on prospective samples obtained from 86 antiretroviral-naive patients with symptomatic infection and CD4+ < 300/mm3, enrolled in a randomized double-blind trial of the HIV protease inhibitor saquinavir (SQV) in combination with zidovudine (ZDV). Subjects were stratified according to plasma virus infectivity and examined for correlations between distinct response categories with respect to CD4 count and HIV RNA copy number changes. Infectious virus could be titrated in 72% of patients at baseline. A significant reduction (< 1 log10) in HIV plasma infectivity titer was observed during the study in 69% of these patients. The reduction in plasma infectivity was a good predictor of sustained CD4+ cell increases and of sustained decrease in HIV RNA plasma copies. A decrease of at least 0.5 log10 in HIV RNA copy number was observed in 82% of the treated patients. A good correlation was found between HIV plasma infectivity titer and plasma HIV RNA copy number variations (p < 0.001). However, 10 of 17 patients with unchanged plasma infectivity titer showed a significant reduction in HIV RNA copies. While a good correlation was found between plasma infectivity and RNA plasma copies variations, only a minor correlation was found between CD4+ cell count variation and plasma infectivity titer variation. However, reduction in plasma infectivity was a very good predictor of high CD4 changes.

  20. HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis.

    PubMed

    Kessl, Jacques J; Kutluay, Sebla B; Townsend, Dana; Rebensburg, Stephanie; Slaughter, Alison; Larue, Ross C; Shkriabai, Nikoloz; Bakouche, Nordine; Fuchs, James R; Bieniasz, Paul D; Kvaratskhelia, Mamuka

    2016-08-25

    While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here, we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity, as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild-type, but not escape mutant, virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs. PMID:27565348

  1. Evaluation of the Aptima(®) HIV-1 Quant Dx assay for HIV-1 RNA viral load detection and quantitation in plasma of HIV-1-infected individuals: A comparison with Abbott RealTime HIV-1 assay.

    PubMed

    Amendola, Alessandra; Pisciotta, Maria; Aleo, Loredana; Ferraioli, Valeria; Angeletti, Claudio; Capobianchi, Maria Rosaria

    2016-09-01

    The Hologic Aptima(®) HIV-1 Quant Dx assay (Aptima HIV) is a real-time transcription-mediated amplification method CE-approved for use in diagnosis and monitoring of HIV-1 infection. The analytical performance of this new assay was compared to the FDA-approved Abbott RealTime HIV-1 (RealTime). The evaluation was performed using 220 clinical plasma samples, the WHO 3rd HIV-1 International Standard, and the QCMD HIV-1 RNA EQA. Concordance on qualitative results, correlation between quantitative results, accuracy, and reproducibility of viral load data were analyzed. The ability to measure HIV-1 subtypes was assessed on the second WHO International Reference Preparation Panel for HIV-1 Subtypes. With clinical samples, inter-assay agreement for qualitative results was high (91.8%) with Cohen's kappa statistic equal to 0.836. For samples with quantitative results in both assays (n = 93), Lin's concordance correlation coefficient was 0.980 (P < 0.0001) and mean differences of measurement, conducted according to Bland-Altman method, was low (0.115 log10  copies/ml). The Aptima HIV quantified the WHO 3rd HIV-1 International Standard diluted from 2000 to 31 cp/ml (5,700-88 IU/ml) at expected values with excellent linearity (R(2)  > 0.970) and showed higher sensitivity compared to RealTime being able to detect HIV-1 RNA in 10 out of 10 replicates containing down to 7 cp/ml (20 IU/ml). Reproducibility was very high, even at low HIV-1 RNA values. The Aptima HIV was able to detect and accurately quantify all the main HIV-1 subtypes in both reference panels and clinical samples. Besides excellent performance, Aptima HIV shows full automation, ease of use, and improved workflow compared to RealTime. J. Med. Virol. 88:1535-1544, 2016. © 2016 Wiley Periodicals, Inc.

  2. Evaluation of the Aptima(®) HIV-1 Quant Dx assay for HIV-1 RNA viral load detection and quantitation in plasma of HIV-1-infected individuals: A comparison with Abbott RealTime HIV-1 assay.

    PubMed

    Amendola, Alessandra; Pisciotta, Maria; Aleo, Loredana; Ferraioli, Valeria; Angeletti, Claudio; Capobianchi, Maria Rosaria

    2016-09-01

    The Hologic Aptima(®) HIV-1 Quant Dx assay (Aptima HIV) is a real-time transcription-mediated amplification method CE-approved for use in diagnosis and monitoring of HIV-1 infection. The analytical performance of this new assay was compared to the FDA-approved Abbott RealTime HIV-1 (RealTime). The evaluation was performed using 220 clinical plasma samples, the WHO 3rd HIV-1 International Standard, and the QCMD HIV-1 RNA EQA. Concordance on qualitative results, correlation between quantitative results, accuracy, and reproducibility of viral load data were analyzed. The ability to measure HIV-1 subtypes was assessed on the second WHO International Reference Preparation Panel for HIV-1 Subtypes. With clinical samples, inter-assay agreement for qualitative results was high (91.8%) with Cohen's kappa statistic equal to 0.836. For samples with quantitative results in both assays (n = 93), Lin's concordance correlation coefficient was 0.980 (P < 0.0001) and mean differences of measurement, conducted according to Bland-Altman method, was low (0.115 log10  copies/ml). The Aptima HIV quantified the WHO 3rd HIV-1 International Standard diluted from 2000 to 31 cp/ml (5,700-88 IU/ml) at expected values with excellent linearity (R(2)  > 0.970) and showed higher sensitivity compared to RealTime being able to detect HIV-1 RNA in 10 out of 10 replicates containing down to 7 cp/ml (20 IU/ml). Reproducibility was very high, even at low HIV-1 RNA values. The Aptima HIV was able to detect and accurately quantify all the main HIV-1 subtypes in both reference panels and clinical samples. Besides excellent performance, Aptima HIV shows full automation, ease of use, and improved workflow compared to RealTime. J. Med. Virol. 88:1535-1544, 2016. © 2016 Wiley Periodicals, Inc. PMID:26864171

  3. 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

  4. Favourable IFNL3 Genotypes Are Associated with Spontaneous Clearance and Are Differentially Distributed in Aboriginals in Canadian HIV-Hepatitis C Co-Infected Individuals

    PubMed Central

    Moqueet, Nasheed; Infante-Rivard, Claire; Platt, Robert W.; Young, Jim; Cooper, Curtis; Hull, Mark; Walmsley, Sharon; Klein, Marina B.

    2015-01-01

    Canadian Aboriginals are reported to clear Hepatitis C (HCV) more frequently. We tested the association of spontaneous clearance and three single nucleotide polymorphisms (SNPs) near the Interferon-lambda 3 (IFNL3) gene (rs12979860, rs8099917, functional variant rs8103142) and compared the SNP frequencies between HIV-HCV co-infected whites and Aboriginals from the Canadian Co-infection Cohort. HCV treatment-naïve individuals with at least two HCV RNA tests were included (n = 538). A spontaneous clearance case was defined as someone with two consecutive HCV RNA-negative tests, at least six months apart. Data were analyzed using Cox proportional hazards adjusted for sex and ethnicity. Advantageous variants and haplotypes were more common in Aboriginals than Caucasians: 57% vs. 46% had the rs12979860 CC genotype, respectively; 58% vs. 48%, rs8103142 TT; 74% vs. 67%, the rs12979860 C allele; and 67% vs. 64% the TCT haplotype with three favourable alleles. The adjusted Hazard Ratios (95% CI) for spontaneous clearance were: rs12979860: 3.80 (2.20, 6.54); rs8099917: 5.14 (2.46, 10.72); and rs8103142: 4.36 (2.49, 7.62). Even after adjusting for rs12979860, Aboriginals and females cleared HCV more often, HR (95% CI) = 1.53 (0.89, 2.61) and 1.42 (0.79, 2.53), respectively. Our results suggest that favourable IFNL3 genotypes are more common among Aboriginals than Caucasians, and may partly explain the higher HCV clearance rates seen among Aboriginals. PMID:25803108

  5. Anti-HIV microRNA expression in a novel Indian cohort

    PubMed Central

    Dey, Rakesh; Soni, Kartik; Saravanan, Shanmugam; Balakrishnan, Pachamuthu; Kumar, Vikram; Boobalan, Jayaseelan; Solomon, Sunil Suhas; Scaria, Vinod; Solomon, Suniti; Brahmachari, Samir K.; Pillai, Beena

    2016-01-01

    HIV-1 replication inside host cells is known to be regulated by various host factors. Host miRNAs, by virtue of its normal functioning, also regulate HIV-1 RNA expression by either directly targeting virus mRNAs or indirectly by regulating host proteins that HIV-1 uses for own replication. Therefore, it is highly possible that with differential miRNA expression, rate of disease progression will vary in HIV-1 infected individuals. In this study we have compared expression of a panel of 13 reported anti-HIV miRNAs in human PBMCs from long term non progressors (LTNPs), regular progressors and rapid progressors. We found that LTNPs have substantial lower expression of miR-382-5p that positively correlates with viral loads. Combinatorial regulation is highly probable in dictating differential disease progression as average expression of miR-382-5p and miR-155-5p can substantially distinguish LTNP individuals from regular progressors. PMID:27320691

  6. HPV genotypes detected in the oropharyngeal mucosa of HIV-infected men who have sex with men in Northern Italy.

    PubMed

    Martinelli, M; Mazza, F; Frati, E R; Fasolo, M M; Colzani, D; Bianchi, S; Fasoli, E; Amendola, A; Orlando, G; Tanzi, E

    2016-09-01

    The aim of this study was to investigate the epidemiological profile of HPV oropharyngeal infections in HIV-infected men who have sex with men. A total of 135 subjects were enrolled at the L. Sacco University Hospital (Milan, Italy) to evaluate their HPV oropharyngeal infection status at baseline and at a follow-up visit at least 12 months later. HPV DNA was detected from oropharyngeal swabs using an in-house nested PCR that amplifies a segment of the L1 gene. The PCR products were then sequenced and genotyped. A greater percentage of high-risk genotypes was identified compared to low-risk genotypes (13·7% vs. 6·9%, P < 0·05), and two uncommon alpha-HPV genotypes were detected, i.e. HPV-102 and HPV-114. HPV infection prevalence was 24·4% and the cumulative incidence was 24·1%. During the follow-up period, one case of HPV infection (HPV-33) persisted, while the overall rate of infection clearance was 58·3%. HPV oropharyngeal infection was widespread in the cohort examined, and most of the infections were transient and cleared within 12 months. These results may help to clarify the role of HPV in the oropharynx and may also improve our understanding of the need to implement preventive strategies in at-risk populations.

  7. Full-length RNA structure prediction of the HIV-1 genome reveals a conserved core domain.

    PubMed

    Sükösd, Zsuzsanna; Andersen, Ebbe S; Seemann, Stefan E; Jensen, Mads Krogh; Hansen, Mathias; Gorodkin, Jan; Kjems, Jørgen

    2015-12-01

    A distance constrained secondary structural model of the ≈10 kb RNA genome of the HIV-1 has been predicted but higher-order structures, involving long distance interactions, are currently unknown. We present the first global RNA secondary structure model for the HIV-1 genome, which integrates both comparative structure analysis and information from experimental data in a full-length prediction without distance constraints. Besides recovering known structural elements, we predict several novel structural elements that are conserved in HIV-1 evolution. Our results also indicate that the structure of the HIV-1 genome is highly variable in most regions, with a limited number of stable and conserved RNA secondary structures. Most interesting, a set of long distance interactions form a core organizing structure (COS) that organize the genome into three major structural domains. Despite overlapping protein-coding regions the COS is supported by a particular high frequency of compensatory base changes, suggesting functional importance for this element. This new structural element potentially organizes the whole genome into three major domains protruding from a conserved core structure with potential roles in replication and evolution for the virus. PMID:26476446

  8. Bioavailable inhibitors of HIV-1 RNA biogenesis identified through a Rev-based screen.

    PubMed

    Prado, Silvia; Beltrán, Manuela; Coiras, Mayte; Bedoya, Luis M; Alcamí, José; Gallego, José

    2016-05-01

    New antiretroviral agents with alternative mechanisms are needed to complement the combination therapies used to treat HIV-1 infections. Here we report the identification of bioavailable molecules that interfere with the gene expression processes of HIV-1. The compounds were detected by screening a small library of FDA-approved drugs with an assay based on measuring the displacement of Rev, and essential virus-encoded protein, from its high-affinity RNA binding site. The antiretroviral activity of two hits was based on interference with post-integration steps of the HIV-1 cycle. Both hits inhibited RRE-Rev complex formation in vitro, and blocked LTR-dependent gene expression and viral transcription in cellular assays. The best compound altered the splicing pattern of HIV-1 transcripts in a manner consistent with Rev inhibition. This mechanism of action is different from those used by current antiretroviral agents. The screening hits recognized the Rev binding site in the viral RNA, and the best compound did so with substantial selectivity, allowing the identification of a new RNA-binding scaffold. These results may be used for developing novel antiretroviral drugs.

  9. Coordination of Genomic RNA Packaging with Viral Assembly in HIV-1.

    PubMed

    Hellmund, Chris; Lever, Andrew M L

    2016-01-01

    The tremendous progress made in unraveling the complexities of human immunodeficiency virus (HIV) replication has resulted in a library of drugs to target key aspects of the replication cycle of the virus. Yet, despite this accumulated wealth of knowledge, we still have much to learn about certain viral processes. One of these is virus assembly, where the viral genome and proteins come together to form infectious progeny. Here we review this topic from the perspective of how the route to production of an infectious virion is orchestrated by the viral genome, and we compare and contrast aspects of the assembly mechanisms employed by HIV-1 with those of other RNA viruses.

  10. Coordination of Genomic RNA Packaging with Viral Assembly in HIV-1.

    PubMed

    Hellmund, Chris; Lever, Andrew M L

    2016-01-01

    The tremendous progress made in unraveling the complexities of human immunodeficiency virus (HIV) replication has resulted in a library of drugs to target key aspects of the replication cycle of the virus. Yet, despite this accumulated wealth of knowledge, we still have much to learn about certain viral processes. One of these is virus assembly, where the viral genome and proteins come together to form infectious progeny. Here we review this topic from the perspective of how the route to production of an infectious virion is orchestrated by the viral genome, and we compare and contrast aspects of the assembly mechanisms employed by HIV-1 with those of other RNA viruses. PMID:27428992

  11. Coordination of Genomic RNA Packaging with Viral Assembly in HIV-1

    PubMed Central

    Hellmund, Chris; Lever, Andrew M. L.

    2016-01-01

    The tremendous progress made in unraveling the complexities of human immunodeficiency virus (HIV) replication has resulted in a library of drugs to target key aspects of the replication cycle of the virus. Yet, despite this accumulated wealth of knowledge, we still have much to learn about certain viral processes. One of these is virus assembly, where the viral genome and proteins come together to form infectious progeny. Here we review this topic from the perspective of how the route to production of an infectious virion is orchestrated by the viral genome, and we compare and contrast aspects of the assembly mechanisms employed by HIV-1 with those of other RNA viruses. PMID:27428992

  12. Sequence and structure requirements for specific recognition of HIV-1 TAR and DIS RNA by the HIV-1 Vif protein.

    PubMed

    Freisz, Séverine; Mezher, Joelle; Hafirassou, Lamine; Wolff, Philippe; Nominé, Yves; Romier, Christophe; Dumas, Philippe; Ennifar, Eric

    2012-07-01

    The HIV-1 Vif protein plays an essential role in the regulation of the infectivity of HIV-1 virion and in vivo pathogenesis. Vif neutralizes the human DNA-editing enzyme APOBEC3 protein, an antiretroviral cellular factor from the innate immune system, allowing the virus to escape the host defence system. It was shown that Vif is packaged into viral particles through specific interactions with the viral genomic RNA. Conserved and structured sequences from the 5'-noncoding region, such as the Tat-responsive element (TAR) or the genomic RNA dimerization initiation site (DIS), are primary binding sites for Vif. In the present study we used isothermal titration calorimetry to investigate sequence and structure determinants important for Vif binding to short viral RNA corresponding to TAR and DIS stem-loops. We showed that Vif specifically binds TAR and DIS in the low nanomolar range. In addition, Vif primarily binds the TAR UCU bulge, but not the apical loop. Determinants for Vif binding to the DIS loop-loop complex are likely more complex and involve the self-complementary loop together with the upper part of the stem. These results suggest that Tat-TAR inhibitors or DIS small molecule binders might be also effective to disturb Vif-TAR and Vif-DIS binding in order to reduce Vif packaging into virions.

  13. Seminal plasma HIV-1 RNA concentration is strongly associated with altered levels of seminal plasma interferon-γ, interleukin-17, and interleukin-5.

    PubMed

    Hoffman, Jennifer C; Anton, Peter A; Baldwin, Gayle Cocita; Elliott, Julie; Anisman-Posner, Deborah; Tanner, Karen; Grogan, Tristan; Elashoff, David; Sugar, Catherine; Yang, Otto O; Hoffman, Risa M

    2014-11-01

    Seminal plasma HIV-1 RNA level is an important determinant of the risk of HIV-1 sexual transmission. We investigated potential associations between seminal plasma cytokine levels and viral concentration in the seminal plasma of HIV-1-infected men. This was a prospective, observational study of paired blood and semen samples from 18 HIV-1 chronically infected men off antiretroviral therapy. HIV-1 RNA levels and cytokine levels in seminal plasma and blood plasma were measured and analyzed using simple linear regressions to screen for associations between cytokines and seminal plasma HIV-1 levels. Forward stepwise regression was performed to construct the final multivariate model. The median HIV-1 RNA concentrations were 4.42 log10 copies/ml (IQR 2.98, 4.70) and 2.96 log10 copies/ml (IQR 2, 4.18) in blood and seminal plasma, respectively. In stepwise multivariate linear regression analysis, blood HIV-1 RNA level (p<0.0001) was most strongly associated with seminal plasma HIV-1 RNA level. After controlling for blood HIV-1 RNA level, seminal plasma HIV-1 RNA level was positively associated with interferon (IFN)-γ (p=0.03) and interleukin (IL)-17 (p=0.03) and negatively associated with IL-5 (p=0.0007) in seminal plasma. In addition to blood HIV-1 RNA level, cytokine profiles in the male genital tract are associated with HIV-1 RNA levels in semen. The Th1 and Th17 cytokines IFN-γ and IL-17 are associated with increased seminal plasma HIV-1 RNA, while the Th2 cytokine IL-5 is associated with decreased seminal plasma HIV-1 RNA. These results support the importance of genital tract immunomodulation in HIV-1 transmission.

  14. HIV-1 pre-mRNA commitment to Rev mediated export through PSF and Matrin 3

    SciTech Connect

    Kula, Anna; Gharu, Lavina; Marcello, Alessandro

    2013-01-20

    Human immunodeficiency virus gene expression and replication are regulated at several levels. Incompletely spliced viral RNAs and full-length genomic RNA contain the RRE element and are bound by the viral trans-acting protein Rev to be transported out of the nucleus. Previously we found that the nuclear matrix protein MATR3 was a cofactor of Rev-mediated RNA export. Here we show that the pleiotropic protein PSF binds viral RNA and is associated with MATR3. PSF is involved in the maintenance of a pool of RNA available for Rev activity. However, while Rev and PSF bind the viral pre-mRNA at the site of viral transcription, MATR3 interacts at a subsequent step. We propose that PSF and MATR3 define a novel pathway for RRE-containing HIV-1 RNAs that is hijacked by the viral Rev protein.

  15. Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells

    PubMed Central

    Zhou, Jiehua; Swiderski, Piotr; Li, Haitang; Zhang, Jane; Neff, C. Preston; Akkina, Ramesh; Rossi, John J.

    2009-01-01

    The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2′-F substituted RNA aptamers that bind to the HIV-1BaL gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer–siRNA chimeras. The aptamers and aptamer–siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a ‘sticky’ sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs. PMID:19304999

  16. Distinct Conformational Transition Patterns of Noncoding 7SK snRNA and HIV TAR RNAs upon Tat Binding

    PubMed Central

    2015-01-01

    Noncoding 7SK snRNA is believed to play an important role in the recruitment of P-TEFb by viral protein Tat to stimulate HIV processive transcription. Because HIV-2 TAR RNA and 7SK both evolved to feature a dinucleotide bulge region, compared to the trinucleotide bulge for HIV-1 TAR, ultrafast time-resolved fluorescence spectroscopy has been used to probe the conformational landscape of HIV-2 TAR and 7SK-SL4 RNA to monitor the conformational changes upon Tat binding. Our studies demonstrate that both HIV-1/2 TAR and 7SK-SL4 sample heterogeneous ensembles in the free state and undergo distinct conformational transitions upon Tat binding. These findings provide exquisite knowledge on the conformational complexity and intricate mechanism of molecular recognition and pave the way for drug design and discovery that incorporate dynamics information. PMID:24422492

  17. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal.

    PubMed

    Mueller, Nancy; Das, Atze T; Berkhout, Ben

    2016-01-01

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations. PMID:27455303

  18. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal

    PubMed Central

    Mueller, Nancy; Das, Atze T.; Berkhout, Ben

    2016-01-01

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5′ splice site (5′ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5′ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5′ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations. PMID:27455303

  19. A Phylogenetic Survey on the Structure of the HIV-1 Leader RNA Domain That Encodes the Splice Donor Signal.

    PubMed

    Mueller, Nancy; Das, Atze T; Berkhout, Ben

    2016-07-21

    RNA splicing is a critical step in the human immunodeficiency virus type 1 (HIV-1) replication cycle because it controls the expression of the complex viral proteome. The major 5' splice site (5'ss) that is positioned in the untranslated leader of the HIV-1 RNA transcript is of particular interest because it is used for the production of the more than 40 differentially spliced subgenomic mRNAs. HIV-1 splicing needs to be balanced tightly to ensure the proper levels of all viral proteins, including the Gag-Pol proteins that are translated from the unspliced RNA. We previously presented evidence that the major 5'ss is regulated by a repressive local RNA structure, the splice donor (SD) hairpin, that masks the 11 nucleotides (nts) of the 5'ss signal for recognition by U1 small nuclear RNA (snRNA) of the spliceosome machinery. A strikingly different multiple-hairpin RNA conformation was recently proposed for this part of the HIV-1 leader RNA. We therefore inspected the sequence of natural HIV-1 isolates in search for support, in the form of base pair (bp) co-variations, for the different RNA conformations.

  20. RRE-dependent HIV-1 Env RNA effects on Gag protein expression, assembly and release.

    PubMed

    López, Claudia S; Sloan, Rachel; Cylinder, Isabel; Kozak, Susan L; Kabat, David; Barklis, Eric

    2014-08-01

    The HIV-1 Gag proteins are translated from the full-length HIV-1 viral RNA (vRNA), whereas the envelope (Env) protein is translated from incompletely spliced Env mRNAs. Nuclear export of vRNAs and Env mRNAs is mediated by the Rev accessory protein which binds to the rev-responsive element (RRE) present on these RNAs. Evidence has shown there is a direct or indirect interaction between the Gag protein, and the cytoplasmic tail (CT) of the Env protein. Our current work shows that env gene expression impacts HIV-1 Gag expression and function in two ways. At the protein level, full-length Env expression altered Gag protein expression, while Env CT-deletion proteins did not. At the RNA level, RRE-containing Env mRNA expression reduced Gag expression, processing, and virus particle release from cells. Our results support models in which Gag is influenced by the Env CT, and Env mRNAs compete with vRNAs for nuclear export. PMID:24971705

  1. Physician experience and rates of plasma HIV-1 RNA suppression among illicit drug users: an observational study

    PubMed Central

    2012-01-01

    Background Despite the availability of antiretroviral therapy (ART), suboptimal treatment outcomes have been observed among HIV-seropositive illicit drug users. As there is an urgent need to improve responses to antiretroviral therapy among this population, we undertook this study to evaluate the role of physician experience on rates of plasma HIV-1 RNA suppression following initiation of ART. Methods Using data from a community-recruited cohort of HIV-positive illicit drug users, we used Cox proportional hazards regression to model the time to plasma viral HIV RNA < 500 copies/mL among antiretroviral-naïve subjects initiating ART. Physician experience was defined as a continuous variable measured per 100 HIV-infected patients previously enrolled in the province-wide HIV treatment registry by that physician at the time a patient was enrolled. Results Between May 1996 and December 2008, 267 individuals initiated ART among whom 227 (85%) achieved a plasma HIV RNA < 500 copies/mL during the study period. In a multivariate analysis, greater physician experience was independently associated with higher rates of plasma HIV RNA suppression (adjusted hazard ratio [AHR] = 1.17, 95% confidence interval [CI]: 1.03-1.34) after adjustment for adherence to ART. Other factors associated with viral suppression included engagement in methadone maintenance therapy (AHR = 1.61, 95% CI: 1.23-2.09), ≥ 95% adherence to ART (AHR = 2.42, 95% CI: 1.80-3.26), baseline CD4 count (AHR = 0.89, 95% CI: 0.83-0.96) and baseline plasma HIV-1 RNA (AHR = 0.65, 95% CI: 0.53-0.81). Conclusions In this setting of universal HIV/AIDS care, illicit drug users with more experienced physicians exhibited faster rates of plasma viral load suppression. These findings argue for specialized services to help optimize HIV treatment outcomes among this population. PMID:22276960

  2. Emergence of a complex relationship between HIV-1 and the microRNA pathway

    PubMed Central

    Ouellet, Dominique L.; Plante, Isabelle; Barat, Corinne; Tremblay, Michel J.; Provost, Patrick

    2010-01-01

    Summary Recent experimental evidences support the existence of an increasingly complex and multifaceted interaction between viruses and the microRNA-guided RNA silencing machinery of human cells. The discovery of small interfering RNAs (siRNAs), which are designed to mediate cleavage of specific messenger RNAs (mRNAs), prompted virologists to establish therapeutic strategies based on siRNAs with the aim to suppress replication of several viruses, including human immunodeficiency virus type 1 (HIV-1). It has been appreciated only recently that viral RNAs can also be processed endogenously by the microRNA-generating enzyme Dicer or recognized by cellular miRNAs, in processes that could be viewed as an adapted antiviral defense mechanism. Known to repress mRNA translation through recognition of specific binding sites usually located in their 3′ untranslated region, miRNAs of host or viral origin may exert regulatory effects towards host and/or viral genes and influence viral replication and/or the host response to viral infection. This article summarizes our current state of knowledge on the relationship between HIV-1 and miRNA-guided RNA silencing, and discusses the different aspects of their interaction. PMID:19301659

  3. A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

    PubMed

    Kenyon, Julia C; Prestwood, Liam J; Lever, Andrew M L

    2015-01-01

    RNA-protein interactions govern many viral and host cell processes. Conventional 'footprinting' to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2' hydroxyl reactivity ('SHAPE') that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. 'XL-SHAPE' was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome.

  4. A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader

    PubMed Central

    Kenyon, Julia C.; Prestwood, Liam J.; Lever, Andrew M. L.

    2015-01-01

    RNA-protein interactions govern many viral and host cell processes. Conventional ‘footprinting’ to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2′ hydroxyl reactivity (‘SHAPE’) that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. ‘XL-SHAPE’ was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome. PMID:26449409

  5. A novel combined RNA-protein interaction analysis distinguishes HIV-1 Gag protein binding sites from structural change in the viral RNA leader.

    PubMed

    Kenyon, Julia C; Prestwood, Liam J; Lever, Andrew M L

    2015-01-01

    RNA-protein interactions govern many viral and host cell processes. Conventional 'footprinting' to examine RNA-protein complex formation often cannot distinguish between sites of RNA-protein interaction and sites of RNA structural remodelling. We have developed a novel technique combining photo crosslinking with RNA 2' hydroxyl reactivity ('SHAPE') that achieves rapid and hitherto unachievable resolution of both RNA structural changes and the sites of protein interaction within an RNA-protein complex. 'XL-SHAPE' was validated using well-characterized viral RNA-protein interactions: HIV-1 Tat/TAR and bacteriophage MS2 RNA/Coat Binding Protein. It was then used to map HIV-1 Gag protein interactions on 2D and 3D models of the viral RNA leader. Distinct Gag binding sites were identified on exposed RNA surfaces corresponding to regions identified by mutagenesis as important for genome packaging. This widely applicable technique has revealed a first view of the stoichiometry and structure of the initial complex formed when HIV captures its genome. PMID:26449409

  6. Molecular mimicry of human tRNALys anti-codon domain by HIV-1 RNA genome facilitates tRNA primer annealing.

    PubMed

    Jones, Christopher P; Saadatmand, Jenan; Kleiman, Lawrence; Musier-Forsyth, Karin

    2013-02-01

    The primer for initiating reverse transcription in human immunodeficiency virus type 1 (HIV-1) is tRNA(Lys3). Host cell tRNA(Lys) is selectively packaged into HIV-1 through a specific interaction between the major tRNA(Lys)-binding protein, human lysyl-tRNA synthetase (hLysRS), and the viral proteins Gag and GagPol. Annealing of the tRNA primer onto the complementary primer-binding site (PBS) in viral RNA is mediated by the nucleocapsid domain of Gag. The mechanism by which tRNA(Lys3) is targeted to the PBS and released from hLysRS prior to annealing is unknown. Here, we show that hLysRS specifically binds to a tRNA anti-codon-like element (TLE) in the HIV-1 genome, which mimics the anti-codon loop of tRNA(Lys) and is located proximal to the PBS. Mutation of the U-rich sequence within the TLE attenuates binding of hLysRS in vitro and reduces the amount of annealed tRNA(Lys3) in virions. Thus, LysRS binds specifically to the TLE, which is part of a larger LysRS binding domain in the viral RNA that includes elements of the Psi packaging signal. Our results suggest that HIV-1 uses molecular mimicry of the anti-codon of tRNA(Lys) to increase the efficiency of tRNA(Lys3) annealing to viral RNA.

  7. Movements of HIV-1 genomic RNA-APOBEC3F complexes and PKR reveal cytoplasmic and nuclear PKR defenses and HIV-1 evasion strategies.

    PubMed

    Marin, Mariana; Golem, Sheetal; Kozak, Susan L; Kabat, David

    2016-02-01

    APOBEC3 cytidine deaminases and viral genomic RNA (gRNA) occur in virions, polysomes, and cytoplasmic granules, but have not been tracked together. Moreover, gRNA traffic is important, but the factors that move it into granules are unknown. Using in situ hybridization of transfected cells and protein synthesis inhibitors that drive mRNAs between locales, we observed APOBEC3F cotrafficking with gRNA without altering its movements. Whereas cells with little cytoplasmic gRNA were translationally active and accumulated Gag, suprathreshold amounts induced autophosphorylation of the cytoplasmic double-stranded RNA (dsRNA)-dependent protein kinase (PKR), causing eIF2α phosphorylation, protein synthesis suppression, and gRNA sequestration in stress granules. Additionally, we confirmed recent evidence that PKR is activated by chromosome-associated cellular dsRNAs after nuclear membranes disperse in prophase. By arresting cells in G2, HIV-1 blocks this mechanism for PKR activation and eIF2α phosphorylation. However, cytopathic membrane damage in CD4- and coreceptor-positive cultures infected with laboratory-adapted fusogenic HIV-1LAI eventually enabled PKR entry and activation in interphase nuclei. These results reveal multiple stages in the PKR-HIV-1 battleground that culminate in cell death. We discuss evidence suggesting that HIV-1s evolve in vivo to prevent or delay PKR activation by all these mechanisms. PMID:26626364

  8. Plasma viremia titration and RNA quantitation in ICD-p24 negative HIV type-1-infected patients.

    PubMed

    Ercoli, L; Sarmati, L; el-Sawaf, G; Cochi, S; Lanti, T; Iudicone, P; Guglielmetti, M; Giannini, G; Galluzzo, C; Tomino, C

    1995-10-01

    Quantitative culture of human immunodeficiency virus (HIV) was performed on 202 plasma samples obtained from asymptomatic and early symptomatic HIV-1 infected patients (mean CD4+ count: 186/mm3) before antiretroviral therapy was started. HIV could be isolated from 84% of the plasma samples (titers ranging from 10(0) to 10(2.75) TCID50/ml). Immune complex dissociated p24 antigen (ICD-p24) was detected in 66% of the samples. Only 23 samples (11%) were negative for both ICD-p24 as well as HIV culture. Discordant results were obtained in 55 samples, and 45 samples negative for ICD-p24 were positive for HIV culture. A significant proportion (42%) of patients that were negative for ICD-p24 belonged to a very advanced group with very low CD4+ cell count. However, almost 90% of these ICD-p24 negative samples were positive for HIV plasma viremia, stressing the value of this virological marker in patients with low CD4+ cell count and without any detectable ICD-p24 antigenemia. HIV-1 RNA was detected in all ICD-p24 negative plasma samples tested by the branched DNA (bDNA) assay. A very good correlation was found between high RNA copy number and HIV plasma isolation in samples obtained from patients with low CD4+ cell count, suggesting that HIV-1 RNA quantitation may also reflect viral infectivity of plasma.

  9. LNA/DNA chimeric oligomers mimic RNA aptamers targeted to the TAR RNA element of HIV-1.

    PubMed

    Darfeuille, Fabien; Hansen, Jens Bo; Orum, Henrik; Di Primo, Carmelo; Toulmé, Jean-Jacques

    2004-01-01

    One of the major limitations of the use of phosphodiester oligonucleotides in cells is their rapid degradation by nucleases. To date, several chemical modifications have been employed to overcome this issue but insufficient efficacy and/or specificity have limited their in vivo usefulness. In this work conformationally restricted nucleotides, locked nucleic acid (LNA), were investigated to design nuclease resistant aptamers targeted against the HIV-1 TAR RNA. LNA/DNA chimeras were synthesized from a shortened version of the hairpin RNA aptamer identified by in vitro selection against TAR. The results indicate that these modifications confer good protection towards nuclease digestion. Electrophoretic mobility shift assays, thermal denaturation monitored by UV-spectroscopy and surface plasmon resonance experiments identified LNA/DNA TAR ligands that bind to TAR with a dissociation constant in the low nanomolar range as the parent RNA aptamer. The crucial G, A residues that close the aptamer loop remain a key structural determinant for stable LNA/DNA chimera-TAR complexes. This work provides evidence that LNA modifications alternated with DNA can generate stable structured RNA mimics for interacting with folded RNA targets. PMID:15181175

  10. Isolation of HIV-1 RNA from plasma: evaluation of seven different methods for extraction (part two).

    PubMed

    Fransen, K; Mortier, D; Heyndrickx, L; Verhofstede, C; Janssens, W; van der Groen, G

    1998-12-01

    Some new commercial methods for the extraction of viral RNA have been introduced recently. In addition to the study published previously (Verhofstede, C., Reniers, S., Van Wanzeele. F., Plum J., 1996. AIDS 8, 1421-1427), seven different methods (four newly developed and three reference methods) for extraction of HIV-1 RNA from plasma have been evaluated. The RNA preparation method that gave the best results (acceptable reproducibility, highest sensitivity, reasonable price, fast and easy to perform), was the QIAamp Viral RNA kit from QIAgen. The High Pure Viral RNA Kit (Boehringer Mannheim) as well as the non-commercialised extraction kits were also very sensitive. The non-commercial tests seem less suitable for routine use and for the processing of large number of samples. Two methods, RNA Insta-Pure LS (Eurogentec) and PANext RNA extraction kit 1 (NTL, PANsystems GmbH) are not adapted for HIV plasma extraction. The single step methods using glass fibre or silica column are rapid (from 60 to 75 min depending on the number of wash steps) and although the price is high they are cheaper than the Boom extraction methods: High Pure Viral RNA Kit (Boehringer Mannheim) ($3.3/sample), QIAamp Viral RNA Kit (Qiagen) ($3.6/sample), Boom extraction ($5/sample). The Qiagen kit is the only kit that combines sensitivity with reproducibility, it is commercialised, rapid and affordable in price and can be automated. For most of the methods evaluated the inter-test variability was acceptable (mean variation coefficient between duplicate extractions varied between 26.4 and 48.6%).

  11. Dynamics of the human and viral m(6)A RNA methylomes during HIV-1 infection of T cells.

    PubMed

    Lichinchi, Gianluigi; Gao, Shang; Saletore, Yogesh; Gonzalez, Gwendolyn Michelle; Bansal, Vikas; Wang, Yinsheng; Mason, Christopher E; Rana, Tariq M

    2016-01-01

    N(6)-methyladenosine (m(6)A) is the most prevalent internal modification of eukaryotic mRNA. Very little is known of the function of m(6)A in the immune system or its role in host-pathogen interactions. Here, we investigate the topology, dynamics and bidirectional influences of the viral-host RNA methylomes during HIV-1 infection of human CD4 T cells. We show that viral infection triggers a massive increase in m(6)A in both host and viral mRNAs. In HIV-1 mRNA, we identified 14 methylation peaks in coding and noncoding regions, splicing junctions and splicing regulatory sequences. We also identified a set of 56 human gene transcripts that were uniquely methylated in HIV-1-infected T cells and were enriched for functions in viral gene expression. The functional relevance of m(6)A for viral replication was demonstrated by silencing of the m(6)A writer or the eraser enzymes, which decreased or increased HIV-1 replication, respectively. Furthermore, methylation of two conserved adenosines in the stem loop II region of HIV-1 Rev response element (RRE) RNA enhanced binding of HIV-1 Rev protein to the RRE in vivo and influenced nuclear export of RNA. Our results identify a new mechanism for the control of HIV-1 replication and its interaction with the host immune system. PMID:27572442

  12. Impact of HIV-1 Subtype and Antiretroviral Therapy on Protease and Reverse Transcriptase Genotype: Results of a Global Collaboration

    PubMed Central

    2005-01-01

    Background The genetic differences among HIV-1 subtypes may be critical to clinical management and drug resistance surveillance as antiretroviral treatment is expanded to regions of the world where diverse non-subtype-B viruses predominate. Methods and Findings To assess the impact of HIV-1 subtype and antiretroviral treatment on the distribution of mutations in protease and reverse transcriptase, a binomial response model using subtype and treatment as explanatory variables was used to analyze a large compiled dataset of non-subtype-B HIV-1 sequences. Non-subtype-B sequences from 3,686 persons with well characterized antiretroviral treatment histories were analyzed in comparison to subtype B sequences from 4,769 persons. The non-subtype-B sequences included 461 with subtype A, 1,185 with C, 331 with D, 245 with F, 293 with G, 513 with CRF01_AE, and 618 with CRF02_AG. Each of the 55 known subtype B drug-resistance mutations occurred in at least one non-B isolate, and 44 (80%) of these mutations were significantly associated with antiretroviral treatment in at least one non-B subtype. Conversely, of 67 mutations found to be associated with antiretroviral therapy in at least one non-B subtype, 61 were also associated with antiretroviral therapy in subtype B isolates. Conclusion Global surveillance and genotypic assessment of drug resistance should focus primarily on the known subtype B drug-resistance mutations. PMID:15839752

  13. HIV-1 sensitivity to zidovudine: a consensus culture technique validated by genotypic analysis of the reverse transcriptase.

    PubMed

    Brun-Vézinet, F; Ingrand, D; Deforges, L; Gochi, K; Ferchal, F; Schmitt, M P; Jung, M; Masquelier, B; Aubert, J; Buffet-Janvresse, C

    1992-05-01

    In order to select and standardize a reliable assay for the analysis of sensitivity of HIV isolates to AZT, we have compared two culture methods. The first assay (Cell-Associated Isolate Sensitivity Assay: CAISA) quantified AZT-resistant HIV isolates by end-point dilution cultures of peripheral blood mononuclear cells (PBMCs) in the presence of various concentrations of AZT. In the second assay (Cell-Free Isolate Sensitivity Assay: CFISA), following a conventional isolation of HIV, dilutions of infected cell-free supernatants were cultivated with fresh normal donor PBMCs in the presence of increasing concentrations of AZT. Samples from 64 untreated and AZT-treated patients were studied by CAISA (41), CFISA (43) or both assays (20). The CFISA, which allows the determination of titration parameters with respect to various kinetics patterns of viral replication was selected, and some of the CFISA phenotypically characterized isolates were further studied by nucleotide sequence analysis of the reverse transcriptase gene. CFISA showed that isolates from untreated patients were susceptible to AZT while the frequency of resistance increased with the duration of therapy. Genotypic analysis of CFISA-resistant isolates exhibited mutations at crucial positions, particularly at residue 215. We consider CFISA as a consensus culture technique for longitudinal studies of isolates from patients receiving AZT or other analogs of nucleosides.

  14. Correlation between genotypes of tRNA-linked short tandem repeats in Entamoeba nuttalli isolates and the geographical distribution of host rhesus macaques.

    PubMed

    Feng, Meng; Komiyama, Tomoyoshi; Yanagi, Tetsuo; Cheng, Xunjia; Sherchand, Jeevan B; Tachibana, Hiroshi

    2014-01-01

    Several polymorphic markers, including serine-rich protein genes, have been used for the genotyping of isolates from the morphologically indistinguishable protozoan parasites Entamoeba histolytica, Entamoeba dispar, and Entamoeba nuttalli. Genotypes of tRNA-linked short tandem repeats (STRs) are highly polymorphic, but the correlation with geographical distribution is unknown. We have recently isolated 15 E. nuttalli strains from wild rhesus macaques in four locations in Kathmandu Valley, Nepal. The sequences of the serine-rich protein genes of the E. nuttalli strains differed among the four locations. In this study, we analyzed tRNA-linked STRs in six loci of the 15 strains. Two genotypes were found in loci N-K2, R-R, and S(TGA)-D, three in locus S-Q, and five in locus D-A. In locus A-L, one major genotype and ten minor genotypes were found, resulting in mixtures of two to six genotypes in eight strains. By combination of the main genotypes in the six loci, the 15 strains were divided into nine genotypes. The genotypes observed in E. nuttalli strains were quite different from those in E. histolytica and E. dispar. A phylogenetic tree constructed from tRNA-linked STRs in the six loci reflected the different places of isolation. These results suggest that sequence diversity of tRNA-linked STRs in E. nuttalli occurs with relatively high frequency and might be a marker of geographical distribution of host rhesus macaques, even in limited areas.

  15. Relationship between hunger, adherence to antiretroviral therapy and plasma HIV RNA suppression among HIV-positive illicit drug users in a Canadian setting.

    PubMed

    Anema, Aranka; Kerr, Thomas; Milloy, M-J; Feng, Cindy; Montaner, Julio S G; Wood, Evan

    2014-04-01

    Food insecurity may be a barrier to achieving optimal HIV treatment-related outcomes among illicit drug users. This study therefore, aimed to assess the impact of severe food insecurity, or hunger, on plasma HIV RNA suppression among illicit drug users receiving antiretroviral therapy (ART). A cross-sectional Multivariate logistic regression model was used to assess the potential relationship between hunger and plasma HIV RNA suppression. A sample of n = 406 adults was derived from a community-recruited open prospective cohort of HIV-positive illicit drug users, in Vancouver, British Columbia (BC), Canada. A total of 235 (63.7%) reported "being hungry and unable to afford enough food," and 241 (59.4%) had plasma HIV RNA < 50 copies/ml. In unadjusted analyses, self-reported hunger was associated with lower odds of plasma HIV RNA suppression (Odds Ratio = 0.59, 95% confidence interval [CI]: 0.39-0.90, p = 0.015). In multivariate analyses, this association was no longer significant after controlling for socio-demographic, behavioral, and clinical characteristics, including 95% adherence (Adjusted Odds Ratio [AOR] = 0.65, 95% CI: 0.37-1.10, p = 0.105). Multivariate models stratified by 95% adherence found that the direction and magnitude of this association was not significantly altered by the adherence level. Hunger was common among illicit drug users in this setting. Although, there was an association between hunger and lower likelihood of plasma HIV RNA suppression, this did not persist in adjusted analyses. Further research is warranted to understand the social-structural, policy, and physical factors shaping the HIV outcomes of illicit drug users.

  16. Human papillomavirus DNA and mRNA prevalence and association with cervical cytological abnormalities in the Irish HIV population.

    PubMed

    Loy, Aisling; McInerney, Jamie; Pilkington, Loretto; Keegan, Helen; Delamere, Sandra; Martin, Cara M; Sheils, Orla; O'Leary, John J; Mulcahy, Fiona

    2015-10-01

    The complex interplay between HIV and human papillomavirus and its link to cervical dysplasia is poorly understood. This is the first study to assess the prevalence of oncogenic human papillomavirus mRNA in HIV-positive women, its relationship to HIV and its potential use in the triage of cervical cancer screening in HIV-positive women. In this cross-sectional study, we included 321 HIV-positive women. In all, 28.7% had abnormal cervical cytology, 51.1% were human papillomavirus DNA-positive and 21.8% tested positive for human papillomavirus mRNA. Women with a CD4 count of <200 × 10(6)/L were more likely to test positive for human papillomavirus DNA and mRNA. Virally suppressed women were less likely to be human papillomavirus DNA-positive; however, the same did not hold true for human papillomavirus mRNA. We found the human papillomavirus mRNA screening to be more specific when screening for low-grade squamous intraepithelial lesion and high-grade squamous intraepithelial lesion than human papillomavirus DNA at 84.53% compared to 57.36%. However, the sensitivity was less at 51.59% versus 91.07% for human papillomavirus DNA. It may be possible in the future to use human papillomavirus mRNA/DNA testing within a triage algorithm for the screening and management of cervical cancer in the HIV-positive patient.

  17. Is HIV-1 RNA dimerization a prerequisite for packaging? Yes, no, probably?

    PubMed Central

    Russell, Rodney S; Liang, Chen; Wainberg, Mark A

    2004-01-01

    During virus assembly, all retroviruses specifically encapsidate two copies of full-length viral genomic RNA in the form of a non-covalently linked RNA dimer. The absolute conservation of this unique genome structure within the Retroviridae family is strong evidence that a dimerized genome is of critical importance to the viral life cycle. An obvious hypothesis is that retroviruses have evolved to preferentially package two copies of genomic RNA, and that dimerization ensures the proper packaging specificity for such a genome. However, this implies that dimerization must be a prerequisite for genome encapsidation, a notion that has been debated for many years. In this article, we review retroviral RNA dimerization and packaging, highlighting the research that has attempted to dissect the intricate relationship between these two processes in the context of HIV-1, and discuss the therapeutic potential of these putative antiretroviral targets. PMID:15345057

  18. An RNAi in silico approach to find an optimal shRNA cocktail against HIV-1

    PubMed Central

    2010-01-01

    Background HIV-1 can be inhibited by RNA interference in vitro through the expression of short hairpin RNAs (shRNAs) that target conserved genome sequences. In silico shRNA design for HIV has lacked a detailed study of virus variability constituting a possible breaking point in a clinical setting. We designed shRNAs against HIV-1 considering the variability observed in naïve and drug-resistant isolates available at public databases. Methods A Bioperl-based algorithm was developed to automatically scan multiple sequence alignments of HIV, while evaluating the possibility of identifying dominant and subdominant viral variants that could be used as efficient silencing molecules. Student t-test and Bonferroni Dunn correction test were used to assess statistical significance of our findings. Results Our in silico approach identified the most common viral variants within highly conserved genome regions, with a calculated free energy of ≥ -6.6 kcal/mol. This is crucial for strand loading to RISC complex and for a predicted silencing efficiency score, which could be used in combination for achieving over 90% silencing. Resistant and naïve isolate variability revealed that the most frequent shRNA per region targets a maximum of 85% of viral sequences. Adding more divergent sequences maintained this percentage. Specific sequence features that have been found to be related with higher silencing efficiency were hardly accomplished in conserved regions, even when lower entropy values correlated with better scores. We identified a conserved region among most HIV-1 genomes, which meets as many sequence features for efficient silencing. Conclusions HIV-1 variability is an obstacle to achieving absolute silencing using shRNAs designed against a consensus sequence, mainly because there are many functional viral variants. Our shRNA cocktail could be truly effective at silencing dominant and subdominant naïve viral variants. Additionally, resistant isolates might be targeted

  19. Isolation of Cognate Cellular and Viral Ribonucleoprotein Complexes of HIV-1 RNA Applicable to Proteomic Discovery and Molecular Investigations.

    PubMed

    Singh, Deepali; Boeras, Ioana; Singh, Gatikrushna; Boris-Lawrie, Kathleen

    2016-01-01

    All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.

  20. DDX1 is an RNA-dependent ATPase Involved in HIV-1 Rev Function and Virus Replication

    PubMed Central

    Edgcomb, Stephen P.; Carmel, Andrew B.; Naji, Souad; Ambrus-Aikelin, Geza; Reyes, Jason R.; Saphire, Andrew C.S.; Gerace, Larry; Williamson, James R.

    2011-01-01

    The HIV-1 Rev protein is essential for the virus because it promotes nuclear export of alternatively-processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in E. coli is a well-behaved folded protein. Binding assays using fluorescently-labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1-Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell based assays of HIV-1 production, and provide the first demonstration that recombinant DDX1 binds Rev and RNA, and has RNA dependent catalytic activity. PMID:22051512

  1. A Conserved Target Site in HIV-1 Gag RNA is Accessible to Inhibition by Both an HDV Ribozyme and a Short Hairpin RNA

    PubMed Central

    Scarborough, Robert J; Lévesque, Michel V; Boudrias-Dalle, Etienne; Chute, Ian C; Daniels, Sylvanne M; Ouellette, Rodney J; Perreault, Jean-Pierre; Gatignol, Anne

    2014-01-01

    Antisense-based molecules targeting HIV-1 RNA have the potential to be used as part of gene or drug therapy to treat HIV-1 infection. In this study, HIV-1 RNA was screened to identify more conserved and accessible target sites for ribozymes based on the hepatitis delta virus motif. Using a quantitative screen for effects on HIV-1 production, we identified a ribozyme targeting a highly conserved site in the Gag coding sequence with improved inhibitory potential compared to our previously described candidates targeting the overlapping Tat/Rev coding sequence. We also demonstrate that this target site is highly accessible to short hairpin directed RNA interference, suggesting that it may be available for the binding of antisense RNAs with different modes of action. We provide evidence that this target site is structurally conserved in diverse viral strains and that it is sufficiently different from the human transcriptome to limit off-target effects from antisense therapies. We also show that the modified hepatitis delta virus ribozyme is more sensitive to a mismatch in its target site compared to the short hairpin RNA. Overall, our results validate the potential of a new target site in HIV-1 RNA to be used for the development of antisense therapies. PMID:25072692

  2. G3BP1 restricts HIV-1 replication in macrophages and T-cells by sequestering viral RNA.

    PubMed

    Cobos Jiménez, Viviana; Martinez, Fernando O; Booiman, Thijs; van Dort, Karel A; van de Klundert, Maarten A A; Gordon, Siamon; Geijtenbeek, Teunis B H; Kootstra, Neeltje A

    2015-12-01

    HIV-1 exploits the cellular machinery for replication and therefore several interactions with cellular factors take place, some of which are yet unknown. We identified GTPase-activating protein-(SH3 domain)-binding protein 1 (G3BP1) as a cellular factor that restricts HIV-1, by analyzing transcriptome profiles of in vitro-cytokine-activated macrophages that are non-permissive to HIV-1 replication. Silencing of G3BP1 by RNA interference resulted in increased HIV-1 replication in primary T-cells and macrophages, but did not affect replication of other retroviruses. G3BP1 specifically interacted with HIV-1 RNA in the cytoplasm, suggesting that it sequesters viral transcripts, thus preventing translation or packaging. G3BP1 was highly expressed in resting naïve or memory T-cells from healthy donors and HIV-1 infected patients, but significantly lower in IL-2-activated T-cells. These results strongly suggest that G3BP1 captures HIV-1 RNA transcripts and thereby restricts mRNA translation, viral protein production and virus particle formation.

  3. Circulating MiRNA-122 Levels Are Associated with Hepatic Necroinflammation and Portal Hypertension in HIV/HCV Coinfection

    PubMed Central

    Jansen, Christian; Reiberger, Thomas; Huang, Jia; Eischeid, Hannah; Schierwagen, Robert; Mandorfer, Mattias; Anadol, Evrim; Schwabl, Philipp; Schwarze-Zander, Carolynne; Warnecke-Eberz, Ute; Strassburg, Christian P.; Rockstroh, Jürgen K.; Peck-Radosavljevic, Markus; Odenthal, Margarete; Trebicka, Jonel

    2015-01-01

    Background Introduction of combined antiretroviral therapy (cART) has improved survival of HIV infected individuals, while the relative contribution of liver-related mortality increased. Especially in HIV/HCV-coinfected patients hepatic fibrosis and portal hypertension represent the main causes of liver-related morbidity and mortality. Circulating miRNA-122 levels are elevated in HIV patients and have been shown to correlate with severity of liver injury. However, the association of miRNA-122 levels and hepatic fibrosis and portal hypertension remains to be explored in HIV/HCV coinfection. Methods From a total of 74 (31% female) patients with HIV/HCV coinfection were included. Serum levels of miRNA-122 were analyzed by quantitative polymerase chain reaction (PCR) and normalized to SV-40 spike-in RNA. Hepatic venous pressure gradient (HVPG) was measured in 52 (70%) patients and the fibrosis stage was determined in 63 (85%) patients using transient elastography. Results The levels of circulating miRNA-122 were increased in HIV/HCV coinfected patients and significantly correlated with the alanine aminotransferase (ALT) (rs = 0.438; p<0.001) and aspartate transaminase AST values (rs = 0.336; p = 0.003), but not with fibrosis stage (p = n.s.). Interestingly, miRNA-122 levels showed an inverse correlation with hepatic venous pressure gradient (HVPG) (rs = −0.302; p = 0.03). Conclusion Elevated miRNA-122 levels are associated with liver injury, and with low HVPG. Though, miRNA-122 levels are not suitable to predict the degree of fibrosis, they might function as indicators for portal hypertension in HIV/HCV coinfected patients. PMID:25646812

  4. Two-metal ion mechanism of RNA cleavage by HIV RNase H and mechanism-based design of selective HIV RNase H inhibitors.

    PubMed

    Klumpp, Klaus; Hang, Julie Qi; Rajendran, Surendran; Yang, Yanli; Derosier, Andre; Wong Kai In, Philippe; Overton, Hilary; Parkes, Kevin E B; Cammack, Nick; Martin, Joseph A

    2003-12-01

    Human immunodeficiency virus (HIV) RNase H activity is essential for the synthesis of viral DNA by HIV reverse transcriptase (HIV-RT). RNA cleavage by RNase H requires the presence of divalent metal ions, but the role of metal ions in the mechanism of RNA cleavage has not been resolved. We measured HIV RNase H activity associated with HIV-RT protein in the presence of different concentrations of either Mg2+, Mn2+, Co2+ or a combination of these divalent metal ions. Polymerase-independent HIV RNase H was similar to or more active with Mn2+ and Co2+ compared with Mg2+. Activation of RNase H by these metal ions followed sigmoidal dose-response curves suggesting cooperative metal ion binding. Titration of Mg2+-bound HIV RNase H with Mn2+ or Co2+ ions generated bell-shaped activity dose-response curves. Higher activity could be achieved through simultaneous binding of more than one divalent metal ion at intermediate Mn2+ and Co2+ concentrations, and complete replacement of Mg2+ occurred at higher Mn2+ or Co2+ concentrations. These results are consistent with a two-metal ion mechanism of RNA cleavage as previously suggested for a number of polymerase-associated nucleases. In contrast, the structurally highly homologous RNase HI from Escherichia coli is most strongly activated by Mg2+, is significantly inhibited by submillimolar concentrations of Mn2+ and most probably cleaves RNA via a one-metal ion mechanism. Based on this difference in active site structure, a series of small molecule N-hydroxyimides was identified with significant enzyme inhibitory potency and selectivity for HIV RNase H.

  5. Performance of commonly used genotypic assays and comparison with phenotypic assays of HIV-1 coreceptor tropism in acutely HIV-1-infected patients

    PubMed Central

    Ceresola, Elisa Rita; Nozza, Silvia; Sampaolo, Michela; Pignataro, Angela Rosa; Saita, Diego; Ferrarese, Roberto; Ripa, Marco; Deng, Wenjie; Mullins, James I.; Boeri, Enzo; Tambussi, Giuseppe; Toniolo, Antonio; Lazzarin, Adriano; Clementi, Massimo; Canducci, Filippo

    2015-01-01

    Objectives Although founder viruses in primary HIV-1 infections (PHIs) typically use the CCR5 coreceptor (R5-tropic), 3%–19% of subjects also harbour CXCR4-using viruses (X4-tropic), making tropism determination before CCR5 antagonist usage mandatory. Genotypic methods can be used to accurately determine HIV-1 tropism in chronically infected patients. Methods We compared the results of genotypic methods [geno2pheno, PSSMx4r5 including a novel nucleotide-input version (ntPSSM) and distant segments (ds)Kernel] to predict coreceptor usage in a cohort of 67 PHIs. Specimens with discrepant results were phenotypically tested after cloning the V3 gene region into proviral backbones. Recombinant viruses were used to infect U87 indicator cell lines bearing CD4 and either CCR5 or CXCR4. Results Geno2pheno10%, PSSMx4r5 and (ds)Kernel gave identical predictions in 85% of cases. Geno2pheno10% predicted the presence of CXCR4 viruses in 18% of patients. Two patients were predicted to carry X4-tropic viruses by all algorithms and X4-tropic viruses were detected in at least one of the recombinant AD8 or NL4-3 backbone-based assays. Ten samples resulted in discordant predictions with at least one algorithm. Full concordance between tropism prediction by using population sequencing and phenotypic assays was observed only with ntPSSM. Geno2pheno prediction and the phenotypic assay gave the same results in a minority of ‘discordant’ patients. Conclusions Compared with both PSSMx4r5 versions, (ds)Kernel and our phenotypic assay, geno2pheno10% overestimated the frequency of X4-tropic viruses (18% versus 3%). ntPSSM was able to detect one additional X4 virus compared with (ds)Kernel that was confirmed with the phenotypic assay. PMID:25608585

  6. Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection.

    PubMed Central

    Purohit, P; Dupont, S; Stevenson, M; Green, M R

    2001-01-01

    The human immunodeficiency virus type-1 matrix protein (HIV-1 MA) is a multifunctional structural protein synthesized as part of the Pr55 gag polyprotein. We have used in vitro genetic selection to identify an RNA consensus sequence that specifically interacts with MA (Kd = 5 x 10(-7) M). This 13-nt MA binding consensus sequence bears a high degree of homology (77%) to a region (nt 1433-1446) within the POL open reading frame of the HIV-1 genome (consensus sequence from 38 HIV-1 strains). Chemical interference experiments identified the nucleotides within the MA binding consensus sequence involved in direct contact with MA. We further demonstrate that this RNA-protein interaction is mediated through a stretch of basic amino acids within MA. Mutations that disrupt the interaction between MA and its RNA binding site within the HIV-1 genome resulted in a measurable decrease in viral replication. PMID:11345436

  7. Post-extraction stabilization of HIV viral RNA for quantitative molecular tests.

    PubMed

    Stevens, Daniel S; Crudder, Christopher H; Domingo, Gonzalo J

    2012-06-01

    Two approaches to stabilize viral nucleic acid in processed clinical specimens were evaluated. HIV-1 RNA extracted from clinical specimens was stabilized in a dry matrix in a commercial product (RNAstable, Biomatrica, San Diego, CA, USA) and in a reverse-transcription reaction mixture in liquid form as cDNA. As few as 145 HIV-1 genome copies of viral RNA are reliably stabilized by RNAstable at 45°C for 92 days and in the cDNA format at 45°C for 7 days as determined by real-time PCR. With RNAstable the R(2) at days 1, 7, and 92 were 0.888, 0.871, and 0.943 when compared to baseline viral load values. The cDNA generated from the same clinical specimens was highly stable with an R(2) value of 0.762 when comparing viral load determinations at day 7 to baseline values. In conclusion viral RNA stabilized in a dry RNAstable matrix is highly stable for long periods of time at high temperatures across a substantial dynamic range. Viral RNA signal can also be stabilized in liquid in the form of cDNA for limited periods of time. Methods that reduce reliance on the cold chain and preserve specimen integrity are critical for extending the reach of molecular testing to low-resource settings. Products based on anhydrobiosis, such as the RNAstable should be evaluated further to support viral pathogen diagnosis. PMID:22433512

  8. Treatment of Genotype 1 HCV Infection in the HIV Coinfected Patient in 2014

    PubMed Central

    Chastain, Cody A.; Naggie, Susanna

    2016-01-01

    Hepatitis C (HCV) coinfection is the leading cause of liver-related morbidity and is a leading cause of mortality in human immunodeficiency virus (HIV)-infected individuals in the antiretroviral therapy era. Direct-acting antiviral (DAA) therapies are transforming how HCV is treated with significant improvements in efficacy and tolerability. In this article, DAA agents expected to be available in 2014 are reviewed, including telaprevir, boceprevir, sofosbuvir, simeprevir, faldaprevir, and daclatasvir. Available data regarding clinical efficacy, adverse effects, and drug interactions in HIV-HCV coinfection are discussed. The management of adverse effects of HCV therapy and treatment considerations in patients with cirrhosis are also reviewed. PMID:24272069

  9. Identification of Novel Recombinant Forms of Hepatitis B Virus Generated from Genotypes Ae and G in HIV-1-Positive Japanese Men Who Have Sex with Men.

    PubMed

    Kojima, Yoko; Kawahata, Takuya; Mori, Haruyo; Furubayashi, Keiichi; Taniguchi, Yasushi; Itoda, Ichiro; Komano, Jun

    2015-07-01

    The rare hepatitis B virus (HBV) genotype G (HBV/G) coinfects HIV-1-positive individuals along with HBV/A and generates recombinants. However, the circulation of HBV A/G recombinants remains poorly understood. This molecular epidemiologic study examined HBV A/G recombinants in Japanese HIV-1-positive men who have sex with men (MSM). Initially, blood specimens submitted for confirmatory tests of HIV infection in Osaka and Tokyo, Japan, from 2006 to 2013 were examined for HIV-1, and HIV-1-positive specimens were screened for HBV. Among 817 specimens from HIV-1-positive individuals, HBsAg was detected in 59 specimens; of these, HBV/Ae (alternatively A2), a subgenotype of HBV/A prevalent in Europe and North America, was identified in 70.2%, HBV/C in 17.5%, and HBV/G in 10.5%, and HBV/E in 1.8% according to the core gene sequence. The full-length genome analysis of HBV was performed on HBV/G-positive specimens because some HBV A/G recombinants were historically overlooked by genotyping based on a partial genome analysis. It revealed that five of the specimens contained novel Ae/G recombinants, the core gene of which had a high sequence similarity to HBV/G. Detailed analyses showed that novel recombinants were coinfected with HBV/Ae in a recombinant-dominant fashion. No major drug-resistant mutations were found in the newly identified HBV Ae/G recombinants. Some of the individuals asymptomatically coinfected with HIV/HBV suffered mild liver injury. This study demonstrated that novel Ae/G HBV recombinants were identified in Japanese HIV-1-positive MSM. The pathogenicity of novel HBV Ae/G recombinants should be examined in a future longitudinal study. Surveillance of such viruses in HIV-1-positive individuals should be emphasized.

  10. Identification of Novel Recombinant Forms of Hepatitis B Virus Generated from Genotypes Ae and G in HIV-1-Positive Japanese Men Who Have Sex with Men

    PubMed Central

    Kawahata, Takuya; Mori, Haruyo; Furubayashi, Keiichi; Taniguchi, Yasushi; Itoda, Ichiro; Komano, Jun

    2015-01-01

    Abstract The rare hepatitis B virus (HBV) genotype G (HBV/G) coinfects HIV-1-positive individuals along with HBV/A and generates recombinants. However, the circulation of HBV A/G recombinants remains poorly understood. This molecular epidemiologic study examined HBV A/G recombinants in Japanese HIV-1-positive men who have sex with men (MSM). Initially, blood specimens submitted for confirmatory tests of HIV infection in Osaka and Tokyo, Japan, from 2006 to 2013 were examined for HIV-1, and HIV-1-positive specimens were screened for HBV. Among 817 specimens from HIV-1-positive individuals, HBsAg was detected in 59 specimens; of these, HBV/Ae (alternatively A2), a subgenotype of HBV/A prevalent in Europe and North America, was identified in 70.2%, HBV/C in 17.5%, and HBV/G in 10.5%, and HBV/E in 1.8% according to the core gene sequence. The full-length genome analysis of HBV was performed on HBV/G-positive specimens because some HBV A/G recombinants were historically overlooked by genotyping based on a partial genome analysis. It revealed that five of the specimens contained novel Ae/G recombinants, the core gene of which had a high sequence similarity to HBV/G. Detailed analyses showed that novel recombinants were coinfected with HBV/Ae in a recombinant-dominant fashion. No major drug-resistant mutations were found in the newly identified HBV Ae/G recombinants. Some of the individuals asymptomatically coinfected with HIV/HBV suffered mild liver injury. This study demonstrated that novel Ae/G HBV recombinants were identified in Japanese HIV-1-positive MSM. The pathogenicity of novel HBV Ae/G recombinants should be examined in a future longitudinal study. Surveillance of such viruses in HIV-1-positive individuals should be emphasized. PMID:25825936

  11. Targeted cleavage of HIV RRE RNA by Rev-coupled transition metal chelates.

    PubMed

    Joyner, Jeff C; Cowan, J A

    2011-06-29

    A series of compounds that target reactive metal chelates to the HIV-1 Rev response element (RRE) mRNA have been synthesized. Dissociation constants and chemical reactivity toward HIV RRE RNA have been determined and evaluated in terms of reduction potential, coordination unsaturation, and overall charge associated with the metal-chelate-Rev complex. Ethylenediaminetetraacetic acid (EDTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were linked to a lysine side chain of a Rev-derived peptide by either EDC/NHS or isothiocyanate coupling. The resulting chelate-Rev (EDTA-Rev, DTPA-Rev, NTA-Rev, and DOTA-Rev) conjugates were used to form coordination complexes with Fe(2+), Co(2+), Ni(2+), and Cu(2+) such that the arginine-rich Rev peptide could mediate localization of the metal chelates to the Rev peptide's high-affinity mRNA binding partner, RRE stem loop IIB. Metal complexes of the extended peptides GGH-Rev and KGHK-Rev, which also contain N-terminal peptidic chelators (ATCUN motifs), were studied for comparison. A fluorescence titration assay revealed high-affinity RRE RNA binding by all 22 metal-chelate-Rev species, with K(D) values ranging from ~0.2 to 16 nM, indicating little to no loss of RNA affinity due to the coupling of the metal chelates to the Rev peptide. Dissociation constants for binding at a previously unobserved low-affinity site are also reported. Rates of RNA modification by each metal-chelate-Rev species were determined and varied from ~0.28 to 4.9 nM/min but were optimal for Cu(2+)-NTA-Rev. Metal-chelate reduction potentials were determined and varied from -228 to +1111 mV vs NHE under similar solution conditions, allowing direct comparison of reactivity with redox thermodynamics. Optimal activity was observed when the reduction potential for the metal center was poised between those of the two principal co-reagents for metal-promoted formation of

  12. Single genome amplification and standard bulk PCR yield HIV-1 envelope products with similar genotypic and phenotypic characteristics.

    PubMed

    Etemad, Behzad; Ghulam-Smith, Melissa; Gonzalez, Oscar; White, Laura F; Sagar, Manish

    2015-03-01

    Recent studies suggest that single genome amplification (SGA) as compared to standard bulk PCR and virus stocks from 293T transfection versus short term passage in peripheral blood mononuclear cells (PBMC) yield a less biased representation of HIV-1 envelope characteristics. In 9 different subjects, genetic diversity, divergence, and population structure were not significantly different among SGA or bulk PCR amplified envelope V1-V3 segments. In these subjects, 293T transfection derived virus stocks with SGA or bulk PCR amplified envelopes have similar infectivity, replication kinetics, co-receptor usage, and neutralization susceptibility. While PBMC passage as compared to the 293T derived virus stocks had similar co-receptor usage, PBMC viruses were less neutralization susceptible to some specific antibodies. Our results suggest that the method of envelope sequence amplification, either SGA or bulk PCR, does not have a significant impact on the genotypic and phenotypic properties of the virus envelope quasispecies.

  13. Single genome amplification and standard bulk PCR yield HIV-1 envelope products with similar genotypic and phenotypic characteristics

    PubMed Central

    Etemad, Behzad; Smith, Melissa Ghulam; Gonzalez, Oscar; White, Laura F; Sagar, Manish

    2015-01-01

    Recent studies suggest that single genome amplification (SGA) as compared to standard bulk PCR and virus stocks from 293T transfection versus short term passage in peripheral blood mononuclear cells (PBMC) yield a less biased representation of HIV-1 envelope characteristics. In 9 different subjects, genetic diversity, divergence, and population structure was not significantly different among SGA or bulk PCR amplified envelope V1–V3 segments. In these subjects, 293T transfection derived virus stocks with SGA or bulk PCR amplified envelopes had similar infectivity, replication kinetics, co-receptor usage, and neutralization susceptibility. While PBMC passage as compared to the 293T derived virus stocks had similar co-receptor usage, PBMC viruses were less neutralization susceptible to some specific antibodies. Our results suggest that the method of envelope sequence amplification, either SGA or bulk PCR, does not have a significant impact on the genotypic and phenotypic properties of the virus envelope quasispecies. PMID:25681527

  14. Single genome amplification and standard bulk PCR yield HIV-1 envelope products with similar genotypic and phenotypic characteristics.

    PubMed

    Etemad, Behzad; Ghulam-Smith, Melissa; Gonzalez, Oscar; White, Laura F; Sagar, Manish

    2015-03-01

    Recent studies suggest that single genome amplification (SGA) as compared to standard bulk PCR and virus stocks from 293T transfection versus short term passage in peripheral blood mononuclear cells (PBMC) yield a less biased representation of HIV-1 envelope characteristics. In 9 different subjects, genetic diversity, divergence, and population structure were not significantly different among SGA or bulk PCR amplified envelope V1-V3 segments. In these subjects, 293T transfection derived virus stocks with SGA or bulk PCR amplified envelopes have similar infectivity, replication kinetics, co-receptor usage, and neutralization susceptibility. While PBMC passage as compared to the 293T derived virus stocks had similar co-receptor usage, PBMC viruses were less neutralization susceptible to some specific antibodies. Our results suggest that the method of envelope sequence amplification, either SGA or bulk PCR, does not have a significant impact on the genotypic and phenotypic properties of the virus envelope quasispecies. PMID:25681527

  15. Cerebrospinal fluid biomarkers in patients with plasma HIV RNA below 20 copies/mL

    PubMed Central

    Calcagno, Andrea; Atzori, Cristiana; Romito, Alessandra; Ecclesia, Sara; Imperiale, Daniele; Audagnotto, Sabrina; Chiara Alberione, Maria; Trentalange, Alice; Di Perri, Giovanni; Bonora, Stefano

    2014-01-01

    Introduction Low level HIV-1 CSF replication (CsfLLV) is often found even in patients with controlled plasma viraemia. The clinical consequences of such finding are uncertain; however, both symptomatic neurological disturbances and neurocognitive disorders may arise in the context of CSF-escape. Two reports suggested that low level replication in the CSF may be associated with increased CSF neopterine although the impact on other markers of neuroinflammation/damage is currently unknown. Materials and Methods Patients with neurocognitive disorders, new neurological symptoms or followed in longitudinal studies were included provided that they were on HAART, with last available viral load below 20 copies/mL and without central nervous system (CNS)-involving infections/neoplasms. After brain Magnetic Resonance (MR) CSF HIV RNA (CAP/CTM HIV-1 v2.0) and biomarkers [total tau (t-tau), phosphorylated tau (p-tau), 1–42 Beta amyloid (Beta42), neopterine and S100beta] were measured through validated methods. Data are presented as medians (IQR); non-parametric tests are used for all analysis. Results 70 patients [66.7% male, median age 47.8 years (40–56), median BMI 22.2 kg/m2 (20–24)] were enrolled. Current and nadir CD4+ cell count were 379 (219–656) and 116 cell/mm3 (46–225); HIV RNA was undetectable since 19.7 months (9–53). CSF HIV RNA was undetectable in 24 (34.3%), below 20 copies/mL in 26 (37.1%), above 20 copies/mL in 25 patients [35.7%, median 69 copies/mL (41–134]). Median (IQR) CSF biomarkers values were as follows: t-tau 109 pg/mL (<75–161), p-tau 31.6 pg/mL (23.4–35.4), Beta42 818 pg/mL (623–973), neopterine 0.58 ng/mL (0.45–0.87) and S100beta 149 pg/mL (110–186). Patients with CsfLLV did not show significant differences as for demographic, therapeutic, virological, radiological variables. t-tau (134 vs 92.6, p=0.05) and Beta42 (953 vs 675, p=0.007) were higher in patients with CsfLLV. Neopterine levels were directly associated with p

  16. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    PubMed Central

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-01-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates. PMID:27405089

  17. Iron(II) supramolecular helicates interfere with the HIV-1 Tat–TAR RNA interaction critical for viral replication

    NASA Astrophysics Data System (ADS)

    Malina, Jaroslav; Hannon, Michael J.; Brabec, Viktor

    2016-07-01

    The interaction between the HIV-1 transactivator protein Tat and TAR (transactivation responsive region) RNA, plays a critical role in HIV-1 transcription. Iron(II) supramolecular helicates were evaluated for their in vitro activity to inhibit Tat–TAR RNA interaction using UV melting studies, electrophoretic mobility shift assay, and RNase A footprinting. The results demonstrate that iron(II) supramolecular helicates inhibit Tat-TAR interaction at nanomolar concentrations by binding to TAR RNA. These studies provide a new insight into the biological potential of metallosupramolecular helicates.

  18. Combination of low producer AA-genotypes in IFN-γ and IL-10 genes makes a high risk genetic variant for HIV disease progression.

    PubMed

    Singh, Sukhvinder; Sharma, Aman; Arora, Sunil K

    2016-01-01

    Rate of HIV disease progression varies considerably among individuals, host genetic makeup be one of the possible reasons. We aimed to determine association of functional single nucleotide polymorphism (SNPs), (-179G/T and +874T/A) in IFN-γ and (-1082A/G, -819C/T and -592C/A) in IL-10 genes, with the rate of disease progression or susceptibility to HIV infection. Therapy naïve HIV infected individuals from North India, categorized as slow progressors or fast progressors and HIV exposed seronegative individuals were recruited for this study. Genotyping results revealed significantly higher frequencies of low producer AA genotype at +874T/A in IFN-γ gene and -592C/A position in IL-10 gene in FPs (p<0.002). Multifactor dimensional reduction (MDR) analysis revealed this to be a high risk combination for faster disease progression in HIV-1 infected individuals. Low producer AA genotype carriers at +874T/A in IFN-γ gene produced significantly low amounts of cellular IFN-γ. Low producing haplotype 'ATA' at -1082, -819 and -592 loci in IL-10 gene was significantly over-represented in FPs as compared to SPs (p<0.01) and these individuals showed poor response to therapy in terms of CD4 count gains after one year of ART, compared to high producing haplotype (GCC) carriers. Thus, a combination of genetic variations in IFN-γ and IL-10 cytokine genes in a single host associate with HIV disease progression and may help clinicians to better manage the HIV disease if known earlier.

  19. Cross-Platform Analysis of HIV-1 RNA Data Generated by a Multicenter Assay Validation Study with Wide Geographic Representation

    PubMed Central

    Harty, Brian; Granger, Suzanne; Wager, Carrie; Crump, John A.; Fiscus, Susan A.; Bremer, James W.

    2012-01-01

    HIV-1 RNA quantitation continues to be extremely important for monitoring patients infected with HIV-1, and a number of assays have been utilized for this purpose. Differences in assay performance with respect to log10 recovery and HIV-1 subtype specificity have been well documented for commercially available assays, although comparisons are usually limited to one or two assay platforms. Two new FDA-approved assays, the Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test (RT) and the Abbott RealTime HIV-1 assay (AR), that utilize real-time PCR have replaced previous HIV-1 RNA platforms. Inadequate detection of some strains of HIV-1 resulted in the addition of a new primer/probe set and the introduction of a second version of the RT assay. In this study, comparisons of assay performance between the different FDA-approved HIV-1 RNA assay platforms (both new and existing) were performed by using validation data that included both well-characterized virus stock and locally collected clinical samples. Laboratories across diverse geographical regions performed the validation testing and submitted data to the Virology Quality Assurance program (VQA) for analysis. Correlation values for clinical sample testing varied across the assay platforms (r = 0.832 to 0.986), and average log10 recoveries for HIV-1 RNA controls (compared to the nominal value) ranged from −0.215 to 0.181. These data demonstrate the need for use of one assay platform for longitudinal patient monitoring, but the data also reinforce the notion that no one assay is superior and that testing across platforms may be required for discordance reconciliation. PMID:22692747

  20. Exosomes from HIV-1-infected Cells Stimulate Production of Pro-inflammatory Cytokines through Trans-activating Response (TAR) RNA.

    PubMed

    Sampey, Gavin C; Saifuddin, Mohammed; Schwab, Angela; Barclay, Robert; Punya, Shreya; Chung, Myung-Chul; Hakami, Ramin M; Zadeh, Mohammad Asad; Lepene, Benjamin; Klase, Zachary A; El-Hage, Nazira; Young, Mary; Iordanskiy, Sergey; Kashanchi, Fatah

    2016-01-15

    HIV-1 infection results in a chronic illness because long-term highly active antiretroviral therapy can lower viral titers to an undetectable level. However, discontinuation of therapy rapidly increases virus burden. Moreover, patients under highly active antiretroviral therapy frequently develop various metabolic disorders, neurocognitive abnormalities, and cardiovascular diseases. We have previously shown that exosomes containing trans-activating response (TAR) element RNA enhance susceptibility of undifferentiated naive cells to HIV-1 infection. This study indicates that exosomes from HIV-1-infected primary cells are highly abundant with TAR RNA as detected by RT-real time PCR. Interestingly, up to a million copies of TAR RNA/μl were also detected in the serum from HIV-1-infected humanized mice suggesting that TAR RNA may be stable in vivo. Incubation of exosomes from HIV-1-infected cells with primary macrophages resulted in a dramatic increase of proinflammatory cytokines, IL-6 and TNF-β, indicating that exosomes containing TAR RNA could play a direct role in control of cytokine gene expression. The intact TAR molecule was able to bind to PKR and TLR3 effectively, whereas the 5' and 3' stems (TAR microRNAs) bound best to TLR7 and -8 and none to PKR. Binding of TAR to PKR did not result in its phosphorylation, and therefore, TAR may be a dominant negative decoy molecule in cells. The TLR binding through either TAR RNA or TAR microRNA potentially can activate the NF-κB pathway and regulate cytokine expression. Collectively, these results imply that exosomes containing TAR RNA could directly affect the proinflammatory cytokine gene expression and may explain a possible mechanism of inflammation observed in HIV-1-infected patients under cART.

  1. Toward a durable anti-HIV gene therapy based on RNA interference.

    PubMed

    Berkhout, Ben

    2009-09-01

    Basic research in the field of molecular biology led to the discovery of the mechanism of RNA interference (RNAi) in Caenorhabditis elegans in 1998. RNAi is now widely appreciated as an important gene control mechanism in mammals, and several RNAi-based gene-silencing applications have already been used in clinical trials. In this review I will discuss RNAi approaches to inhibit the pathogenic human immunodeficiency virus type 1 (HIV-1), which establishes a chronic infection that would most likely require a durable gene therapy approach. Viruses, such as HIV-1, are particularly difficult targets for RNAi attack because they mutate frequently, which allows viral escape by mutation of the RNAi target sequence. Combinatorial RNAi strategies are required to prevent viral escape. PMID:19796072

  2. HIV-1 Nef-associated Factor 1 Enhances Viral Production by Interacting with CRM1 to Promote Nuclear Export of Unspliced HIV-1 gag mRNA.

    PubMed

    Ren, Xiao-Xin; Wang, Hai-Bo; Li, Chuan; Jiang, Jin-Feng; Xiong, Si-Dong; Jin, Xia; Wu, Li; Wang, Jian-Hua

    2016-02-26

    HIV-1 depends on host-cell-encoded factors to complete its life cycle. A comprehensive understanding of how HIV-1 manipulates host machineries during viral infection can facilitate the identification of host targets for antiviral drugs or gene therapy. The cellular protein Naf1 (HIV-1 Nef-associated factor 1) is a CRM1-dependent nucleo-cytoplasmic shuttling protein, and has been identified to regulate multiple receptor-mediated signal pathways in inflammation. The cytoplasm-located Naf1 can inhibit NF-κB activation through binding to A20, and the loss of Naf1 controlled NF-κB activation is associated with multiple autoimmune diseases. However, the effect of Naf1 on HIV-1 mRNA expression has not been characterized. In this study we found that the nucleus-located Naf1 could promote nuclear export of unspliced HIV-1 gag mRNA. We demonstrated that the association between Naf1 and CRM1 was required for this function as the inhibition or knockdown of CRM1 expression significantly impaired Naf1-promoted HIV-1 production. The mutation of Naf1 nuclear export signals (NESs) that account for CRM1 recruitment for nuclear export decreased Naf1 function. Additionally, the mutation of the nuclear localization signal (NLS) of Naf1 diminished its ability to promote HIV-1 production, demonstrating that the shuttling property of Naf1 is required for this function. Our results reveal a novel role of Naf1 in enhancing HIV-1 production, and provide a potential therapeutic target for controlling HIV-1 infection.

  3. N-Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA.

    PubMed

    Hilimire, Thomas A; Bennett, Ryan P; Stewart, Ryan A; Garcia-Miranda, Pablo; Blume, Alex; Becker, Jordan; Sherer, Nathan; Helms, Eric D; Butcher, Samuel E; Smith, Harold C; Miller, Benjamin L

    2016-01-15

    Human Immunodeficiency Virus (HIV) type 1 uses a -1 programmed ribosomal frameshift (-1 PRF) event to translate its enzymes from the same transcript used to encode the virus' structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5-10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides. PMID:26496521

  4. N-Methylation as a Strategy for Enhancing the Affinity and Selectivity of RNA-binding Peptides: Application to the HIV-1 Frameshift-Stimulating RNA

    PubMed Central

    2015-01-01

    Human Immunodeficiency Virus (HIV) type 1 uses a −1 programmed ribosomal frameshift (−1 PRF) event to translate its enzymes from the same transcript used to encode the virus’ structural proteins. The frequency of this event is highly regulated, and significant deviation from the normal 5–10% frequency has been demonstrated to decrease viral infectivity. Frameshifting is primarily regulated by the Frameshift Stimulatory Signal RNA (FSS-RNA), a thermodynamically stable, highly conserved stem loop that has been proposed as a therapeutic target. We describe the design, synthesis, and testing of a series of N-methyl peptides able to bind the HIV-1 FSS RNA stem loop with low nanomolar affinity and high selectivity. Surface plasmon resonance (SPR) data indicates increased affinity is a reflection of a substantially enhanced on rate. Compounds readily penetrate cell membranes and inhibit HIV infectivity in a pseudotyped virus assay. Viral infectivity inhibition correlates with compound-dependent changes in the ratios of Gag and Gag-Pol in virus particles. As the first compounds with both single digit nanomolar affinities for the FSS RNA and an ability to inhibit HIV in cells, these studies support the use of N-methylation for enhancing the affinity, selectivity, and bioactivity of RNA-binding peptides. PMID:26496521

  5. Association between KIR genotypes and HLA-B alleles on viral load in Southern Brazilian individuals infected by HIV-1 subtypes B and C.

    PubMed

    Fernandes-Cardoso, Juliana; Süffert, Theodoro Armando; Correa, Maria da Gloria; Jobim, Luiz Fernando Job; Jobim, Mariana; Salim, Patricia Hartstein; Arruda, Monica Barcelos; Boullosa, Lidia Theodoro; Tanuri, Amilcar; Porto, Luis Cristóvão; Ferreira, Orlando C

    2016-10-01

    There is a great variety of HIV-1 subtypes circulating in Brazil, including subtype C, whose prevalence is on the rise, particularly in the southern region. Many host and viral genetic factors may be involved in this trend. We evaluated the influence of human leukocyte antigen (HLA) class I alleles and killer-cell immunoglobulin-like receptor (KIR) genotypes on viral set point and T-CD4(+) parameters in 84 treatment-naïve HIV-1-positive individuals. Frequency data in the infected group were compared to data of 548 healthy control subjects. Individuals with the KIR AA genotype had a higher viral load (VL) than individuals with the KIR Bx genotype. The HIV-1 group was subdivided into three subgroups according to HLA-B allele presence: those with protection to disease alleles (HLA-B(+)), accelerated disease progression alleles (HLA-B(-)), or neither (HLA-B(o)) were grouped. We observed a significant effect of the HLA-B allele presence on VL. The HLA-B(+) group had significantly lower VL than the HLA-B(-) group and trended toward a lower VL than the HLA-B(o) group. There were significant differences between groups expressing extreme VL values: KIR-AA+HLA-B(-) vs. KIR Bx+HLA-B(+) and KIR-AA+HLA-B(o)vs. KIR Bx+HLA-B(+). The relationship of KIR/HLA host genetics with slow HIV disease progression in southern Brazil may be useful for vaccine developers, epidemiologists, and clinicians.

  6. Anal Human Papillomavirus Genotype Distribution in HIV-Infected Men Who Have Sex with Men by Geographical Origin, Age, and Cytological Status in a Spanish Cohort

    PubMed Central

    Torres, Montserrat; González, Cristina; del Romero, Jorge; Viciana, Pompeyo; Ocampo, Antonio; Rodríguez-Fortúnez, Patricia; Masiá, Mar; Blanco, José Ramón; Portilla, Joaquín; Rodríguez, Carmen; Hernández-Novoa, Beatriz; del Amo, Julia

    2013-01-01

    Knowledge of human papillomavirus (HPV) type distribution in populations at risk for anal cancer is needed. Here, we describe the anal HPV genotype distribution in a large Spanish cohort (Cohort of the Spanish HIV Research Network HPV [CoRIS-HPV]) of HIV-positive men who have sex with men (MSM) according to geographical origin, age, and cytological status. A cross-sectional analysis of baseline data from 1,439 HIV-infected MSM (2007 to 2012) was performed. Anal HPV genotyping was performed using the Linear Array HPV genotyping test. Descriptive analyses of subject characteristics, prevalences, and 95% confidence intervals (CI) were performed. The global prevalences of HPV, high-risk HPV (HR-HPV), and low-risk HPV (LR-HPV) types were 95.8%, 83.0%, and 72.7%, respectively. Among the HR-HPV types, HPV16 was the most common, followed by HPV59, -39, -51, -18, and -52. The prevalence of multiple HR-HPV infections was 58.5%. There were no differences in the crude analyses between Spanish and Latin-American MSM for most HPV types, and a peak in prevalence for most HPV types was seen in patients in their late thirties. Globally and by specific HPV groups, men with abnormal anal cytologies had a higher prevalence of infection than those with normal cytologies. This study has the largest number of HIV-positive MSM with HPV genotype data analyzed according to cytological status as far as we know. The information gained from this study can help with the design of anal cancer prevention strategies in HIV-positive patients. PMID:23966501

  7. Anal human papillomavirus genotype distribution in HIV-infected men who have sex with men by geographical origin, age, and cytological status in a Spanish cohort.

    PubMed

    Torres, Montserrat; González, Cristina; del Romero, Jorge; Viciana, Pompeyo; Ocampo, Antonio; Rodríguez-Fortúnez, Patricia; Masiá, Mar; Blanco, José Ramón; Portilla, Joaquín; Rodríguez, Carmen; Hernández-Novoa, Beatriz; del Amo, Julia; Ortiz, Marta

    2013-11-01

    Knowledge of human papillomavirus (HPV) type distribution in populations at risk for anal cancer is needed. Here, we describe the anal HPV genotype distribution in a large Spanish cohort (Cohort of the Spanish HIV Research Network HPV [CoRIS-HPV]) of HIV-positive men who have sex with men (MSM) according to geographical origin, age, and cytological status. A cross-sectional analysis of baseline data from 1,439 HIV-infected MSM (2007 to 2012) was performed. Anal HPV genotyping was performed using the Linear Array HPV genotyping test. Descriptive analyses of subject characteristics, prevalences, and 95% confidence intervals (CI) were performed. The global prevalences of HPV, high-risk HPV (HR-HPV), and low-risk HPV (LR-HPV) types were 95.8%, 83.0%, and 72.7%, respectively. Among the HR-HPV types, HPV16 was the most common, followed by HPV59, -39, -51, -18, and -52. The prevalence of multiple HR-HPV infections was 58.5%. There were no differences in the crude analyses between Spanish and Latin-American MSM for most HPV types, and a peak in prevalence for most HPV types was seen in patients in their late thirties. Globally and by specific HPV groups, men with abnormal anal cytologies had a higher prevalence of infection than those with normal cytologies. This study has the largest number of HIV-positive MSM with HPV genotype data analyzed according to cytological status as far as we know. The information gained from this study can help with the design of anal cancer prevention strategies in HIV-positive patients.

  8. Genotypic Prediction of Co-receptor Tropism of HIV-1 Subtypes A and C.

    PubMed

    Riemenschneider, Mona; Cashin, Kieran Y; Budeus, Bettina; Sierra, Saleta; Shirvani-Dastgerdi, Elham; Bayanolhagh, Saeed; Kaiser, Rolf; Gorry, Paul R; Heider, Dominik

    2016-01-01

    Antiretroviral treatment of Human Immunodeficiency Virus type-1 (HIV-1) infections with CCR5-antagonists requires the co-receptor usage prediction of viral strains. Currently available tools are mostly designed based on subtype B strains and thus are in general not applicable to non-B subtypes. However, HIV-1 infections caused by subtype B only account for approximately 11% of infections worldwide. We evaluated the performance of several sequence-based algorithms for co-receptor usage prediction employed on subtype A V3 sequences including circulating recombinant forms (CRFs) and subtype C strains. We further analysed sequence profiles of gp120 regions of subtype A, B and C to explore functional relationships to entry phenotypes. Our analyses clearly demonstrate that state-of-the-art algorithms are not useful for predicting co-receptor tropism of subtype A and its CRFs. Sequence profile analysis of gp120 revealed molecular variability in subtype A viruses. Especially, the V2 loop region could be associated with co-receptor tropism, which might indicate a unique pattern that determines co-receptor tropism in subtype A strains compared to subtype B and C strains. Thus, our study demonstrates that there is a need for the development of novel algorithms facilitating tropism prediction of HIV-1 subtype A to improve effective antiretroviral treatment in patients. PMID:27126912

  9. miRNA Profiles of Monocyte-Lineage Cells Are Consistent with Complicated Roles in HIV-1 Restriction

    PubMed Central

    Sisk, Jeanne M.; Clements, Janice E.; Witwer, Kenneth W.

    2012-01-01

    Long-lived HIV-1 reservoirs include tissue macrophages. Monocyte-derived macrophages are more susceptible to infection and more permissive to HIV-1 replication than monocytes for reasons that may include the effects of different populations of miRNAs in these two cell classes. Specifically, miRs-28-3p, -150, -223, -198, and -382 exert direct or indirect negative effects on HIV-1 and are reportedly downmodulated during monocyte-to-macrophage differentiation. Here, new experimental results are presented along with reviews and analysis of published studies and publicly available datasets, supporting a broader role of miRNAs in HIV-1 restriction than would be suggested by a simple and uniform downregulation of anti-HIV miRNAs during monocyte-to-macrophage differentiation. Although miR-223 is downregulated in macrophages, other putatively antiviral miRNAs are more abundant in macrophages than in monocytes or are rare and/or variably present in both cell classes. Our analyses point to the need for further studies to determine miRNA profiles of monocytes and macrophages, including classic and newly identified subpopulations; examine the sensitivity of miRNA profiling to cell isolation and differentiation protocols; and characterize rigorously the antiviral effects of previously reported and novel predicted miRNA-HIV-1 interactions in cell-specific contexts. PMID:23202444

  10. HIV-1 reverse transcriptase specifically interacts with the anticodon domain of its cognate primer tRNA.

    PubMed Central

    Barat, C; Lullien, V; Schatz, O; Keith, G; Nugeyre, M T; Grüninger-Leitch, F; Barré-Sinoussi, F; LeGrice, S F; Darlix, J L

    1989-01-01

    The virion cores of the replication competent type 1 human immunodeficiency virus (HIV-1), a retrovirus, contain and RNA genome associated with nucleocapsid (NC) and reverse transcriptase (RT p66/p51) molecules. In vitro reconstructions of these complexes with purified components show that NC is required for efficient annealing of the primer tRNALys,3. In the absence of NC, HIV-1 RT is unable to retrotranscribe the viral RNA template from the tRNA primer. We demonstrate that the HIV-1 RT p66/p51 specifically binds to its cognate primer tRNALys,3 even in the presence of a 100-fold molar excess of other tRNAs. Cross-linking analysis of this interaction locates the contact site to a region within the heavily modified anti-codon domain of tRNALys,3. Images PMID:2479543

  11. Does the HIV-1 primer activation signal interact with tRNA3Lys during the initiation of reverse transcription?

    PubMed Central

    Goldschmidt, Valérie; Ehresmann, Chantal; Ehresmann, Bernard; Marquet, Roland

    2003-01-01

    Reverse transcription of HIV-1 RNA is primed by a tRNA3Lys molecule bound at the primer binding site (PBS). Complex intermolecular interactions were proposed between tRNA3Lys and the RNA of the HIV-1 Mal isolate. Recently, an alternative interaction was proposed between the TΨC stem of tRNA3Lys and a primer activation signal (PAS) of the Lai and Hxb2 RNAs, suggesting major structural variations in the reverse transcription complex of different HIV-1 strains. Here, we analyzed mutants of the Hxb2 RNA that prevent the interaction between the PAS and tRNA3Lys or/and a complementary sequence in the viral RNA. We compared the kinetics of reverse transcription of the wild type and mutant Hxb2 RNAs, using either tRNA3Lys or an 18mer oligoribonucleotide complementary to the PBS, which cannot interact with the PAS, as primers. We also used chemical probing to test the structure of the mutant and wild type RNAs, as well as the complex formed between the later RNA and tRNA3Lys. These experiments, together with the analysis of long term replication data of mutant viruses obtained by C. Morrow and coworkers (Birmingham, USA) that use alternate tRNAs as primers, strongly suggest that the interaction between the Hxb2 PAS and tRNA3Lys does not exist. Instead, the effects of the vRNA mutations on reverse transcription seem to be linked to incorrect folding of the mutant RNAs. PMID:12560480

  12. Health care industries' perspective of viral load assays: the VERSANT HIV-1 RNA 3.0 assay.

    PubMed

    Elbeik, Tarek; Loftus, Richard A; Beringer, Scott

    2002-05-01

    This article will compare the VERSANT HIV-1 RNA 3.0 (bDNA 3.0) assay with other HIV-1 viral load assays, particularly the AMPLICOR HIV-1 MONITOR (Amplicor 1.5), the industry standard. The discussion will cover the history of viral load assay development and challenges to the field. It will finish with a description of the evolving markets for viral load assays in the developing world and the impact of variations in the different assays on their ability to reach those markets and patient populations.

  13. The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA

    PubMed Central

    Booth, David S; Cheng, Yifan; Frankel, Alan D

    2014-01-01

    The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-RanGTP nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression. DOI: http://dx.doi.org/10.7554/eLife.04121.001 PMID:25486595

  14. The export receptor Crm1 forms a dimer to promote nuclear export of HIV RNA.

    PubMed

    Booth, David S; Cheng, Yifan; Frankel, Alan D

    2014-12-08

    The HIV Rev protein routes viral RNAs containing the Rev Response Element (RRE) through the Crm1 nuclear export pathway to the cytoplasm where viral proteins are expressed and genomic RNA is delivered to assembling virions. The RRE assembles a Rev oligomer that displays nuclear export sequences (NESs) for recognition by the Crm1-Ran(GTP) nuclear receptor complex. Here we provide the first view of an assembled HIV-host nuclear export complex using single-particle electron microscopy. Unexpectedly, Crm1 forms a dimer with an extensive interface that enhances association with Rev-RRE and poises NES binding sites to interact with a Rev oligomer. The interface between Crm1 monomers explains differences between Crm1 orthologs that alter nuclear export and determine cellular tropism for viral replication. The arrangement of the export complex identifies a novel binding surface to possibly target an HIV inhibitor and may point to a broader role for Crm1 dimerization in regulating host gene expression.

  15. Predicting the duration of antiviral treatment needed to suppress plasma HIV-1 RNA

    PubMed Central

    Rizzardi, G. Paolo; De Boer, Rob J.; Hoover, Shelley; Tambussi, Giuseppe; Chapuis, Aude; Halkic, Nermin; Bart, Pierre-Alexandre; Miller, Veronica; Staszewski, Schlomo; Notermans, Daan W.; Perrin, Luc; Fox, Cecil H.; Lange, Joep M.A.; Lazzarin, Adriano; Pantaleo, Giuseppe

    2000-01-01

    Effective therapeutic interventions and clinical care of adults infected with HIV-1 require an understanding of factors that influence time of response to antiretroviral therapy. We have studied a cohort of 118 HIV-1–infected subjects naive to antiretroviral therapy and have correlated the time of response to treatment with a series of virological and immunological measures, including levels of viral load in blood and lymph node, percent of CD4 T cells in lymph nodes, and CD4 T-cell count in blood at study entry. Suppression of viremia below the limit of detection, 50 HIV-1 RNA copies/mL of plasma, served as a benchmark for a successful virological response. We employed these correlations to predict the length of treatment required to attain a virological response in each patient. Baseline plasma viremia emerged as the factor most tightly correlated with the duration of treatment required, allowing us to estimate the required time as a function of this one measure. PMID:10727446

  16. Visualising single molecules of HIV-1 and miRNA nucleic acids

    PubMed Central

    2013-01-01

    Background The scarcity of certain nucleic acid species and the small size of target sequences such as miRNA, impose a significant barrier to subcellular visualization and present a major challenge to cell biologists. Here, we offer a generic and highly sensitive visualization approach (oligo fluorescent in situ hybridization, O-FISH) that can be used to detect such nucleic acids using a single-oligonucleotide probe of 19–26 nucleotides in length. Results We used O-FISH to visualize miR146a in human and avian cells. Furthermore, we reveal the sensitivity of O-FISH detection by using a HIV-1 model system to show that as little as 1–2 copies of nucleic acids can be detected in a single cell. We were able to discern newly synthesized viral cDNA and, moreover, observed that certain HIV RNA sequences are only transiently available for O-FISH detection. Conclusions Taken together, these results suggest that the O-FISH method can potentially be used for in situ probing of, as few as, 1–2 copies of nucleic acid and, additionally, to visualize small RNA such as miRNA. We further propose that the O-FISH method could be extended to understand viral function by probing newly transcribed viral intermediates; and discern the localisation of nucleic acids of interest. Additionally, interrogating the conformation and structure of a particular nucleic acid in situ might also be possible, based on the accessibility of a target sequence. PMID:23590669

  17. The bulge region of HIV-1 TAR RNA binds metal ions in solution.

    PubMed

    Olejniczak, Mikołaj; Gdaniec, Zofia; Fischer, Artur; Grabarkiewicz, Tomasz; Bielecki, Lukasz; Adamiak, Ryszard W

    2002-10-01

    Binding of Mg2+, Ca2+ and Co(NH3)6(3+) ions to the HIV-1 TAR RNA in solution was analysed by 19F NMR spectroscopy, metal ion-induced RNA cleavages and Brownian dynamics (BD) simulations. Chemically synthesised 29mer oligoribonucleotides of the TAR sequence labelled with 5-fluorouridine (FU) were used for 19F NMR-monitored metal ion titration. The chemical shift changes of fluorine resonances FU-23, FU-25 and FU-40 upon titration with Mg2+ and Ca2+ ions indicated specific, although weak, binding at the bulge region with the dissociation constants (K(d)) of 0.9 +/- 0.6 and 2.7 +/- 1.7 mM, respectively. Argininamide, inducing largest (19)F chemical shifts changes at FU-23, was used as a reference ligand (K(d) = 0.3 +/- 0.1 mM). In the Pb2+-induced TAR RNA cleavage experiment, strong and selective cleavage of the C24-U25 phosphodiester bond was observed, while Mg2+ and Ca2+ induced cuts at all 3-nt residues of the bulge. The inhibition of Pb2+-specific TAR cleavage by di- and trivalent metal ions revealed a binding specificity [in the order Co(NH3)6(3+) > Mg2+ > Ca2+] at the bulge site. A BD simulation search of potential magnesium ion sites within the NMR structure of HIV-1 TAR RNA was conducted on a set of 20 conformers (PDB code 1ANR). For most cases, the bulge region was targeted by magnesium cations.

  18. Hepatitis B Virus Sub-genotype A1 Infection Is Characterized by High Replication Levels and Rapid Emergence of Drug Resistance in HIV-Positive Adults Receiving First-line Antiretroviral Therapy in Malawi

    PubMed Central

    Aoudjane, Samir; Chaponda, Mas; González del Castillo, Antonio Adrián; O'Connor, Jemma; Noguera, Marc; Beloukas, Apostolos; Hopkins, Mark; Khoo, Saye; van Oosterhout, Joep J.; Geretti, Anna Maria

    2014-01-01

    Background. It has been proposed that hepatitis B virus (HBV) sub-genotype A1 infections have mild outcomes and a low risk of drug-resistance among patients infected with human immunodeficiency virus (HIV) receiving lamivudine-containing antiretroviral therapy (ART) without tenofovir in Africa. Methods. The virologic expression of HBV sub-genotype A1 coinfection was studied over 12 months in HIV-positive adults starting stavudine/lamivudine/nevirapine in Malawi, using Sanger, deep, clonal, and single full-genome sequencing for the sensitive characterization of HBV resistance-associated mutations (RAMs). Results. Among 1117 subjects, 133 (12%) tested HBsAg-positive. After starting ART, retention rates were 96/133 (72%) at 6 months and 54/133 (41%) at 12 months. Based upon the last available follow-up, 92/96 (96%) subjects achieved HIV-1 RNA <40 copies/mL, 48/96 (50%) showed HBV DNA <14 IU/mL, and 24/96 (25%) acquired HBV RAMs. At 6 months, M204I was detected in 8/46 (17%) and 16/17 (94%) subjects using Sanger and deep sequencing, respectively. At 12 months, all viremic patients had multiple resistance and compensatory mutations coexisting on the same HBV genomes. Comparing HBeA-positive (67/133, 50%) with HBeAg-negative subjects, 64/67 (96%) vs 35/66 (55%) showed baseline HBV DNA >2000 IU/mL (P = .0006), 39/47 (17%) vs 9/49 (82%) had persistent HBV DNA detection during follow-up (P < .0001), and 23/47 (49%) vs 2/49 (4%) acquired HBV RAMs (P < .0001). Baseline HBV DNA levels were median 8.1 vs 5.3 log10 IU/mL in subjects with vs those without treatment-emergent RAMs (P < .0001). Conclusions. HBV sub-genotype A1 infections showed a severe virologic expression in HIV-positive Malawians. The findings strengthen the urgency of interventions to improve ascertainment and management of chronic hepatitis B in the region. PMID:25100867

  19. HIV-1 Vif binds to APOBEC3G mRNA and inhibits its translation.

    PubMed

    Mercenne, Gaëlle; Bernacchi, Serena; Richer, Delphine; Bec, Guillaume; Henriet, Simon; Paillart, Jean-Christophe; Marquet, Roland

    2010-01-01

    The HIV-1 viral infectivity factor (Vif) allows productive infection of non-permissive cells (including most natural HIV-1 targets) by counteracting the cellular cytosine deaminases APOBEC-3G (hA3G) and hA3F. The Vif-induced degradation of these restriction factors by the proteasome has been extensively studied, but little is known about the translational repression of hA3G and hA3F by Vif, which has also been proposed to participate in Vif function. Here, we studied Vif binding to hA3G mRNA and its role in translational repression. Filter binding assays and fluorescence titration curves revealed that Vif tightly binds to hA3G mRNA. Vif overall binding affinity was higher for the 3'UTR than for the 5'UTR, even though this region contained at least one high affinity Vif binding site (apparent K(d) = 27 +/- 6 nM). Several Vif binding sites were identified in 5' and 3'UTRs using RNase footprinting. In vitro translation evidenced that Vif inhibited hA3G translation by two mechanisms: a main time-independent process requiring the 5'UTR and an additional time-dependent, UTR-independent process. Results using a Vif protein mutated in the multimerization domain suggested that the molecular mechanism of translational control is more complicated than a simple physical blockage of scanning ribosomes.

  20. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA.

    PubMed

    Sarafianos, S G; Das, K; Tantillo, C; Clark, A D; Ding, J; Whitcomb, J M; Boyer, P L; Hughes, S H; Arnold, E

    2001-03-15

    We have determined the 3.0 A resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The 'RNase H primer grip', consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual 'unzipping' of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage.

  1. Crystal structure of HIV-1 reverse transcriptase in complex with a polypurine tract RNA:DNA

    PubMed Central

    Sarafianos, Stefan G.; Das, Kalyan; Tantillo, Chris; Clark, Arthur D.; Ding, Jianping; Whitcomb, Jeannette M.; Boyer, Paul L.; Hughes, Stephen H.; Arnold, Edward

    2001-01-01

    We have determined the 3.0 Å resolution structure of wild-type HIV-1 reverse transcriptase in complex with an RNA:DNA oligonucleotide whose sequence includes a purine-rich segment from the HIV-1 genome called the polypurine tract (PPT). The PPT is resistant to ribonuclease H (RNase H) cleavage and is used as a primer for second DNA strand synthesis. The ‘RNase H primer grip’, consisting of amino acids that interact with the DNA primer strand, may contribute to RNase H catalysis and cleavage specificity. Cleavage specificity is also controlled by the width of the minor groove and the trajectory of the RNA:DNA, both of which are sequence dependent. An unusual ‘unzipping’ of 7 bp occurs in the adenine stretch of the PPT: an unpaired base on the template strand takes the base pairing out of register and then, following two offset base pairs, an unpaired base on the primer strand re-establishes the normal register. The structural aberration extends to the RNase H active site and may play a role in the resistance of PPT to RNase H cleavage. PMID:11250910

  2. HIV Viral RNA Extraction in Wax Immiscible Filtration Assisted by Surface Tension (IFAST) Devices

    PubMed Central

    Berry, Scott M.; LaVanway, Alex J.; Pezzi, Hannah M.; Guckenberger, David J.; Anderson, Meghan A.; Loeb, Jennifer M.; Beebe, David J.

    2015-01-01

    The monitoring of viral load is critical for proper management of antiretroviral therapy for HIV-positive patients. Unfortunately, in the developing world, significant economic and geographical barriers exist, limiting access to this test. The complexity of current viral load assays makes them expensive and their access limited to advanced facilities. We attempted to address these limitations by replacing conventional RNA extraction, one of the essential processes in viral load quantitation, with a simplified technique known as immiscible filtration assisted by surface tension (IFAST). Furthermore, these devices were produced via the embossing of wax, enabling local populations to produce and dispose of their own devices with minimal training or infrastructure, potentially reducing the total assay cost. In addition, IFAST can be used to reduce cold chain dependence during transportation. Viral RNA extracted from raw samples stored at 37°C for 1 week exhibited nearly complete degradation. However, IFAST-purified RNA could be stored at 37°C for 1 week without significant loss. These data suggest that RNA isolated at the point of care (eg, in a rural clinic) via IFAST could be shipped to a central laboratory for quantitative RT-PCR without a cold chain. Using this technology, we have demonstrated accurate and repeatable measurements of viral load on samples with as low as 50 copies per milliliter of sample. PMID:24613822

  3. The Influence of Genotype and Environment on Small RNA Profiles in Grapevine Berry

    PubMed Central

    Paim Pinto, Daniela Lopes; Brancadoro, Lucio; Dal Santo, Silvia; De Lorenzis, Gabriella; Pezzotti, Mario; Meyers, Blake C.; Pè, Mario E.; Mica, Erica

    2016-01-01

    Understanding the molecular mechanisms involved in the interaction between the genetic composition and the environment is crucial for modern viticulture. We approached this issue by focusing on the small RNA transcriptome in grapevine berries of the two varieties Cabernet Sauvignon and Sangiovese, growing in adjacent vineyards in three different environments. Four different developmental stages were studied and a total of 48 libraries of small RNAs were produced and sequenced. Using a proximity-based pipeline, we determined the general landscape of small RNAs accumulation in grapevine berries. We also investigated the presence of known and novel miRNAs and analyzed their accumulation profile. The results showed that the distribution of small RNA-producing loci is variable between the two cultivars, and that the level of variation depends on the vineyard. Differently, the profile of miRNA accumulation mainly depends on the developmental stage. The vineyard in Riccione maximizes the differences between the varieties, promoting the production of more than 1000 specific small RNA loci and modulating their expression depending on the cultivar and the maturation stage. In total, 89 known vvi-miRNAs and 33 novel vvi-miRNA candidates were identified in our samples, many of them showing the accumulation profile modulated by at least one of the factors studied. The in silico prediction of miRNA targets suggests their involvement in berry development and in secondary metabolites accumulation such as anthocyanins and polyphenols. PMID:27761135

  4. The Contribution of Viral Genotype to Plasma Viral Set-Point in HIV Infection

    PubMed Central

    Hodcroft, Emma; Hadfield, Jarrod D.; Fearnhill, Esther; Phillips, Andrew; Dunn, David; O'Shea, Siobhan; Pillay, Deenan; Leigh Brown, Andrew J.

    2014-01-01

    Disease progression in HIV-infected individuals varies greatly, and while the environmental and host factors influencing this variation have been widely investigated, the viral contribution to variation in set-point viral load, a predictor of disease progression, is less clear. Previous studies, using transmission-pairs and analysis of phylogenetic signal in small numbers of individuals, have produced a wide range of viral genetic effect estimates. Here we present a novel application of a population-scale method based in quantitative genetics to estimate the viral genetic effect on set-point viral load in the UK subtype B HIV-1 epidemic, based on a very large data set. Analyzing the initial viral load and associated pol sequence, both taken before anti-retroviral therapy, of 8,483 patients, we estimate the proportion of variance in viral load explained by viral genetic effects to be 5.7% (CI 2.8–8.6%). We also estimated the change in viral load over time due to selection on the virus and environmental effects to be a decline of 0.05 log10 copies/mL/year, in contrast to recent studies which suggested a reported small increase in viral load over the last 20 years might be due to evolutionary changes in the virus. Our results suggest that in the UK epidemic, subtype B has a small but significant viral genetic effect on viral load. By allowing the analysis of large sample sizes, we expect our approach to be applicable to the estimation of the genetic contribution to traits in many organisms. PMID:24789308

  5. The CCL3L1-CCR5 genotype influences the development of AIDS, but not HIV susceptibility or the response to HAART

    SciTech Connect

    Bhattacharya, Tanmoy; Stanton, Jennifer; Kim, Eun - Young; Kunstman, Kevin; Phair, John; Jacobson, Lisa P; Wolinsky, Steven M

    2008-01-01

    A selective advantage against infectious diseases such as HIV/AIDS is associated with differences in the genes relevant to immunity and virus replication. The CC chemokine receptor 5 (CCR5), the principal coreceptor for HIV, and its chemokine ligands, including CCL3L1, influences the CD4+ target cells susceptibility to infection. The CCL3L1 gene is in a region of segmental duplication on the q-arm of human chromosome 17. Increased numbers of CCL3L1 gene copies that affect the gene expression phenotype might have substantial protective effects. Here we show that the population-specific CCL3L1 gene copy number and the CCR5 {Delta}32 protein-inactivating deletion that categorizes the CCL3L1-CCR5 genotype do not influence HIV/AIDS susceptibility or the robustness of immune recovery after the initiation of highly active antiretroviral therapy (HAART).

  6. Evolutionary constraints acting on DDX3X protein potentially interferes with Rev-mediated nuclear export of HIV-1 RNA.

    PubMed

    Sharma, Deepak; Bhattacharya, Jayanta

    2010-01-01

    Differential host-pathogen interactions direct viral replication in infected cells. In HIV-1 infected cells, nuclear export of viral RNA transcripts into cellular cytoplasm is governed by interaction of HIV-1 Rev, Exportin-1 (CRM-1) and DDX3X. Knock down of DDX3X has been shown to drastically impair HIV replication. Here we show that evolutionary forces are responsible for demarking previously unidentified critical functionally important residues on the surface of DDX3X. Using computational approaches, we show that these functional residues, depending on their location, are capable of regulating ATPase and RNA helicase functions of DDX3X. The potential of these residues in designing better blockers against HIV-1 replication was also assessed. Also, using stepwise docking simulations, we could identify DDX3X-CRM-1 interface and its critical functional residues. Our data would help explain the role of DDX3X in HIV-1 Rev function with potential to design new intervention strategies against HIV-1 replication.

  7. Plasma HIV-2 RNA According to CD4 Count Strata among HIV-2-Infected Adults in the IeDEA West Africa Collaboration

    PubMed Central

    Ekouévi, Didier K.; Avettand-Fènoël, Véronique; Tchounga, Boris K.; Coffie, Patrick A.; Sawadogo, Adrien; Minta, Daouda; Minga, Albert; Eholie, Serge P.; Plantier, Jean-Christophe; Damond, Florence; Dabis, François; Rouzioux, Christine

    2015-01-01

    Background Plasma HIV-1 RNA monitoring is one of the standard tests for the management of HIV-1 infection. While HIV-1 RNA can be quantified using several commercial tests, no test has been commercialized for HIV-2 RNA quantification. We studied the relationship between plasma HIV-2 viral load (VL) and CD4 count in West African patients who were either receiving antiretroviral therapy (ART) or treatment-naïve. Method A cross sectional survey was conducted among HIV-2-infected individuals followed in three countries in West Africa from March to December 2012. All HIV-2 infected-patients who attended one of the participating clinics were proposed a plasma HIV-2 viral load measurement. HIV-2 RNA was quantified using the new ultrasensitive in-house real-time PCR assay with a detection threshold of 10 copies/ mL (cps/mL). Results A total of 351 HIV-2-infected individuals participated in this study, of whom 131 (37.3%) were treatment naïve and 220 (62.7%) had initiated ART. Among treatment-naïve patients, 60 (46.5%) had undetectable plasma HIV-2 viral load (<10 cps/mL), it was detectable between 10-100 cps/mL in 35.8%, between 100-1000 cps/mL in 11.7% and >1000 cps/mL in 6.0% of the patients. Most of the treatment-naïve patients (70.2%) had CD4-T cell count ≥500 cells/mm3 and 43 (46.7%) of these patients had a detectable VL (≥10 cps/mL). Among the 220 patients receiving ART, the median CD4-T cell count rose from 231 to 393 cells/mm3 (IQR [259-561]) after a median follow-up duration of 38 months and 145 (66.0%) patients had CD4-T cell count ≤ 500 cells/mm3 with a median viral load of 10 cps/mL (IQR [10-33]). Seventy five (34.0%) patients had CD4-T cell count ≥ 500 cells/mm3, among them 14 (18.7%) had a VL between 10-100 cps/mL and 2 (2.6%) had VL >100 cps/mL. Conclusion This study suggests that the combination of CD4-T cell count and ultrasensitive HIV-2 viral load quantification with a threshold of 10 cps/mL, could improve ART initiation among treatment na

  8. Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function

    PubMed Central

    Urcuqui-Inchima, Silvio; Castaño, Maria Eugenia; Hernandez-Verdun, Danièle; St-Laurent, Georges; Kumar, Ajit

    2006-01-01

    Background The HIV Rev protein is known to facilitate export of incompletely spliced and unspliced viral transcripts to the cytoplasm, a necessary step in virus life cycle. The Rev-mediated nucleo-cytoplasmic transport of nascent viral transcripts, dependents on interaction of Rev with the RRE RNA structural element present in the target RNAs. The C-terminal variant of dsRNA-binding nuclear protein 90 (NF90ctv) has been shown to markedly attenuate viral replication in stably transduced HIV-1 target cell line. Here we examined a mechanism of interference of viral life cycle involving Rev-NF90ctv interaction. Results Since Rev:RRE complex formations depend on protein:RNA and protein:protein interactions, we investigated whether the expression of NF90ctv might interfere with Rev-mediated export of RRE-containing transcripts. When HeLa cells expressed both NF90ctv and Rev protein, we observed that NF90ctv inhibited the Rev-mediated RNA transport. In particular, three regions of NF90ctv protein are involved in blocking Rev function. Moreover, interaction of NF90ctv with the RRE RNA resulted in the expression of a reporter protein coding sequences linked to the RRE structure. Moreover, Rev influenced the subcellular localization of NF90ctv, and this process is leptomycin B sensitive. Conclusion The dsRNA binding protein, NF90ctv competes with HIV Rev function at two levels, by competitive protein:protein interaction involving Rev binding to specific domains of NF90ctv, as well as by its binding to the RRE-RNA structure. Our results are consistent with a model of Rev-mediated HIV-1 RNA export that envisions Rev-multimerization, a process interrupted by NF90ctv. PMID:17125513

  9. Types of HIV/AIDS Antiretroviral Drugs

    MedlinePlus

    ... reverse transcriptase (RT) from converting single-stranded HIV RNA into double-stranded HIV DNA―a process called ... RT, interfering with its ability to convert HIV RNA into HIV DNA Integrase Inhibitors block the HIV ...

  10. From RNA-seq to large-scale genotyping - genomics resources for rye (Secale cereale L.)

    PubMed Central

    2011-01-01

    Background The improvement of agricultural crops with regard to yield, resistance and environmental adaptation is a perpetual challenge for both breeding and research. Exploration of the genetic potential and implementation of genome-based breeding strategies for efficient rye (Secale cereale L.) cultivar improvement have been hampered by the lack of genome sequence information. To overcome this limitation we sequenced the transcriptomes of five winter rye inbred lines using Roche/454 GS FLX technology. Results More than 2.5 million reads were assembled into 115,400 contigs representing a comprehensive rye expressed sequence tag (EST) resource. From sequence comparisons 5,234 single nucleotide polymorphisms (SNPs) were identified to develop the Rye5K high-throughput SNP genotyping array. Performance of the Rye5K SNP array was investigated by genotyping 59 rye inbred lines including the five lines used for sequencing, and five barley, three wheat, and two triticale accessions. A balanced distribution of allele frequencies ranging from 0.1 to 0.9 was observed. Residual heterozygosity of the rye inbred lines varied from 4.0 to 20.4% with higher average heterozygosity in the pollen compared to the seed parent pool. Conclusions The established sequence and molecular marker resources will improve and promote genetic and genomic research as well as genome-based breeding in rye. PMID:21951788

  11. Development and application of a broadly sensitive dried-blood-spot-based genotyping assay for global surveillance of HIV-1 drug resistance.

    PubMed

    Yang, Chunfu; McNulty, Amanda; Diallo, Karidia; Zhang, Jing; Titanji, Boghuma; Kassim, Sidibe; Wadonda-Kabondo, Nellie; Aberle-Grasse, John; Kibuka, Tabitha; Ndumbe, Peter M; Vedapuri, Shanmugam; Zhou, Zhiyong; Chilima, Benson; Nkengasong, John N

    2010-09-01

    As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.

  12. HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells.

    PubMed

    Reeder, Jonathan E; Kwak, Youn-Tae; McNamara, Ryan P; Forst, Christian V; D'Orso, Iván

    2015-01-01

    HIV encodes Tat, a small protein that facilitates viral transcription by binding an RNA structure (trans-activating RNA [TAR]) formed on nascent viral pre-messenger RNAs. Besides this well-characterized mechanism, Tat appears to modulate cellular transcription, but the target genes and molecular mechanisms remain poorly understood. We report here that Tat uses unexpected regulatory mechanisms to reprogram target immune cells to promote viral replication and rewire pathways beneficial for the virus. Tat functions through master transcriptional regulators bound at promoters and enhancers, rather than through cellular 'TAR-like' motifs, to both activate and repress gene sets sharing common functional annotations. Despite the complexity of transcriptional regulatory mechanisms in the cell, Tat precisely controls RNA polymerase II recruitment and pause release to fine-tune the initiation and elongation steps in target genes. We propose that a virus with a limited coding capacity has optimized its genome by evolving a small but 'multitasking' protein to simultaneously control viral and cellular transcription.

  13. HIV Tat controls RNA Polymerase II and the epigenetic landscape to transcriptionally reprogram target immune cells

    PubMed Central

    McNamara, Ryan P; Forst, Christian V; D'Orso, Iván

    2015-01-01

    HIV encodes Tat, a small protein that facilitates viral transcription by binding an RNA structure (trans-activating RNA [TAR]) formed on nascent viral pre-messenger RNAs. Besides this well-characterized mechanism, Tat appears to modulate cellular transcription, but the target genes and molecular mechanisms remain poorly understood. We report here that Tat uses unexpected regulatory mechanisms to reprogram target immune cells to promote viral replication and rewire pathways beneficial for the virus. Tat functions through master transcriptional regulators bound at promoters and enhancers, rather than through cellular ‘TAR-like’ motifs, to both activate and repress gene sets sharing common functional annotations. Despite the complexity of transcriptional regulatory mechanisms in the cell, Tat precisely controls RNA polymerase II recruitment and pause release to fine-tune the initiation and elongation steps in target genes. We propose that a virus with a limited coding capacity has optimized its genome by evolving a small but ‘multitasking’ protein to simultaneously control viral and cellular transcription. DOI: http://dx.doi.org/10.7554/eLife.08955.001 PMID:26488441

  14. Cervical human papillomavirus infection among young women engaged in sex work in Phnom Penh, Cambodia: prevalence, genotypes, risk factors and association with HIV infection

    PubMed Central

    2012-01-01

    Background Although cervical cancer is the leading cancer in Cambodia, most women receive no routine screening for cervical cancer and few treatment options exist. Moreover, nothing is known regarding the prevalence of cervical HPV or the genotypes present among women in the country. Young sexually active women, especially those with multiple sex partners are at highest risk of HPV infection. We examine the prevalence and genotypes of cervical HPV, as well as the associated risk factors among young women engaged in sex work in Phnom Penh, Cambodia. Methods We conducted a cross-sectional study among 220 young women (15–29 years) engaged in sex work in different venues including brothels or entertainment establishments, and on a freelance basis in streets, parks and private apartments. Cervical specimens were collected using standard cytobrush technique. HPV DNA was tested for by polymerase chain reaction (PCR) and genotyping using type-specific probes for 29 individual HPV types, as well as for a mixture of 10 less common HPV types. All participants were also screened for HIV status using blood samples. Multivariate logistic regression analyses were conducted to assess risk factors for any or multiple HPV infection. Results The prevalence of cervical HPV 41.1%. HPV 51 and 70 were the most common (5.0%), followed by 16 (4.6%), 71 (4.1%) and 81 (3.7%). Thirty-six women (16.4%) were infected with multiple genotypes and 23.3% were infected with at least one oncogenic HPV type. In multivariate analyses, having HIV infection and a higher number of sexual partners were associated with cervical HPV infection. Risk factors for infection with multiple genotypes included working as freelance female sex workers (FSW) or in brothels, recent binge use of drugs, high number of sexual partners, and HIV infection. Conclusions This is the first Cambodian study on cervical HPV prevalence and genotypes. We found that HPV infection was common among young FSW, especially among women

  15. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    SciTech Connect

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.

  16. The Interaction of RNA Helicase DDX3 with HIV-1 Rev-CRM1-RanGTP Complex during the HIV Replication Cycle

    PubMed Central

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-01-01

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNA transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP. PMID:25723178

  17. Molecular analysis of the 18S rRNA gene of Cryptosporidium parasites from patients with or without human immunodeficiency virus infections living in Kenya, Malawi, Brazil, the United Kingdom, and Vietnam.

    PubMed

    Gatei, Wangeci; Greensill, Julie; Ashford, Richard W; Cuevas, Luis E; Parry, Christopher M; Cunliffe, Nigel A; Beeching, Nicholas J; Hart, C Anthony

    2003-04-01

    An 840-bp fragment of the 18S rRNA gene was used to identify Cryptosporidium spp. recovered from human immunodeficiency virus (HIV)-infected and -uninfected patients from Kenya, Malawi, Brazil, the United Kingdom, and Vietnam. Initial identification was by Ziehl-Neelsen acid-fast staining. Confirmation was by nested PCR, targeting the most polymorphic region of the 18S rRNA gene. Genotyping was by restriction endonuclease digestion of the PCR product followed by nucleotide sequencing. Among 63 isolates analyzed, four genotypes of Cryptosporidium were identified; 75% of the isolates were of the C. parvum human genotype, while the potentially zoonotic species were of the C. parvum bovine genotype (21.7%), the C. meleagridis genotype (1.6% [one isolate]), and the C. muris genotype (1.6% [one case]). HIV-infected individuals were more likely to have zoonotic genotypes than the HIV-uninfected individuals. Among the C. parvum group, strains clustered distinctly into either human or bovine genotypes regardless of the geographical origin, age, or HIV status of the patients. The intragenotypic variation observed in the C. parvum human genotype was extensive compared to that within the C. parvum bovine genotype group. The variation within genotypes was conserved in all geographical regions regardless of the patients' HIV status. The extensive diversity within genotypes at the 18S rRNA gene locus may limit its application to phylogenetic analyses.

  18. Conformations of flanking bases in HIV-1 RNA DIS kissing complexes studied by molecular dynamics.

    PubMed

    Réblová, Kamila; Fadrná, Eva; Sarzynska, Joanna; Kulinski, Tadeusz; Kulhánek, Petr; Ennifar, Eric; Koca, Jaroslav; Sponer, Jirí

    2007-12-01

    Explicit solvent molecular dynamics simulations (in total almost 800 ns including locally enhanced sampling runs) were applied with different ion conditions and with two force fields (AMBER and CHARMM) to characterize typical geometries adopted by the flanking bases in the RNA kissing-loop complexes. We focus on flanking base positions in multiple x-ray and NMR structures of HIV-1 DIS kissing complexes and kissing complex from the large ribosomal subunit of Haloarcula marismortui. An initial x-ray open conformation of bulged-out bases in HIV-1 DIS complexes, affected by crystal packing, tends to convert to a closed conformation formed by consecutive stretch of four stacked purine bases. This is in agreement with those recent crystals where the packing is essentially avoided. We also observed variants of the closed conformation with three stacked bases, while nonnegligible populations of stacked geometries with bulged-in bases were detected, too. The simulation results reconcile differences in positions of the flanking bases observed in x-ray and NMR studies. Our results suggest that bulged-out geometries are somewhat more preferred, which is in accord with recent experiments showing that they may mediate tertiary contacts in biomolecular assemblies or allow binding of aminoglycoside antibiotics.

  19. Insights into the mechanisms of RNA secondary structure destabilization by the HIV-1 nucleocapsid protein.

    PubMed

    Belfetmi, Anissa; Zargarian, Loussiné; Tisné, Carine; Sleiman, Dona; Morellet, Nelly; Lescop, Ewen; Maskri, Ouerdia; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2016-04-01

    The mature HIV-1 nucleocapsid protein NCp7 (NC) plays a key role in reverse transcription facilitating the two obligatory strand transfers. Several properties contribute to its efficient chaperon activity: preferential binding to single-stranded regions, nucleic acid aggregation, helix destabilization, and rapid dissociation from nucleic acids. However, little is known about the relationships between these different properties, which are complicated by the ability of the protein to recognize particular HIV-1 stem-loops, such as SL1, SL2, and SL3, with high affinity and without destabilizing them. These latter properties are important in the context of genome packaging, during which NC is part of the Gag precursor. We used NMR to investigate destabilization of the full-length TAR (trans activating response element) RNA by NC, which is involved in the first strand transfer step of reverse transcription. NC was used at a low protein:nucleotide (nt) ratio of 1:59 in these experiments. NMR data for the imino protons of TAR identified most of the base pairs destabilized by NC. These base pairs were adjacent to the loops in the upper part of the TAR hairpin rather than randomly distributed. Gel retardation assays showed that conversion from the initial TAR-cTAR complex to the fully annealed form occurred much more slowly at the 1:59 ratio than at the higher ratios classically used. Nevertheless, NC significantly accelerated the formation of the initial complex at a ratio of 1:59.

  20. Repeated HIV-1 resistance genotyping external quality assessments improve virology laboratory performance.

    PubMed

    Descamps, Diane; Delaugerre, Constance; Masquelier, Bernard; Ruffault, Annick; Marcelin, Anne-Geneviève; Izopet, Jacques; Chaix, Marie-Laure; Calvez, Vincent; Brun-Vézinet, Françoise; Costagliola, Dominique

    2006-02-01

    The performance of French virology laboratories belonging to the ANRS network has been assessed annually for 3 years. The performance of these laboratories was compared between the years 2002 and 2003. Ten and 7 coded samples were sent to 38 virology laboratories in 2002 and 45 virology laboratories in 2003, respectively. Each panel of coded samples included at least one HIV-negative control, a pair of duplicate specimens, samples with a wide range of viral loads, and samples with a large number of resistance mutations. The laboratories used their standard sequencing procedures and were asked to report the amino acids at codons associated with resistance mutations, based on the IAS-USA expert panel list. The reference amino acid sequences were defined as those most frequently reported by the participants. The specificity of detection of RT mutations was significantly better in 2003 (99.9%) than in 2002 (99.7%) (P = 0.05). There was no difference between 2002 and 2003 in the specificity of detection of protease mutations (99.6% and 99.8%) or the sensitivity of detection of RT mutations (98.8% and 98.2%). The sensitivity of detection of protease mutations improved significantly between 2002 and 2003 (97.6% and 99.0%, respectively; P = 0.037). The proportion of laboratories reporting fully accurate results, in terms of amplification, specificity, sensitivity, and reproducibility, tended to increase between 2002 and 2003 (P = 0.077). No errors were made by 19% of laboratories in 2002, compared to 42% in 2003. These results show the value of repeated external quality assessments.

  1. Distinct binding interactions of HIV-1 Gag to Psi and non-Psi RNAs: implications for viral genomic RNA packaging.

    PubMed

    Webb, Joseph A; Jones, Christopher P; Parent, Leslie J; Rouzina, Ioulia; Musier-Forsyth, Karin

    2013-08-01

    Despite the vast excess of cellular RNAs, precisely two copies of viral genomic RNA (gRNA) are selectively packaged into new human immunodeficiency type 1 (HIV-1) particles via specific interactions between the HIV-1 Gag and the gRNA psi (ψ) packaging signal. Gag consists of the matrix (MA), capsid, nucleocapsid (NC), and p6 domains. Binding of the Gag NC domain to ψ is necessary for gRNA packaging, but the mechanism by which Gag selectively interacts with ψ is unclear. Here, we investigate the binding of NC and Gag variants to an RNA derived from ψ (Psi RNA), as well as to a non-ψ region (TARPolyA). Binding was measured as a function of salt to obtain the effective charge (Zeff) and nonelectrostatic (i.e., specific) component of binding, Kd(1M). Gag binds to Psi RNA with a dramatically reduced Kd(1M) and lower Zeff relative to TARPolyA. NC, GagΔMA, and a dimerization mutant of Gag bind TARPolyA with reduced Zeff relative to WT Gag. Mutations involving the NC zinc finger motifs of Gag or changes to the G-rich NC-binding regions of Psi RNA significantly reduce the nonelectrostatic component of binding, leading to an increase in Zeff. These results show that Gag interacts with gRNA using different binding modes; both the NC and MA domains are bound to RNA in the case of TARPolyA, whereas binding to Psi RNA involves only the NC domain. Taken together, these results suggest a novel mechanism for selective gRNA encapsidation.

  2. Highly divergent 18S rRNA gene paralogs in a Cryptosporidium genotype from eastern chipmunks (Tamias striatus)1

    PubMed Central

    Stenger, Brianna L.S.; Clark, Mark E.; Kváč, Martin; Khan, Eakalak; Giddings, Catherine W.; Dyer, Neil W.; Schultz, Jessie L.; McEvoy, John M.

    2015-01-01

    Cryptosporidium is an apicomplexan parasite that causes the disease cryptosporidiosis in humans, livestock, and other vertebrates. Much of the knowledge on Cryptosporidium diversity is derived from 18S rRNA gene (18S rDNA) phylogenies. Eukaryote genomes generally have multiple 18S rDNA copies that evolve in concert, which is necessary for the accurate inference of phylogenetic relationships. However, 18S rDNA copies in some genomes evolve by a birth-and-death process that can result in sequence divergence among copies. Most notably, divergent 18S rDNA paralogs in the apicomplexan Plasmodium share only 89–95% sequence similarity, encode structurally distinct rRNA molecules, and are expressed at different life cycle stages. In the present study, Cryptosporidium 18S rDNA was amplified from 28/72 (38.9%) eastern chipmunks (Tamias striatus). Phylogenetic analyses showed the co-occurrence of two 18S rDNA types, Type A and Type B, in 26 chipmunks, and Type B clustered with a sequence previously identified as Cryptosporidium chipmunk genotype II. Types A and B had a sister group relationship but shared less than 93% sequence similarity. In contrast, actin and heat shock protein 70 gene sequences were homogeneous in samples with both Types A and B present. It was therefore concluded that Types A and B are divergent 18S rDNA paralogs in Cryptosporidium chipmunk genotype II. Substitution patterns in Types A and B were consistent with functionally constrained evolution; however, Type B evolved more rapidly than Type A and had a higher G+C content (46.3% versus 41.0%). Oocysts of Cryptosporidium chipmunk genotype II measured 4.17 μm (3.73–5.04 μm) × 3.94 μm (3.50–4.98 μm) with a length-to-width ratio of 1.06 ± 0.06 μm, and infection occurred naturally in the jejunum, cecum, and colon of eastern chipmunks. The findings of this study have implications for the use of 18S rDNA sequences to infer phylogenetic relationships. PMID:25772204

  3. Highly divergent 18S rRNA gene paralogs in a Cryptosporidium genotype from eastern chipmunks (Tamias striatus).

    PubMed

    Stenger, Brianna L S; Clark, Mark E; Kváč, Martin; Khan, Eakalak; Giddings, Catherine W; Dyer, Neil W; Schultz, Jessie L; McEvoy, John M

    2015-06-01

    Cryptosporidium is an apicomplexan parasite that causes the disease cryptosporidiosis in humans, livestock, and other vertebrates. Much of the knowledge on Cryptosporidium diversity is derived from 18S rRNA gene (18S rDNA) phylogenies. Eukaryote genomes generally have multiple 18S rDNA copies that evolve in concert, which is necessary for the accurate inference of phylogenetic relationships. However, 18S rDNA copies in some genomes evolve by a birth-and-death process that can result in sequence divergence among copies. Most notably, divergent 18S rDNA paralogs in the apicomplexan Plasmodium share only 89-95% sequence similarity, encode structurally distinct rRNA molecules, and are expressed at different life cycle stages. In the present study, Cryptosporidium 18S rDNA was amplified from 28/72 (38.9%) eastern chipmunks (Tamias striatus). Phylogenetic analyses showed the co-occurrence of two 18S rDNA types, Type A and Type B, in 26 chipmunks, and Type B clustered with a sequence previously identified as Cryptosporidium chipmunk genotype II. Types A and B had a sister group relationship but shared less than 93% sequence similarity. In contrast, actin and heat shock protein 70 gene sequences were homogeneous in samples with both Types A and B present. It was therefore concluded that Types A and B are divergent 18S rDNA paralogs in Cryptosporidium chipmunk genotype II. Substitution patterns in Types A and B were consistent with functionally constrained evolution; however, Type B evolved more rapidly than Type A and had a higher G+C content (46.3% versus 41.0%). Oocysts of Cryptosporidium chipmunk genotype II measured 4.17 μm (3.73-5.04 μm) × 3.94 μm (3.50-4.98 μm) with a length-to-width ratio of 1.06 ± 0.06 μm, and infection occurred naturally in the jejunum, cecum, and colon of eastern chipmunks. The findings of this study have implications for the use of 18S rDNA sequences to infer phylogenetic relationships.

  4. HIV-1 genomic RNA diversification following sexual and parenteral virus transmission.

    PubMed

    Wolfs, T F; Zwart, G; Bakker, M; Goudsmit, J

    1992-07-01

    Human immunodeficiency virus type 1 (HIV-1) genomic RNA variation was studied in seven presumed donor-recipient pairs directly following sexual (6/7) or parenteral (1/7) transmission. The first RNA-positive serum sample of each recipient and the serum sample of the virus transmitter, identified by epidemiological history and taken within a time bracket of three months of the recipient seroconversion, were analyzed by polymerase chain reaction amplification followed by sequencing of eight cDNA clones of 276 bp, including the V3 coding region. The sequence populations of the recipients were without exception homogeneous, while the sequence populations of the transmitters showed varying degrees of heterogeneity. Nucleotide distance between consensus sequences of unrelated individuals from the Amsterdam population (interpatient variation) averaged 11% (range 7-15%). The largest distance between two clonal sequences of one individual (intrapatient variation) was also 11%. Consensus sequences of five recipients differed by only 0-1% from the consensus sequence of the presumed transmitter, including two pairs of which the transmission was either proven or highly probable. This contrasted with a difference of 10-12% in two pairs, casting doubt on the epidemiological relatedness. Antibody reactivity to a panel of V3 peptides with varying degrees of similarity to the V3 sequences obtained did not augment the discriminatory power of sequence analysis. Results of the sequential sequencing of samples of one transmitter suggest that this was due to an anamnestic antibody response of the transmitter to early variants. From the loss of sequence heterogeneity following transmission and the consensus sequence similarities observed within five transmitter-recipient pairs, we conclude that HIV-1 transmission results in the selection of a limited number of genomes carrying on the infection in the new host, but does not generally lead to a shift in the sequence population as defined by

  5. Clinical Evaluation of Rega 8: An Updated Genotypic Interpretation System That Significantly Predicts HIV-Therapy Response

    PubMed Central

    Vercauteren, Jurgen; Beheydt, Gertjan; Prosperi, Mattia; Libin, Pieter; Imbrechts, Stijn; Camacho, Ricardo; Clotet, Bonaventura; De Luca, Andrea; Grossman, Zehava; Kaiser, Rolf; Sönnerborg, Anders; Torti, Carlo; Van Wijngaerden, Eric; Schmit, Jean-Claude; Zazzi, Maurizio; Geretti, Anna-Maria; Vandamme, Anne-Mieke; Van Laethem, Kristel

    2013-01-01

    Introduction Clinically evaluating genotypic interpretation systems is essential to provide optimal guidance in designing potent individualized HIV-regimens. This study aimed at investigating the ability of the latest Rega algorithm to predict virological response on a short and longer period. Materials & Methods 9231 treatment changes episodes were extracted from an integrated patient database. The virological response after 8, 24 and 48 weeks was dichotomized to success and failure. Success was defined as a viral load below 50 copies/ml or alternatively, a 2 log decrease from the baseline viral load at 8 weeks. The predictive ability of Rega version 8 was analysed in comparison with that of previous evaluated version Rega 5 and two other algorithms (ANRS v2011.05 and Stanford HIVdb v6.0.11). A logistic model based on the genotypic susceptibility score was used to predict virological response, and additionally, confounding factors were added to the model. Performance of the models was compared using the area under the ROC curve (AUC) and a Wilcoxon signed-rank test. Results Per unit increase of the GSS reported by Rega 8, the odds on having a successful therapy response on week 8 increased significantly by 81% (OR = 1.81, CI = [1.76–1.86]), on week 24 by 73% (OR = 1.73, CI = [1.69–1.78]) and on week 48 by 85% (OR = 1.85, CI = [1.80–1.91]). No significant differences in AUC were found between the performance of Rega 8 and Rega 5, ANRS v2011.05 and Stanford HIVdb v6.0.11, however Rega 8 had the highest sensitivity: 76.9%, 76.5% and 77.2% on 8, 24 and 48 weeks respectively. Inclusion of additional factors increased the performance significantly. Conclusion Rega 8 is a significant predictor for virological response with a better sensitivity than previously, and with rules for recently approved drugs. Additional variables should be taken into account to ensure an effective regimen. PMID:23613852

  6. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

    PubMed Central

    Pocock, Ginger M.; Becker, Jordan T.; Swanson, Chad M.; Ahlquist, Paul; Sherer, Nathan M.

    2016-01-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

  7. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

    PubMed

    Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

    2016-04-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. PMID:27070420

  8. HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.

    PubMed

    Pocock, Ginger M; Becker, Jordan T; Swanson, Chad M; Ahlquist, Paul; Sherer, Nathan M

    2016-04-01

    Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent "burst-like" transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm.

  9. Viroids: from genotype to phenotype just relying on RNA sequence and structural motifs.

    PubMed

    Flores, Ricardo; Serra, Pedro; Minoia, Sofía; Di Serio, Francesco; Navarro, Beatriz

    2012-01-01

    As a consequence of two unique physical properties, small size and circularity, viroid RNAs do not code for proteins and thus depend on RNA sequence/structural motifs for interacting with host proteins that mediate their invasion, replication, spread, and circumvention of defensive barriers. Viroid genomes fold up on themselves adopting collapsed secondary structures wherein stretches of nucleotides stabilized by Watson-Crick pairs are flanked by apparently unstructured loops. However, compelling data show that they are instead stabilized by alternative non-canonical pairs and that specific loops in the rod-like secondary structure, characteristic of Potato spindle tuber viroid and most other members of the family Pospiviroidae, are critical for replication and systemic trafficking. In contrast, rather than folding into a rod-like secondary structure, most members of the family Avsunviroidae adopt multibranched conformations occasionally stabilized by kissing-loop interactions critical for viroid viability in vivo. Besides these most stable secondary structures, viroid RNAs alternatively adopt during replication transient metastable conformations containing elements of local higher-order structure, prominent among which are the hammerhead ribozymes catalyzing a key replicative step in the family Avsunviroidae, and certain conserved hairpins that also mediate replication steps in the family Pospiviroidae. Therefore, different RNA structures - either global or local - determine different functions, thus highlighting the need for in-depth structural studies on viroid RNAs.

  10. Dysregulation Of Innate Immunity In HCV Genotype 1 IL28B Unfavorable Genotype Patients: Impaired Viral Kinetics And Therapeutic Response

    PubMed Central

    Naggie, Susanna; Osinusi, Anu; Katsounas, Antonios; Lempicki, Richard; Herrmann, Eva; Thompson, Alexander J; Clark, Paul J; Patel, Keyur; Muir, Andrew J; McHutchison, John G; Schlaak, Joerg F; Trippler, Martin; Shivakumar, Bhavana; Masur, Henry; Polis, Michael A.; Kottilil, Shyam

    2012-01-01

    Recent studies have shown that a single nucleotide polymorphism upstream of the interleukin (IL)-28B gene plays a major role in predicting therapeutic response in HCV-infected patients treated with pegylated interferon-alpha (IFN)/ribavirin. We sought to investigate the mechanism of the IL28B polymorphism, specifically as it relates to early HCV viral kinetics (VK), IFN pharmacokinetics (PK), IFN pharmacodynamics (PD), and gene expression profiles. Two prospective cohorts (HIV/HCV co-infected and HCV mono-infected) completing treatment with IFN/ribavirin were enrolled. Patients (N=88; 44 HIV/HCV and 44 HCV) were genotyped at the polymorphic site rs12979860. In the HIV/HCV cohort frequent serum sampling was completed for HCV RNA and IFN-levels. DNA microarray of PBMCs and individual expression of interferon stimulated genes (ISGs) were quantified on IFN-therapy. The IL28B favorable (CC) genotype was associated with improved therapeutic response compared to unfavorable (CT/TT) genotypes. Patients with favorable genotype had greater first and second phase VK (P=0.004 and P=0.036, respectively), IFN maximum anti-viral efficiency (P=0.007) and infected cell death loss (P=0.009) compared to unfavorable genotypes. Functional annotation analysis of DNA microarray data was consistent with depressed innate immune function, particularly of NK cells, from patients with unfavorable genotypes (P<0.004). Induction of innate immunity genes was also lower in unfavorable genotype. ISG expression at baseline and induction with IFN was independent of IL28B genotype. Conclusions Carriers of the IL28B favorable genotype were more likely to have superior innate immune response to IFN-therapy compared to unfavorable genotypes, this suggests the unfavorable genotype has aberrant baseline induction of innate immune response pathways resulting in impaired virologic response. IL28B genotype is associated with more rapid viral kinetics and improved treatment response outcomes independent of

  11. CCL3L1-CCR5 genotype influences durability of immune recovery during antiretroviral therapy of HIV-1–infected individuals

    PubMed Central

    Ahuja, Sunil K; Kulkarni, Hemant; Catano, Gabriel; Agan, Brian K; Camargo, Jose F; He, Weijing; O'Connell, Robert J; Marconi, Vincent C; Delmar, Judith; Eron, Joseph; Clark, Robert A; Frost, Simon; Martin, Jeffrey; Ahuja, Seema S; Deeks, Steven G; Little, Susan; Richman, Douglas; Hecht, Frederick M; Dolan, Matthew J

    2008-01-01

    The basis for the extensive variability seen in the reconstitution of CD4+ T cell counts in HIV-infected individuals receiving highly active antiretroviral therapy (HAART) is not fully known. Here, we show that variations in CCL3L1 gene dose and CCR5 genotype, but not major histocompatibility complex HLA alleles, influence immune reconstitution, especially when HAART is initiated at <350 CD4+ T cells/mm3. The CCL3L1-CCR5 genotypes favoring CD4+ T cell recovery are similar to those that blunted CD4+ T cell depletion during the time before HAART became available (pre-HAART era), suggesting that a common CCL3L1-CCR5 genetic pathway regulates the balance between pathogenic and reparative processes from early in the disease course. Hence, CCL3L1-CCR5 variations influence HIV pathogenesis even in the presence of HAART and, therefore, may prospectively identify subjects in whom earlier initiation of therapy is more likely to mitigate immunologic failure despite viral suppression by HAART. Furthermore, as reconstitution of CD4+ cells during HAART is more sensitive to CCL3L1 dose than to CCR5 genotypes, CCL3L1 analogs might be efficacious in supporting immunological reconstitution. PMID:18376407

  12. Optimized virus disruption improves detection of HIV-1 p24 in particles and uncovers a p24 reactivity in patients with undetectable HIV-1 RNA under long-term HAART.

    PubMed

    Schüpbach, Jörg; Tomasik, Zuzana; Knuchel, Marlyse; Opravil, Milos; Günthard, Huldrych F; Nadal, David; Böni, Jürg

    2006-08-01

    HIV-1 p24 antigen (p24) measurement by signal amplification-boosted ELISA of heat-denatured plasma is being evaluated as an alternative to HIV-1 RNA quantitation in resource-poor settings. Some observations suggested that virion-associated p24 is suboptimally detected using Triton X-100-based virus dissociation buffer (kit buffer). A new reagent (SNCR buffer) containing both denaturing and non-denaturing detergents was therefore developed and evaluated. The SNCR buffer increased the measured p24 concentration about 1.5- to 3-fold in HIV-negative plasma reconstituted with purified HIV-1 particles, while not increasing the background. Among 127 samples of HIV-1-positive patients with moderate to high concentrations of HIV-1 RNA the increase was about threefold across the entire concentration range (P < 0.0001). Specificity before neutralization among prospectively tested clinical samples ruled HIV-negative was 828 of 845 (98.0%) for the SNCR buffer and 464 of 479 (96.9%) for kit buffer. Specificity after confirmatory neutralization of reactive samples or a follow-up test was 100% with either buffer. Surprisingly, the SNCR buffer revealed a p24 reactivity in 115 of 187 samples (61.5%) from adult patients exhibiting undetectable HIV-1 RNA below 5 copies/ml for a duration of 6-30 months under HAART (3.7% with kit buffer). The rate of p24 reactivity in these patients did not decrease with duration of HAART. In conclusion, the SNCR buffer improves the detection of particle-associated HIV-1 p24, thereby increasing the measured p24 concentration in samples with medium to high HIV-1 RNA. It also uncovers the presence of a p24 reactivity, whose identity remains to be determined, in a significant fraction of samples with undetectable HIV-1 RNA under long-term HAART.

  13. The interaction of RNA helicase DDX3 with HIV-1 Rev-CRM1-RanGTP complex during the HIV replication cycle

    DOE PAGES

    Mahboobi, Seyed Hanif; Javanpour, Alex A.; Mofrad, Mohammad R. K.

    2015-02-27

    Molecular traffic between the nucleus and the cytoplasm is regulated by the nuclear pore complex (NPC), which acts as a highly selective channel perforating the nuclear envelope in eukaryotic cells. The human immunodeficiency virus (HIV) exploits the nucleocytoplasmic pathway to export its RNA transcripts across the NPC to the cytoplasm. Despite extensive study on the HIV life cycle and the many drugs developed to target this cycle, no current drugs have been successful in targeting the critical process of viral nuclear export, even though HIV’s reliance on a single host protein, CRM1, to export its unspliced and partially spliced RNAmore » transcripts makes it a tempting target. Due to recent findings implicating a DEAD-box helicase, DDX3, in HIV replication and a member of the export complex, it has become an appealing target for anti-HIV drug inhibition. In the present research, we have applied a hybrid computational protocol to analyze protein-protein interactions in the HIV mRNA export cycle. This method is based on molecular docking followed by molecular dynamics simulation and accompanied by approximate free energy calculation (MM/GBSA), computational alanine scanning, clustering, and evolutionary analysis. We highlight here some of the most likely binding modes and interfacial residues between DDX3 and CRM1 both in the absence and presence of RanGTP. This work shows that although DDX3 can bind to free CRM1, addition of RanGTP leads to more concentrated distribution of binding modes and stronger binding between CRM1 and RanGTP.« less

  14. Intracellular interactions between APOBEC3G, RNA, and HIV-1 Gag: APOBEC3G multimerization is dependent on its association with RNA

    PubMed Central

    Friew, Yeshitila N; Boyko, Vitaly; Hu, Wei-Shau; Pathak, Vinay K

    2009-01-01

    Background Host restriction factor APOBEC3G (A3G) blocks human immunodeficiency virus type 1 (HIV-1) replication by G-to-A hypermutation, and by inhibiting DNA synthesis and provirus formation. Previous reports have suggested that A3G is a dimer and its virion incorporation is mediated through interactions with viral or nonviral RNAs and/or HIV-1 Gag. We have now employed a bimolecular fluorescence complementation assay (BiFC) to analyze the intracellular A3G-A3G, A3G-RNA, and A3G-Gag interactions in living cells by reconstitution of yellow fluorescent protein (YFP) from its N- or C-terminal fragments. Results The results obtained with catalytic domain 1 and 2 (CD1 and CD2) mutants indicate that A3G-A3G and A3G-Gag multimerization is dependent on an intact CD1 domain, which is required for RNA binding. A mutant HIV-1 Gag that exhibits reduced RNA binding also failed to reconstitute BiFC with wild-type A3G, indicating a requirement for both HIV-1 Gag and A3G to bind to RNA for their multimerization. Addition of a non-specific RNA binding peptide (P22) to the N-terminus of a CD1 mutant of A3G restored BiFC and virion incorporation, but failed to inhibit viral replication, indicating that the mutations in CD1 resulted in additional defects that interfere with A3G's antiviral activity. Conclusion These studies establish a robust BiFC assay for analysis of intracellular interactions of A3G with other macromolecules. The results indicate that in vivo A3G is a monomer that forms multimers upon binding to RNA. In addition, we observed weak interactions between wild-type A3G molecules and RNA binding-defective mutants of A3G, which could explain previously described protein-protein interactions between purified A3G molecules. PMID:19497112

  15. HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA

    PubMed Central

    Ajamian, Lara; Abel, Karen; Rao, Shringar; Vyboh, Kishanda; García-de-Gracia, Francisco; Soto-Rifo, Ricardo; Kulozik, Andreas E.; Gehring, Niels H.; Mouland, Andrew J.

    2015-01-01

    Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking. PMID:26492277

  16. HIV-1 Recruits UPF1 but Excludes UPF2 to Promote Nucleocytoplasmic Export of the Genomic RNA.

    PubMed

    Ajamian, Lara; Abel, Karen; Rao, Shringar; Vyboh, Kishanda; García-de-Gracia, Francisco; Soto-Rifo, Ricardo; Kulozik, Andreas E; Gehring, Niels H; Mouland, Andrew J

    2015-01-01

    Unspliced, genomic HIV-1 RNA (vRNA) is a component of several ribonucleoprotein complexes (RNP) during the viral replication cycle. In earlier work, we demonstrated that the host upframeshift protein 1 (UPF1), a key factor in nonsense-mediated mRNA decay (NMD), colocalized and associated to the viral structural protein Gag during viral egress. In this work, we demonstrate a new function for UPF1 in the regulation of vRNA nuclear export. OPEN ACCESS Biomolecules 2015, 5 2809 We establish that the nucleocytoplasmic shuttling of UPF1 is required for this function and demonstrate that UPF1 exists in two essential viral RNPs during the late phase of HIV-1 replication: the first, in a nuclear export RNP that contains Rev, CRM1, DDX3 and the nucleoporin p62, and the second, which excludes these nuclear export markers but contains Gag in the cytoplasm. Interestingly, we observed that both UPF2 and the long isoform of UPF3a, UPF3aL, but not the shorter isoforms UPF3aS and UPF3b, are excluded from the UPF1-Rev-CRM1-DDX3 complex as they are negative regulators of vRNA nuclear export. In silico protein-protein docking analyses suggest that Rev binds UPF1 in a region that overlaps the UPF2 binding site, thus explaining the exclusion of this negative regulatory factor by HIV-1 that is necessary for vRNA trafficking. This work uncovers a novel and unique regulatory circuit involving several UPF proteins that ultimately regulate vRNA nuclear export and trafficking. PMID:26492277

  17. Protective genotypes in HIV infection reflect superior function of KIR3DS1+ over KIR3DL1+ CD8+ T cells.

    PubMed

    Zipperlen, Katrin; Gallant, Maureen; Stapleton, Staci; Heath, John; Barrett, Lisa; Grant, Michael

    2015-01-01

    Certain human class I histocompatibility-linked leukocyte antigen (HLA)/killer cell immunoglobulin-like receptor (KIR) genotypic combinations confer more favourable prognoses upon exposure to human immunodeficiency virus (HIV). These combinations influence natural killer (NK) cell function, thereby implicating NK cells in protection from HIV infection or disease progression. Because CD8(+) T cells restrict HIV replication, depend upon HLA class I antigen presentation and can also express KIR molecules, we investigated how these HLA/KIR combinations relate to the phenotype and function of CD8(+) T cells from uninfected controls and individuals with chronic HIV infection. CD8(+) T cells from KIR3DL1 and KIR3DS1 homozygous individuals, and expressing the corresponding KIR, were enumerated and phenotyped for CD127, CD57 and CD45RA expression. Ex vivo and in vitro responsiveness to antigen-specific and polyclonal stimulation was compared between KIR-expressing and non-expressing CD8(+) T cells by interferon-γ production. There were higher numbers and fractions of KIR3DL1-expressing CD8(+) T cells in HIV-infected individuals independent of HLA-Bw4 co-expression, whereas expansion of KIR3DS1-expressing CD8(+) T cells reflected HLA-Bw4*80I co-expression. KIR3DL1(+) and S1(+) CD8(+) T cells were predominantly CD127(-)CD57(+)CD45RA(+). KIR3DL1-expressing CD8(+) T cells were insensitive to ex vivo stimulation with peptides from HIV or common viruses, but responded to anti-CD3 and recovered responsiveness to common viruses in vitro. Ex vivo non-responsiveness of KIR3DL1-expressing CD8(+) T cells was also independent of HLA-Bw4. KIR3DS1-expressing T cells responded normally to ex vivo antigenic stimulation, illustrating functional superiority over KIR3DL1(+) CD8(+) T cells.

  18. Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis.

    PubMed

    Ouyang, Wei; Okaine, Stephen; McPike, Mark P; Lin, Yong; Borer, Philip N

    2013-05-14

    The highly conserved nucleocapsid protein domain in HIV-1 recognizes and binds SL3 in genomic RNA. In this work, we used the structure of the NCp7-SL3 RNA complex to guide the construction of 16 NCp7 mutants to probe the RNA binding surface of the protein [De Guzman, R. N., et al. (1998) Science 279, 384-388]. Thirteen residues with functional or structural significance were mutated individually to Ala (Asn(5), Phe(6), Val(13), Phe(16), Asn(17), Gly(19), Glu(21), Ile(24), Gln(45), Met(46), Gly(22), Pro(31), and Gly(40)), and three salt bridge switch mutants exchanged Lys and Glu (Lys(14)-Glu(21), Lys(33)-Glu(42), and Lys(38)-Glu(51)). Dissociation constants (Kd) determined by fluorescence titration and isothermal titration calorimetry were used to compare affinities of SL3 for the variant proteins to that for the wild type. The F16A (Phe(16) to Ala) variant showed a 25-fold reduction in affinity, consistent with a loss of organized structure in f1, the protein's first zinc finger. I24A, Q45A, and M46A reduced affinity by 2-5-fold; these residues occupy nearly equivalent positions in f1 and f2. E21A increased affinity by 3-fold, perhaps because of the mutant's increased net positive charge. Among the salt bridge switch mutants, only K14E/E21K in f1 caused a substantial change in affinity (5-fold reduction), binding SL3 with a biphasic binding isotherm. Aside from these six variants, most of the mutations studied have relatively minor effects on the stability of the complex. We conclude that many side chain interactions in the wild-type complex contribute little to stability or can be compensated by new contacts in the mutants.

  19. The Liver MicroRNA Expression Profiles Associated With Chronic Hepatitis C Virus (HCV) Genotype-4 Infection: A Preliminary Study

    PubMed Central

    El-Guendy, Nadia Mohamed; Helwa, Reham; El-Halawany, Medhat Salah; Abdel Rahman Ali, Shimaa; Tantawy Aly, Marwa; Hasan Alieldin, Nelly; Fouad, Shawky Abdel Hamid; Saeid, Hany; Abdel-Wahab, Abdel-Hady Ali

    2016-01-01

    Background MicroRNAs (miRNAs) have been repeatedly shown to play important roles in liver pathologies, including hepatitis, liver cirrhosis, and liver cancer. Egypt has the highest hepatitis C virus (HCV) infection rate worldwide, predominantly involving genotype-4. Objectives In this study, we attempted to characterize the miRNA profile of the poorly studied genotype 4 of HCV in chronically infected Egyptian patients to obtain a better understanding of the disease and its complications and help in the design of better management protocols. Patients and Methods We analyzed the expression levels of a selected panel of 94 miRNAs in fresh liver biopsies collected from 50 Egyptian patients diagnosed with chronic HCV infection using quantitative real-time polymerase chain reaction (PCR) assay. Non-parametric tests were used to analyze the expression level of each miRNA and association with the clinicopathological features of enrolled patients in this study. Results Our results revealed differential expression levels of the analyzed miRNAs compared to the normal controls. Twenty-seven miRNAs (including miR-105, miR-147, miR-149-3p, and miR-196b) showed up-regulation, while 17 miRNAs (including miR-21, miR-122, miR-199a-3p, and miR-223) showed down-regulation. An inverse correlation was observed between levels of miR-95, miR-130a, and miR-142-5p with the blood albumin level. Increased expression levels of seven miRNAs (miR-29c, miR-30c, miR-126, miR-145, miR-199a, miR-199a-3p, and miR-222) were observed with severe chronic hepatic inflammation. Several deregulated miRNAs found in this study have been previously linked to chronic liver inflammation and the risk of hepatocellular carcinoma (HCC) development. Conclusions The identified expression profiles of some examined miRNAs might offer important points to consider for the treatment of naive patients and the management of chronically infected HCV patients in Egypt and around the world. PMID:27275163

  20. A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models.

    PubMed

    Boyapalle, Sandhya; Xu, Weidong; Raulji, Payal; Mohapatra, Subhra; Mohapatra, Shyam S

    2015-01-01

    Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections. PMID:26407080

  1. A Multiple siRNA-Based Anti-HIV/SHIV Microbicide Shows Protection in Both In Vitro and In Vivo Models

    PubMed Central

    Raulji, Payal; Mohapatra, Subhra; Mohapatra, Shyam S

    2015-01-01

    Human immunodeficiency virus (HIV) types 1 and 2 (HIV-1 and HIV-2) are the etiologic agents of AIDS. Most HIV-1 infected individuals worldwide are women, who acquire HIV infections during sexual contact. Blocking HIV mucosal transmission and local spread in the female lower genital tract is important in preventing infection and ultimately eliminating the pandemic. Microbicides work by destroying the microbes or preventing them from establishing an infection. Thus, a number of different types of microbicides are under investigation, however, the lack of their solubility and bioavailability, and toxicity has been major hurdles. Herein, we report the development of multifunctional chitosan-lipid nanocomplexes that can effectively deliver plasmids encoding siRNA(s) as microbicides without adverse effects and provide significant protection against HIV in both in vitro and in vivo models. Chitosan or chitosan-lipid (chlipid) was complexed with a cocktail of plasmids encoding HIV-1-specific siRNAs (psiRNAs) and evaluated for their efficacy in HEK-293 cells, PBMCs derived from nonhuman primates, 3-dimensional human vaginal ectocervical tissue (3D-VEC) model and also in non-human primate model. Moreover, prophylactic administration of the chlipid to deliver a psiRNA cocktail intravaginally with a cream formulation in a non-human primate model showed substantial reduction of SHIV (simian/human immunodeficiency virus SF162) viral titers. Taken together, these studies demonstrate the potential of chlipid-siRNA nanocomplexes as a potential genetic microbicide against HIV infections. PMID:26407080

  2. Epidemiology, Risk Factors and Genotypes of HBV in HIV-Infected Patients in the Northeast Region of Colombia: High Prevalence of Occult Hepatitis B and F3 Subgenotype Dominance

    PubMed Central

    Bautista-Amorocho, Henry; Castellanos-Domínguez, Yeny Zulay; Rodríguez-Villamizar, Laura Andrea; Velandia-Cruz, Sindi Alejandra; Becerra-Peña, Jeysson Andrey; Farfán-García, Ana Elvira

    2014-01-01

    Introduction Chronic hepatitis B virus (HBV) infection is an increasing cause of morbidity and mortality in human immunodeficiency virus (HIV)-infected individuals. HIV-positive patients are commonly co-infected with HBV due to shared routes of transmission. Objectives Our aim was to determine the risk factors, prevalence, genotypes, and mutations of the Surface S gene of HBV, and occult hepatitis B infection (OBI) among patients infected with HIV in a northeastern Colombian city. Methods A cross-sectional study was conducted with 275 HIV-positive patients attending an outpatient clinic in Bucaramanga, Colombia during 2009–2010. Blood samples were collected and screened for serological markers of HBV (anti-HBs, anti-HBc and HBsAg) through ELISA assay. Regardless of their serological profile, all samples were tested for the HBV S gene by nested-PCR and HBV genotypes were determined by phylogenetic inference. Clinical records were used to examine demographic, clinical, virological, immunological and antiretroviral therapy (ART) variables of HIV infection. Results Participants were on average 37±11 years old and 65.1% male. The prevalence of HIV-HBV coinfection was 12% (95%CI 8.4–16.4) of which 3.3% had active HBV infection and 8.7% OBI. The prevalence of HIV-HBV coinfection was associated with AIDS stage and ART treatment. Sequence analysis identified genotype F, subgenotype F3 in 93.8% of patients and genotype A in 6.2% of patients. A C149R mutation, which may have resulted from failure in HBsAg detection, was found in one patient with OBI. Conclusions The present study found a high prevalence of HIV-HBV coinfection with an incidence of OBI 2.6-fold higher compared to active HBV infection. These findings suggest including HBV DNA testing to detect OBI in addition to screening for HBV serological markers in HIV patients. PMID:25462190

  3. Identification of Species and Sources of Cryptosporidium Oocysts in Storm Waters with a Small-Subunit rRNA-Based Diagnostic and Genotyping Tool

    PubMed Central

    Xiao, Lihua; Alderisio, Kerri; Limor, Josef; Royer, Michael; Lal, Altaf A.

    2000-01-01

    The identification of Cryptosporidium oocysts in environmental samples is largely made by the use of an immunofluorescent assay. In this study, we have used a small-subunit rRNA-based PCR-restriction fragment length polymorphism technique to identify species and sources of Cryptosporidium oocysts present in 29 storm water samples collected from a stream in New York. A total of 12 genotypes were found in 27 positive samples; for 4 the species and probable origins were identified by sequence analysis, whereas the rest represent new genotypes from wildlife. Thus, this technique provides an alternative method for the detection and differentiation of Cryptosporidium parasites in environmental samples. PMID:11097935

  4. The role of chemokine C-C motif ligand 2 genotype and cerebrospinal fluid chemokine C-C motif ligand 2 in neurocognition among HIV-infected patients

    PubMed Central

    Thames, April D.; Briones, Marisa S.; Magpantay, Larry I.; Martinez-Maza, Otoniel; Singer, Elyse J.; Hinkin, Charles H.; Morgello, Susan; Gelman, Benjamin B.; Moore, David J.; Heizerling, Keith; Levine, Andrew J.

    2015-01-01

    Objectives We examined interrelationships between chemokine C-C motif ligand 2 (CCL2) genotype and expression of inflammatory markers in the cerebrospinal fluid (CSF), plasma viral load, CD4+ cell count and neurocognitive functioning among HIV-infected adults. We hypothesized that HIV-positive carriers of the ‘risk’ CCL2 −2578G allele, caused by a single nucleotide polymorphism (SNP) at rs1024611, would have a higher concentration of CCL2 in CSF, and that CSF CCL2 would be associated with both higher concentrations of other proinflammatory markers in CSF and worse neurocognitive functioning. Design A cross-sectional study of 145 HIV-infected individuals enrolled in the National NeuroAIDS Tissue Consortium cohort for whom genotyping, CSF and neurocognitive data were available. Methods Genomic DNA was extracted from peripheral blood mononuclear cells and/or frozen tissue specimens. CSF levels of CCL2, interleukin (IL)-2, IL-6, tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), soluble tumor necrosis factor receptor 2, sIL-6Rα, sIL-2, sCD14 and B-cell activating factor were quantified. Neurocognitive functioning was measured using a comprehensive battery of neuropsychological tests. Results Carriers of the CCL2 −2578G allele had a significantly higher concentration of CCL2 in CSF. CSF CCL2 level was positively and significantly associated with other CSF neuroinflammatory markers and worse cognitive functioning. There was a significant association between genotype and plasma viral load, such that carriers of the CCL2 −2578G allele with high viral load expressed greater levels of CCL2 and had higher neurocognitive deficit scores than other genotype/viral load groups. Conclusion Individuals with the CCL2 −2578G allele had higher levels of CCL2 in CSF, which was associated with increased pro-inflammatory markers in CSF and worse neurocognitive functioning. The results highlight the potential role of intermediate phenotypes in studies of

  5. Higher HIV RNA Viral Load in Recent Patients with Symptomatic Acute HIV Infection in Lyon University Hospitals

    PubMed Central

    Girerd-Genessay, Isabelle; Baratin, Dominique; Ferry, Tristan; Chidiac, Christian; Ronin, Vincent; Vanhems, Philippe

    2016-01-01

    Introduction Increased human immunodeficiency virus (HIV) virulence at infection has been suggested by a meta-analysis based on viral load and CD4 T lymphocytes (CD4) count during acute infection. This result was obtained after secondary analyses of large databases, facilitating the detection of differences. Similar finding in cohorts of more modest sample size would indicate that the effect could be more substantial. Methods Change from initial CD4 count and HIV viral load after acute HIV infection by calendar year was explored in patients treated at Lyon University hospitals. All patients admitted to our hospitals with acute HIV infection between 1996 and 2013 were included in our study. Initial CD4 count and viral load before the start of anti-retroviral treatment were analyzed. Trends over time were assessed in linear models. Results Initial CD4 count remained similar over time. However, in 2006–2013, initial viral load rose significantly (+1.12 log10/ml/year, p = 0.01). Conclusion Our data, obtained from a single hospital cohort, confirmed findings from a large meta-analysis, showed increased initial viremia at acute HIV infection since 2006 and suggesting potentially higher HIV virulence in recent years. PMID:26799390

  6. Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

    PubMed Central

    Ocwieja, Karen E.; Sherrill-Mix, Scott; Mukherjee, Rithun; Custers-Allen, Rebecca; David, Patricia; Brown, Michael; Wang, Susana; Link, Darren R.; Olson, Jeff; Travers, Kevin; Schadt, Eric; Bushman, Frederic D.

    2012-01-01

    Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-189.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing. PMID:22923523

  7. Characterization and in vitro activity of a branched peptide boronic acid that interacts with HIV-1 RRE RNA.

    PubMed

    Wynn, Jessica E; Zhang, Wenyu; Tebit, Denis M; Gray, Laurie R; Hammarskjold, Marie-Louise; Rekosh, David; Santos, Webster L

    2016-09-01

    A branched peptide containing multiple boronic acids was found to bind RRE IIB selectively and inhibit HIV-1 p24 capsid production in a dose-dependent manner. Structure-activity relationship studies revealed that branching in the peptide is crucial for the low micromolar binding towards RRE IIB, and the peptide demonstrates selectivity towards RRE IIB in the presence of tRNA. Footprinting studies suggest a binding site on the upper stem and internal loop regions of the RNA, which induces enzymatic cleavage of the internal loops of RRE IIB upon binding. PMID:27091070

  8. Characterization of Mycobacterium leprae Genotypes in China--Identification of a New Polymorphism C251T in the 16S rRNA Gene.

    PubMed

    Yuan, Youhua; Wen, Yan; You, Yuangang; Xing, Yan; Li, Huanying; Weng, Xiaoman; Wu, Nan; Liu, Shuang; Zhang, Shanshan; Zhang, Wenhong; Zhang, Ying

    2015-01-01

    Leprosy continues to be prevalent in some mountainous regions of China, and genotypes of leprosy strains endemic to the country are not known. Mycobacterium lepromatosis is a new species that was discovered in Mexico in 2008, and it remains unclear whether this species exists in China. Here, we conducted PCR- restriction fragment length polymorphism (RFLP) analysis to classify genotypes of 85 DNA samples collected from patients from 18 different provinces. All 171 DNA samples from skin biopsies of leprosy patients were tested for the presence of Mycobacterium leprae and Mycobacterium lepromatosis by amplifying the 16S rRNA gene using nested PCR, followed by DNA sequencing. The new species M. lepromatosis was not found among the 171 specimens from leprosy patients in 22 provinces in China. However, we found three SNP genotypes among 85 leprosy patients. A mutation at C251T in the 16S rRNA gene was found in 76% of the strains. We also found that the strains that showed the 16S rRNA C251T mutation belonged to SNP type 3, whereas strains without the point mutation belonged to SNP type 1. The SNP type 3 leprosy strains were observed in patients from both the inner and coastal regions of China, but the SNP type 1 strains were focused only in the coastal region. This indicated that the SNP type 3 leprosy strains were more prevalent than the SNP type 1 strains in China. In addition, the 16S rRNA gene sequence mutation at C251T also indicated a difference in the geographical distribution of the strains. To our knowledge, this is the first report of a new polymorphism in 16S rRNA gene in M. leprae in China. Our findings shed light on the prevalent genotypes and provide insight about leprosy transmission that are important for leprosy control in China.

  9. Rapid suppression of HIV-RNA is associated with improved control of immune activation in Mozambican adults initiating antiretroviral therapy with low CD4 counts.

    PubMed

    Almeida, Jose M; Letang, Emilio; Nhampossa, Tacilta; Ayala, Edgar; David, Catarina; Menendez, Clara; Gascon, Joaquim; Alonso, Pedro; Naniche, Denise

    2011-07-01

    The rapidity of HIV-RNA suppression after initiation of combined antiretroviral therapy (cART) may impact immune reconstitution in developing countries, where patients initiate cART at low CD4 T cell counts. One hundred and thirty-five HIV-1 Mozambican adults initiating cART were prospectively followed over 16 months within a larger observational study. Plasma HIV-RNA, CD4 counts, and CD8 T cell activation were monitored at the pre-cART visit and at 4, 10, and 16 months during cART. Of the 89 patients with available HIV-RNA data at pre-cART and 4 and 10 months post-cART, 68% (60/89) suppressed HIV-RNA at 4 months and were defined as "early virological controllers"(EC). Twenty of the 29 remaining patients who did not control HIV-RNA at 4 months did so at 10 months and were classified as "late virological controllers"(LC). Nine (10%) patients did not control HIV-RNA at either time point. Both initiating an EFV-containing cART regimen and having pre-cART tuberculosis were significantly associated with early HIV-RNA suppression if locked into a multivariate model [EFV OR: 13.6 (95% CI 1.7; 108.1) p = 0.014) tuberculosis OR: 11.0 (95% CI 1.4; 87.9) p = 0.024]. EC demonstrated significantly lower median activated CD8 T cells at 4, 10, and 16 months post-cART than did LC. Approximately 63% (12/19) of LC experienced reappearance of detectable HIV-RNA at 6 months postcontrol as compared to 15% (2/60) of EC (p = 0.001). This study suggests that rapid suppression of HIV-RNA may lead to a lower rate of reappearance of HIV-RNA, which could impact CD8 T cell activation levels in patients initiating cART at low CD4 counts.

  10. Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.

    PubMed

    Jayaraman, Bhargavi; Mavor, David; Gross, John D; Frankel, Alan D

    2015-10-27

    The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function. PMID:26422686

  11. Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.

    PubMed

    Jayaraman, Bhargavi; Mavor, David; Gross, John D; Frankel, Alan D

    2015-10-27

    The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.

  12. Mapping the RNA binding sites for human immunodeficiency virus type-1 gag and NC proteins within the complete HIV-1 and -2 untranslated leader regions.

    PubMed Central

    Damgaard, C K; Dyhr-Mikkelsen, H; Kjems, J

    1998-01-01

    Encapsidation of HIV-1 genomic RNA is mediated by specific interactions between the RNA packaging signal and the Gag protein. During maturation of the virion, the Gag protein is processed into smaller fragments, including the nucleocapsid (NC) domain which remains associated with the viral genomic RNA. We have investigated the binding of glutathione- S -transferase (GST) Gag and NC fusion proteins from HIV-1, to the entire HIV-1 and -2 leader RNAencompassing the packaging signal. We have mapped the binding sites at conditions where only about two complexes are formed and find that GST-Gag and GST-NC fusion proteins bind specifically to discrete sites within the leader. Analysis of the HIV-1 leader indicated that GST-Gag strongly associates with the PSI stem-loop and to a lesser extent with regions near the primer binding site. GST-NC binds the same regions but with reversed preferences. The HIV-1 proteins also interact specifically with the 5'-leader of HIV-2 and the major site of interaction mapped to a stem-loop, with homology to the HIV-1 PSI stem-loop structure. The different specificities of Gag and NC may reflect functionally distinct roles in the viral replication, and suggest that the RNA binding specificity of NC is modulated by its structural context. PMID:9685481

  13. Persistent infection and promiscuous recombination of multiple genotypes of an RNA virus within a single host generate extensive diversity.

    PubMed

    Weng, Ziming; Barthelson, Roger; Gowda, Siddarame; Hilf, Mark E; Dawson, William O; Galbraith, David W; Xiong, Zhongguo

    2007-01-01

    Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses. PMID:17878952

  14. HIV-1 drug resistance genotyping from antiretroviral therapy (ART) naïve and first-line treatment failures in Djiboutian patients

    PubMed Central

    2012-01-01

    Abstract In this study we report the prevalence of antiretroviral drug resistant HIV-1 genotypes of virus isolated from Djiboutian patients who failed first-line antiretroviral therapy (ART) and from ART naïve patients. Patients and methods A total of 35 blood samples from 16 patients who showed first-line ART failure (>1000 viral genome copies/ml) and 19 ART-naïve patients were collected in Djibouti from October 2009 to December 2009. Both the protease (PR) and reverse transcriptase (RT) genes were amplified and sequenced using National Agency for AIDS Research (ANRS) protocols. The Stanford HIV database algorithm was used for interpretation of resistance data and genotyping. Results Among the 16 patients with first-line ART failure, nine (56.2%) showed reverse transcriptase inhibitor-resistant HIV-1 strains: two (12.5%) were resistant to nucleoside (NRTI), one (6.25%) to non-nucleoside (NNRTI) reverse transcriptase inhibitors, and six (37.5%) to both. Analysis of the DNA sequencing data indicated that the most common mutations conferring drug resistance were M184V (38%) for NRTI and K103N (25%) for NNRTI. Only NRTI primary mutations K101Q, K103N and the PI minor mutation L10V were found in ART naïve individuals. No protease inhibitor resistant strains were detected. In our study, we found no detectable resistance in ∼ 44% of all patients who experienced therapeutic failure which was explained by low compliance, co-infection with tuberculosis and malnutrition. Genotyping revealed that 65.7% of samples were infected with subtype C, 20% with CRF02_AG, 8.5% with B, 2.9% with CRF02_AG/C and 2.9% with K/C. Conclusion The results of this first study about drug resistance mutations in first-line ART failures show the importance of performing drug resistance mutation test which guides the choice of a second-line regimen. This will improve the management of HIV-infected Djiboutian patients. Virtual slides The virtual slide(s) for this article can be found here

  15. Differences in HCV Viral Decline between Low and Standard-Dose Pegylated-Interferon-Alpha-2a with Ribavirin in HIV/HCV Genotype 3 Patients

    PubMed Central

    Rivero-Juárez, Antonio; Lopez-Cortes, Luis F.; Camacho, Angela; Torres-Cornejo, Almudena; Pineda, Juan A.; Marquez-Solero, Manuel; Caruz, Antonio; Ruiz-Valderas, Rosa; Torre-Cisneros, Julian; Gutierrez-Valencia, Alicia; Rivero, Antonio

    2012-01-01

    Background The aim of the study was to analyze the different impact of standard and low-dose Peg-IFN-α2a/RBV therapies on HCV viral decline in HIV/HCV genotype 3 co-infected patients during the first weeks of treatment. Methods Plasma HCV viral decline was analyzed between baseline and weeks 1, 2 and 4 in two groups of treatment-naïve HCV genotype 3 patients with HIV co-infection. The Standard Dose Group (SDG) included patients who received Peg-IFN at 180 µg/per week with a weight-adjusted dose of ribavirin; Low-Dose Group (LDG) patients received Peg-IFN at 135 µg/per week with 800 mg/day ribavirin. The effect of IL28B genotype on HCV viral decline was evaluated in both groups. HCV viral decline was analyzed using a multivariate linear regression model. Results One hundred and six patients were included: 48 patients in the SDG and 58 in the LDG. HCV viral decline for patients in the LDG was less than for those in the SDG (week 1∶1.72±0.74 log10 IU/mL versus 1.78±0.67 log10 IU/mL, p = 0.827; week 2∶2.3±0.89 log10 IU/mL versus 3.01±1.02 log10 IU/mL, p = 0.013; week 4∶3.52±1.2 log10 IU/mL versus 4.09±1.1 log10 IU/mL, p = 0.005). The linear regression model identified the Peg-IFN/RBV dose as an independent factor for HCV viral decline at week 4. Conclusions Our results showed that HCV viral decline was less for patients in the low-dose group compared to those receiving the standard dose. Until a randomized clinical trial is conducted, clinicians should be cautious about using lower doses of Peg-IFN/RBV in HIV/HCV genotype 3 co-infected patients. PMID:23145040

  16. Comparison of Ahlstrom grade 226, Munktell TFN, and Whatman 903 filter papers for dried blood spot specimen collection and subsequent HIV-1 load and drug resistance genotyping analysis.

    PubMed

    Rottinghaus, Erin; Bile, Ebi; Modukanele, Mosetsanagape; Maruping, Maruping; Mine, Madisa; Nkengasong, John; Yang, Chunfu

    2013-01-01

    Dried blood spots (DBS) collected onto filter paper have eased the difficulty of blood collection in resource-limited settings. Currently, Whatman 903 (W-903) filter paper is the only filter paper that has been used for HIV load and HIV drug resistance (HIVDR) testing. We therefore evaluated two additional commercially available filter papers, Ahlstrom grade 226 (A-226) and Munktell TFN (M-TFN), for viral load (VL) testing and HIVDR genotyping using W-903 filter paper as a comparison group. DBS specimens were generated from 344 adult patients on antiretroviral therapy (ART) in Botswana. The VL was measured with NucliSENS EasyQ HIV-1 v2.0, and genotyping was performed for those specimens with a detectable VL (≥ 2.90 log(10) copies/ml) using an in-house method. Bland-Altman analysis revealed a strong concordance in quantitative VL analysis between W-903 and A-226 (bias = -0.034 ± 0.246 log(10) copies/ml [mean difference ± standard deviation]) and W-903 and M-TFN (bias = -0.028 ± 0.186 log(10) copies/ml) filter papers, while qualitative VL analysis for virological failure determination, defined as a VL of ≥ 3.00 log(10) copies/ml, showed low sensitivities for A-266 (71.54%) and M-TFN (65.71%) filter papers compared to W-903 filter paper. DBS collected on M-TFN filter paper had the highest genotyping efficiency (100%) compared to W-903 and A-226 filter papers (91.7%) and appeared more sensitive in detecting major HIVDR mutations. DBS collected on A-226 and M-TFN filter papers performed similarly to DBS collected on W-903 filter paper for quantitative VL analysis and HIVDR detection. Together, the encouraging genotyping results and the variability observed in determining virological failure from this small pilot study warrant further investigation of A-226 and M-TFN filter papers as specimen collection devices for HIVDR monitoring surveys.

  17. Identification of virulence factors in 16S-23S rRNA intergenic spacer genotyped Staphylococcus aureus isolated from water buffaloes and small ruminants.

    PubMed

    Cremonesi, P; Zottola, T; Locatelli, C; Pollera, C; Castiglioni, B; Scaccabarozzi, L; Moroni, P

    2013-01-01

    Staphylococcus aureus is an important human and animal pathogen, and is regarded as an important cause of intramammary infection (IMI) in ruminants. Staphylococcus aureus genetic variability and virulence factors have been well studied in veterinary medicine, especially in cows as support for control and management of IMI. The aim of the present study was to genotype 71 Staph. aureus isolates from the bulk tank and foremilk of water buffaloes (n=40) and from udder tissue (n=7) and foremilk (n=24) from small ruminants. The method used was previously applied to bovine Staph. aureus and is based on the amplification of the 16S-23S rRNA intergenic spacer region. The technique applied was able to identify different Staph. aureus genotypes isolated from dairy species other than the bovine species, and cluster the genotypes according to species and herds. Virulence gene distribution was consistent with genotype differentiation. The isolates were also characterized through determination of the presence of 19 virulence-associated genes by specific PCR. Enterotoxins A, C, D, G, I, J, and L were associated with Staph. aureus isolates from buffaloes, whereas enterotoxins C and L were linked to small ruminants. Genes coding for methicillin resistance, Panton-Valentine leukocidin, exfoliative toxins A and B, and enterotoxins B, E, and H were undetected. These findings indicate that RNA template-specific PCR is a valid technique for typing Staph. aureus from buffaloes and small ruminants and is a useful tool for understanding udder infection epidemiology.

  18. Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

    PubMed Central

    Sánchez-Luque, Francisco J.; Stich, Michael; Manrubia, Susanna; Briones, Carlos; Berzal-Herranz, Alfredo

    2014-01-01

    The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5′-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5′-CCCCGGCAAGGAGGGG-3′. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication. PMID:25175101

  19. Efficient HIV-1 inhibition by a 16 nt-long RNA aptamer designed by combining in vitro selection and in silico optimisation strategies

    NASA Astrophysics Data System (ADS)

    Sánchez-Luque, Francisco J.; Stich, Michael; Manrubia, Susanna; Briones, Carlos; Berzal-Herranz, Alfredo

    2014-09-01

    The human immunodeficiency virus type-1 (HIV-1) genome contains multiple, highly conserved structural RNA domains that play key roles in essential viral processes. Interference with the function of these RNA domains either by disrupting their structures or by blocking their interaction with viral or cellular factors may seriously compromise HIV-1 viability. RNA aptamers are amongst the most promising synthetic molecules able to interact with structural domains of viral genomes. However, aptamer shortening up to their minimal active domain is usually necessary for scaling up production, what requires very time-consuming, trial-and-error approaches. Here we report on the in vitro selection of 64 nt-long specific aptamers against the complete 5'-untranslated region of HIV-1 genome, which inhibit more than 75% of HIV-1 production in a human cell line. The analysis of the selected sequences and structures allowed for the identification of a highly conserved 16 nt-long stem-loop motif containing a common 8 nt-long apical loop. Based on this result, an in silico designed 16 nt-long RNA aptamer, termed RNApt16, was synthesized, with sequence 5'-CCCCGGCAAGGAGGGG-3'. The HIV-1 inhibition efficiency of such an aptamer was close to 85%, thus constituting the shortest RNA molecule so far described that efficiently interferes with HIV-1 replication.

  20. The crystal structure of HIV reverse-transcription primer tRNA(Lys,3) shows a canonical anticodon loop.

    PubMed Central

    Bénas, P; Bec, G; Keith, G; Marquet, R; Ehresmann, C; Ehresmann, B; Dumas, P

    2000-01-01

    We have solved to 3.3 A resolution the crystal structure of the HIV reverse-transcription primer tRNA(Lys,3). The overall structure is exactly comparable to the well-known L-shape structure first revealed by yeast tRNA(Phe). In particular, it unambiguously shows a canonical anticodon loop. This contradicts previous results in short RNA fragment studies and leads us to conclude that neither frameshifting specificities of tRNA(Lys) nor tRNA(Lys,3) primer selection by HIV are due to a specific three-dimensional anticodon structure. Comparison of our structure with the results of an NMR study on a hairpin representing a nonmodified anticodon stem-loop makes plausible the conclusion that chemical modifications of the wobble base U34 to 5-methoxycarbonyl-methyl-2-thiouridine and of A37 to 2-methylthio-N-6-threonylcarbamoyl-adenosine would be responsible for a canonical 7-nt anticodon-loop structure, whereas the unmodified form would result in a noncanonical UUU short triloop. The hexagonal crystal packing is remarkable and shows tight dimers of tRNAs forming a right-handed double superhelix. Within the dimers, the tRNAs are associated head-to-tail such that the CCA end of one tRNA interacts with the anticodon of the symmetry-related tRNA. This provides us with a partial view of a codon-anticodon interaction and gives insights into the positioning of residue 37, and of its posttranscriptional modifications, relative to the first base of the codon. PMID:11073212

  1. De Novo Polymerase Activity and Oligomerization of Hepatitis C Virus RNA-Dependent RNA-Polymerases from Genotypes 1 to 5

    PubMed Central

    Bellón-Echeverría, Itxaso; Encinar, José Antonio; Martínez-Alfaro, Elisa; Pérez-Flores, Ricardo; Mas, Antonio

    2011-01-01

    Hepatitis C virus (HCV) shows a great geographical diversity reflected in the high number of circulating genotypes and subtypes. The response to HCV treatment is genotype specific, with the predominant genotype 1 showing the lowest rate of sustained virological response. Virally encoded enzymes are candidate targets for intervention. In particular, promising antiviral molecules are being developed to target the viral NS3/4A protease and NS5B polymerase. Most of the studies with the NS5B polymerase have been done with genotypes 1b and 2a, whilst information about other genotypes is scarce. Here, we have characterized the de novo activity of NS5B from genotypes 1 to 5, with emphasis on conditions for optimum activity and kinetic constants. Polymerase cooperativity was determined by calculating the Hill coefficient and oligomerization through a new FRET-based method. The Vmax/Km ratios were statistically different between genotype 1 and the other genotypes (p<0.001), mainly due to differences in Vmax values, but differences in the Hill coefficient and NS5B oligomerization were noted. Analysis of sequence changes among the studied polymerases and crystal structures show the αF helix as a structural component probably involved in NS5B-NS5B interactions. The viability of the interaction of αF and αT helixes was confirmed by docking studies and calculation of electrostatic surface potentials for genotype 1 and point mutants corresponding to mutations from different genotypes. Results presented in this study reveal the existence of genotypic differences in NS5B de novo activity and oligomerization. Furthermore, these results allow us to define two regions, one consisting of residues Glu128, Asp129, and Glu248, and the other consisting of residues of αT helix possibly involved in NS5B-NS5B interactions. PMID:21490973

  2. High-risk oncogenic HPV genotype infection associates with increased immune activation and T cell exhaustion in ART-suppressed HIV-1-infected women.

    PubMed

    Papasavvas, Emmanouil; Surrey, Lea F; Glencross, Deborah K; Azzoni, Livio; Joseph, Jocelin; Omar, Tanvier; Feldman, Michael D; Williamson, Anna-Lise; Siminya, Maureen; Swarts, Avril; Yin, Xiangfan; Liu, Qin; Firnhaber, Cynthia; Montaner, Luis J

    2016-05-01

    Persistence of human papillomavirus (HPV) and cervical disease in the context of HIV co-infection can be influenced by introduction of antiretroviral therapy (ART) and sustained immune activation despite ART. We conducted a cross-sectional study in order to evaluate immune activation/exhaustion in ART-suppressed HIV(+) women with or without high-risk (HR) HPV-related cervical intraepithelial neoplasia (CIN). 55 South African women were recruited in three groups: HR (-) (n = 16) and HR (+) (n = 15) HPV with negative cervical histopathology, and HR (+) HPV with CIN grade 1/2/3 (n = 24). Sampling included endocervical brushing (HPV DNA genotyping), Pap smear (cytology), colposcopic punch biopsy (histopathology, histochemical evaluation of immune cells), and peripheral blood (clinical assessment, flow cytometry-based immune subset characterization). Statistics were done using R2.5.1. Irrespective of the presence of CIN, HR (+) HPV women had higher circulating levels of T cells expressing markers of activation/exhaustion (CD38, PD1, CTLA-4, BTLA, CD160), Tregs, and myeloid subsets expressing corresponding ligands (PDL1, PDL2, CD86, CD40, HVEM) than HR (-) HPV women. A decrease in circulating NK cells was associated with CIN grade. CD4(+) T cell count associated negatively with T cell exhaustion and expression of negative regulators on myeloid cells. Women with CIN when compared to HR (-) HPV women, had higher cervical cell density in stroma and epithelium for CD4(+), CD68(+), and CD11c(+) cells, and only in stroma for CD8(+) cells. We conclude that in ART-suppressed HIV-infected women with HPV co-infection the levels of T and myeloid cell activation/exhaustion are associated with the presence of HR HPV genotypes. PMID:27467943

  3. Diagnostic accuracy of the genotype MTBDRsl assay for rapid diagnosis of extensively drug-resistant tuberculosis in HIV-coinfected patients.

    PubMed

    Kontsevaya, Irina; Ignatyeva, Olga; Nikolayevskyy, Vladyslav; Balabanova, Yanina; Kovalyov, Alexander; Kritsky, Andrey; Matskevich, Olesya; Drobniewski, Francis

    2013-01-01

    The Russian Federation is a high-tuberculosis (TB)-burden country with high rates of Mycobacterium tuberculosis multidrug resistance (MDR) and extensive drug resistance (XDR), especially in HIV-coinfected patients. Rapid and reliable diagnosis for detection of resistance to second-line drugs is vital for adequate patient management. We evaluated the performance of the GenoType MTBDRsl (Hain Lifescience GmbH, Nehren, Germany) assay on smear-positive sputum specimens obtained from 90 HIV-infected MDR TB patients from Russia. Test interpretability was over 98%. Specificity was over 86% for all drugs, while sensitivity varied, being the highest (71.4%) for capreomycin and lowest (9.4%) for kanamycin, probably due to the presence of mutations in the eis gene. The sensitivity of detection of XDR TB was 13.6%, increasing to 42.9% if kanamycin (not commonly used in Western Europe) was excluded. The assay is a highly specific screening tool for XDR detection in direct specimens from HIV-coinfected TB patients but cannot be used to rule out XDR TB. PMID:23152552

  4. Distribution of natural resistance to NS3 protease inhibitors in hepatitis C genotype 1a separated into clades 1 and 2 and in genotype 1b of HIV-infected patients.

    PubMed

    Bagaglio, S; Uberti-Foppa, C; Messina, E; Merli, M; Hasson, H; Andolina, A; Galli, A; Lazzarin, A; Morsica, G

    2016-04-01

    Naturally occurring resistance-associated variants (RAVs) within the protease domain of hepatitis C virus (HCV) genotype (G) 1a separated into clades 1 and 2, and G1b were investigated in 59 HIV/HCV coinfected patients. RAVs were detected in 10/23 G1a/clade 1 and 1/19 G1b (p 0.0059). A similar frequency of RAVs was found when comparing G1a/clade 2 and G1b (p 0.1672). A cross-resistance to the macrocyclic compounds simeprevir and paritaprevir was detected in two G1a/clade 2 and 1 G1b sequences and none of G1a/clade 1 sequences. The simultaneous characterization of subtype and natural RAVs by population analysis of the NS3 domain by may add important information for anti-HCV treatment strategies including protease inhibitors.

  5. HIV-1 Rev protein specifies the viral RNA export pathway by suppressing TAP/NXF1 recruitment

    PubMed Central

    Taniguchi, Ichiro; Mabuchi, Naoto; Ohno, Mutsuhito

    2014-01-01

    Nuclear RNA export pathways in eukaryotes are often linked to the fate of a given RNA. Therefore, the choice of export pathway should be well-controlled to avoid an unfavorable effect on gene expression. Although some RNAs could be exported by more than one pathway, little is known about how the choice is regulated. This issue is highlighted when the human immunodeficiency virus type 1 (HIV-1) Rev protein induces the export of singly spliced and unspliced HIV-1 transcripts. How these RNAs are exported is not well understood because such transcripts should have the possibility of utilizing CRM1-dependent export via Rev or cellular TAP/NXF1-dependent export via the transcription/export (TREX) complex, or both. Here we found that Rev suppressed TAP/NXF1-dependent export of model RNA substrates that recapitulated viral transcripts. In this effect, Rev interacted with the cap-binding complex and inhibited the recruitment of the TREX complex. Thus, Rev controls the identity of the factor occupying the cap-proximal region that determines the RNA export pathway. This ribonucleoprotein remodeling activity of Rev may favor viral gene expression. PMID:24753416

  6. Persistent CSF but not plasma HIV RNA is associated with increased risk of new-onset moderate-to-severe depressive symptoms; a prospective cohort study.

    PubMed

    Hammond, Edward R; Crum, Rosa M; Treisman, Glenn J; Mehta, Shruti H; Clifford, David B; Ellis, Ronald J; Gelman, Benjamin B; Grant, Igor; Letendre, Scott L; Marra, Christina M; Morgello, Susan; Simpson, David M; Mcarthur, Justin C

    2016-08-01

    Major depressive disorder is the most common neuropsychiatric complication in human immunodeficiency virus (HIV) infections and is associated with worse clinical outcomes. We determined if detectable cerebrospinal fluid (CSF) HIV ribonucleic acid (RNA) at threshold ≥50 copies/ml is associated with increased risk of depression. The CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort is a six-center US-based prospective cohort with bi-annual follow-up of 674 participants. We fit linear mixed models (N = 233) and discrete-time survival models (N = 154; 832 observations) to evaluate trajectories of Beck Depression Inventory (BDI) II scores and the incidence of new-onset moderate-to-severe depressive symptoms (BDI ≥ 17) among participants on combination antiretroviral therapy (cART), who were free of depression at study entry and received a minimum of three CSF examinations over 2496 person-months follow-up. Detectable CSF HIV RNA (threshold ≥50 copies/ml) at any visit was associated with a 4.7-fold increase in new-onset depression at subsequent visits adjusted for plasma HIV RNA and treatment adherence; hazard ratio (HR) = 4.76, (95 % CI 1.58-14.3); P = 0.006. Depression (BDI) scores were 2.53 points higher (95 % CI 0.47-4.60; P = 0.02) over 6 months if CSF HIV RNA was detectable at a prior study visit in fully adjusted models including age, sex, race, education, plasma HIV RNA, duration and adherence of CART, and lifetime depression diagnosis by Diagnostic Statistical Manual (DSM-IV) criteria. Persistent CSF but not plasma HIV RNA is associated with an increased risk for new-onset depression. Further research evaluating the role of immune activation and inflammatory markers may improve our understanding of this association. PMID:26727907

  7. Persistent CSF but not plasma HIV RNA is associated with increased risk of new-onset moderate-to-severe depressive symptoms; a prospective cohort study.

    PubMed

    Hammond, Edward R; Crum, Rosa M; Treisman, Glenn J; Mehta, Shruti H; Clifford, David B; Ellis, Ronald J; Gelman, Benjamin B; Grant, Igor; Letendre, Scott L; Marra, Christina M; Morgello, Susan; Simpson, David M; Mcarthur, Justin C

    2016-08-01

    Major depressive disorder is the most common neuropsychiatric complication in human immunodeficiency virus (HIV) infections and is associated with worse clinical outcomes. We determined if detectable cerebrospinal fluid (CSF) HIV ribonucleic acid (RNA) at threshold ≥50 copies/ml is associated with increased risk of depression. The CNS HIV Anti-Retroviral Therapy Effects Research (CHARTER) cohort is a six-center US-based prospective cohort with bi-annual follow-up of 674 participants. We fit linear mixed models (N = 233) and discrete-time survival models (N = 154; 832 observations) to evaluate trajectories of Beck Depression Inventory (BDI) II scores and the incidence of new-onset moderate-to-severe depressive symptoms (BDI ≥ 17) among participants on combination antiretroviral therapy (cART), who were free of depression at study entry and received a minimum of three CSF examinations over 2496 person-months follow-up. Detectable CSF HIV RNA (threshold ≥50 copies/ml) at any visit was associated with a 4.7-fold increase in new-onset depression at subsequent visits adjusted for plasma HIV RNA and treatment adherence; hazard ratio (HR) = 4.76, (95 % CI 1.58-14.3); P = 0.006. Depression (BDI) scores were 2.53 points higher (95 % CI 0.47-4.60; P = 0.02) over 6 months if CSF HIV RNA was detectable at a prior study visit in fully adjusted models including age, sex, race, education, plasma HIV RNA, duration and adherence of CART, and lifetime depression diagnosis by Diagnostic Statistical Manual (DSM-IV) criteria. Persistent CSF but not plasma HIV RNA is associated with an increased risk for new-onset depression. Further research evaluating the role of immune activation and inflammatory markers may improve our understanding of this association.

  8. Factors associated with high transmission risk and detectable plasma HIV RNA in HIV-infected MSM on ART.

    PubMed

    Blumenthal, Jill; Haubrich, Richard; Jain, Sonia; Sun, Xiaoying; Dube, Michael; Daar, Eric; Milam, Joel; Morris, Sheldon

    2014-09-01

    Summary HIV transmission risk is increased during antiretroviral therapy (ART) use if individuals are not virologically suppressed and engage in high risk transmission behaviour. Baseline data of HIV-infected men who have sex with men (MSM) with recent history of risky behaviour on ART for ≥3 months (n = 139) were evaluated to assess predictors of detectable viraemia and HIV transmission risk-taking behaviour. Twenty-four subjects had viral load (VL) >75 copies/mL and 12 had VL >1000 copies/mL. In multivariable regression analyses, subjects with VL >75 copies/mL were more likely to be Black (OR = 4.48, p = 0.007), have lower CD4 cell counts (OR = 0.727, p = 0.005) and have used methamphetamines in the last month (OR = 6.64, p = 0.019). Subjects with VL >1000 copies/mL were more likely to have lower CD4 cell counts (OR = 0.494, p = 0.004), report <90% adherence (OR = 7.94; p = 0.046) and have used methamphetamines in the last month (OR = 10.01, p = 0.034). Subjects with VL >75 copies/mL with the greatest transmission risk behaviour (n = 14) were more likely to be Black (OR = 8.00, p = 0.006), have lower CD4 cell counts (OR = 0.657, p = 0.009) and have used methamphetamines in the last month (OR = 5.20, p = 0.042). High risk HIV transmission behaviour with viraemia occurred in 10% of the cohort. Future efforts to reduce HIV transmission among MSM on ART will require combined interventions that target risk-taking behaviours and substance use.

  9. Basal microRNA expression patterns in reward circuitry of selectively bred high-responder and low-responder rats vary by brain region and genotype

    PubMed Central

    Hamilton, David E.; Cooke, Christopher L.; Carter, Bradley S.; Akil, Huda; Watson, Stanley J.

    2014-01-01

    Mental health disorders involving altered reward, emotionality, and anxiety are thought to result from the interaction of individual predisposition (genetic factors) and personal experience (environmental factors), although the mechanisms that contribute to an individual's vulnerability to these disorders remain poorly understood. We used an animal model of individual variation [inbred high-responder/low-responder (bHR-bLR) rodents] known to vary in reward, anxiety, and emotional processing to examine neuroanatomical expression patterns of microRNAs (miRNAs). Laser capture microdissection was used to dissect the prelimbic cortex and the nucleus accumbens core and shell prior to analysis of basal miRNA expression in bHR and bLR male rats. These studies identified 187 miRNAs differentially expressed by genotype in at least one brain region, 10 of which were validated by qPCR. Four of these 10 qPCR-validated miRNAs demonstrated differential expression across multiple brain regions, and all miRNAs with validated differential expression between genotypes had lower expression in bHR animals compared with bLR animals. microRNA (miR)-484 and miR-128a expression differences between the prelimbic cortex of bHR and bLR animals were validated by semiquantitative in situ hybridization. miRNA expression analysis independent of genotype identified 101 miRNAs differentially expressed by brain region, seven of which validated by qPCR. Dnmt3a mRNA, a validated target of miR-29b, varied in a direction opposite that of miR-29b's differential expression between bHR and bLR animals. These data provide evidence that basal central nervous system miRNA expression varies in the bHR-bLR model, implicating microRNAs as potential epigenetic regulators of key neural circuits and individual differences associated with mental health disorders. PMID:24569673

  10. Correlation between human immunodeficiency virus type 1 (HIV-1) RNA measurements obtained with dried blood spots and those obtained with plasma by use of Nuclisens EasyQ HIV-1 and Abbott RealTime HIV load tests.

    PubMed

    Garrido, Carolina; Zahonero, Natalia; Corral, Angélica; Arredondo, Miguel; Soriano, Vincent; de Mendoza, Carmen

    2009-04-01

    The plasma human immunodeficiency virus (HIV) RNA load is used in the clinical routine for the monitoring of HIV infection and the patient's response to antiretroviral therapy. Other body fluids or dried blood spots (DBS) can be used, however, to assess the level of viremia. The use of DBS may be especially helpful for the monitoring of HIV-infected patients in resource-poor settings, where access to adequate laboratory facilities is often difficult. However, the correlation between the HIV RNA levels in plasma and those in DBSs has not been well established. Paired plasma and DBS samples obtained from HIV type 1 (HIV-1)-infected patients were tested for HIV RNA copy numbers by using two different commercial assays, the Nuclisens EasyQ HIV-1 (version 1.1) test (the Nuclisens test; Biomerieux) and the m2000rt RealTime HIV test (the m2000rt test; Abbott). Nucleic acid extraction was performed manually by using either the Nuclisens isolation kit (which uses the Boom methodology) or the m2000rt sample preparation kit (an iron particle-based method). A total of 103 paired plasma and DBS samples were tested. Viral load results were obtained for 97 (94.2%) samples with the Nuclisens isolation kit and 81 (78.6%) samples with the m2000rt kit. The overall correlation between the RNA loads in plasma and DBS was good, although better results were obtained by the Nuclisens test (R(2) = 0.87, P < 0.001) than by the m2000rt test (R(2) = 0.70, P < 0.001). While the specificities were excellent and similar for both the Nuclisens and the m2000rt tests (97.1% and 100%, respectively), the sensitivity was greater by the Nuclisens test than by the m2000rt test (75.8% and 56.6%, respectively). Overall, the viral loads in DBS tended to be lower than those in plasma, with mean differences of 0.3 log unit (standard deviation, 0.5 log unit) and 0.76 log unit (standard deviation, 0.8 log unit) for the Nuclisens and the m2000rt tests, respectively. The levels of agreement between the

  11. The lncRNA NRON modulates HIV-1 replication in a NFAT-dependent manner and is differentially regulated by early and late viral proteins.

    PubMed

    Imam, Hasan; Bano, Aalia Shahr; Patel, Paresh; Holla, Prasida; Jameel, Shahid

    2015-03-02

    A majority of the human genome is transcribed into noncoding RNAs, of which the functions of long noncoding RNAs (lncRNAs) are poorly understood. Many host proteins and RNAs have been characterized for their roles in HIV/AIDS pathogenesis, but there is only one lncRNA, NEAT1, which is shown to affect the HIV-1 life cycle. We profiled 90 disease-related lncRNAs and found NRON (noncoding repressor of Nuclear Factor of Activated T cells [NFAT]) to be one of several lncRNAs whose expression was significantly altered following HIV-1 infection. The regulation of NRON expression during the HIV-1 life cycle was complex; its levels were reduced by the early viral accessory protein Nef and increased by the late protein Vpu. Consequently, Nef and Vpu also modulated activity of the transcription factor NFAT. The knockdown of NRON enhanced HIV-1 replication through increased activity of NFAT and the viral LTR. Using siRNA-mediated NFAT knockdown, we show the effects of NRON on HIV-1 replication to be mediated by NFAT, and the viral Nef and Vpu proteins to modulate NFAT activity through their effects on NRON. These findings add the lncRNA, NRON to the vast repertoire of host factors utilized by HIV for infection and persistence.

  12. Immunization of HIV-1-Infected Persons With Autologous Dendritic Cells Transfected With mRNA Encoding HIV-1 Gag and Nef: Results of a Randomized, Placebo-Controlled Clinical Trial

    PubMed Central

    Kwon, Douglas S.; Macklin, Eric A.; Shopis, Janet R.; McLean, Anna P.; McBrine, Nicole; Flynn, Theresa; Peter, Lauren; Sbrolla, Amy; Kaufmann, Daniel E.; Porichis, Filippos; Walker, Bruce D.; Bhardwaj, Nina; Barouch, Dan H.; Kavanagh, Daniel G.

    2016-01-01

    Background: HIV-1 eradication may require reactivation of latent virus along with stimulation of HIV-1-specific immune responses to clear infected cells. Immunization with autologous dendritic cells (DCs) transfected with viral mRNA is a promising strategy for eliciting HIV-1-specific immune responses. We performed a randomized controlled clinical trial to evaluate the immunogenicity of this approach in HIV-1-infected persons on antiretroviral therapy. Methods: Fifteen participants were randomized 2:1 to receive intradermal immunization with HIV-1 Gag- and Nef-transfected DCs (vaccine) or mock-transfected DCs (placebo) at weeks 0, 2, 6, and 10. All participants also received DCs pulsed with keyhole limpet hemocyanin (KLH) to assess whether responses to a neo-antigen could be induced. Results: After immunization, there were no differences in interferon-gamma enzyme-linked immunospot responses to HIV-1 Gag or Nef in the vaccine or placebo group. CD4 proliferative responses to KLH increased 2.4-fold (P = 0.026) and CD8 proliferative responses to KLH increased 2.5-fold (P = 0.053) after vaccination. There were increases in CD4 proliferative responses to HIV-1 Gag (2.5-fold vs. baseline, 3.4-fold vs. placebo, P = 0.054) and HIV-1 Nef (2.3-fold vs. baseline, 6.3-fold vs. placebo, P = 0.009) among vaccine recipients, but these responses were short-lived. Conclusion: Immunization with DCs transfected with mRNA encoding HIV-1 Gag and Nef did not induce significant interferon-gamma enzyme-linked immunospot responses. There were increases in proliferative responses to HIV-1 antigens and to a neo-antigen, KLH, but the effects were transient. Dendritic cell vaccination should be optimized to elicit stronger and long-lasting immune responses for this strategy to be effective as an HIV-1 therapeutic vaccine. PMID:26379068

  13. Identification of Genetic Variation between Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli Using RNA Sequencing and Genotyping-By-Sequencing

    PubMed Central

    Summers, Carly F.; Gulliford, Colwyn M.; Carlson, Craig H.; Lillis, Jacquelyn A.; Carlson, Maryn O.; Cadle-Davidson, Lance; Gent, David H.; Smart, Christine D.

    2015-01-01

    RNA sequencing (RNA-seq) and genotyping-by-sequencing (GBS) were used for single nucleotide polymorphism (SNP) identification from two economically important obligate plant pathogens, Pseudoperonospora cubensis and P. humuli. Twenty isolates of P. cubensis and 19 isolates of P. humuli were genotyped using RNA-seq and GBS. Principle components analysis (PCA) of each data set showed genetic separation between the two species. Additionally, results supported previous findings that P. cubensis isolates from squash are genetically distinct from cucumber and cantaloupe isolates. A PCA-based procedure was used to identify SNPs correlated with the separation of the two species, with 994 and 4,231 PCA-correlated SNPs found within the RNA-seq and GBS data, respectively. The corresponding unigenes (n = 800) containing these potential species-specific SNPs were then annotated and 135 putative pathogenicity genes, including 3 effectors, were identified. The characterization of genes containing SNPs differentiating these two closely related downy mildew species may contribute to the development of improved detection and diagnosis strategies and improve our understanding of host specificity pathways. PMID:26599440

  14. Identification of Genetic Variation between Obligate Plant Pathogens Pseudoperonospora cubensis and P. humuli Using RNA Sequencing and Genotyping-By-Sequencing.

    PubMed

    Summers, Carly F; Gulliford, Colwyn M; Carlson, Craig H; Lillis, Jacquelyn A; Carlson, Maryn O; Cadle-Davidson, Lance; Gent, David H; Smart, Christine D

    2015-01-01

    RNA sequencing (RNA-seq) and genotyping-by-sequencing (GBS) were used for single nucleotide polymorphism (SNP) identification from two economically important obligate plant pathogens, Pseudoperonospora cubensis and P. humuli. Twenty isolates of P. cubensis and 19 isolates of P. humuli were genotyped using RNA-seq and GBS. Principle components analysis (PCA) of each data set showed genetic separation between the two species. Additionally, results supported previous findings that P. cubensis isolates from squash are genetically distinct from cucumber and cantaloupe isolates. A PCA-based procedure was used to identify SNPs correlated with the separation of the two species, with 994 and 4,231 PCA-correlated SNPs found within the RNA-seq and GBS data, respectively. The corresponding unigenes (n = 800) containing these potential species-specific SNPs were then annotated and 135 putative pathogenicity genes, including 3 effectors, were identified. The characterization of genes containing SNPs differentiating these two closely related downy mildew species may contribute to the development of improved detection and diagnosis strategies and improve our understanding of host specificity pathways. PMID:26599440

  15. Structural characterization of a 2'F-RNA aptamer that binds a HIV-1 SU glycoprotein, gp120.

    PubMed

    Sayer, N; Ibrahim, J; Turner, K; Tahiri-Alaoui, A; James, W

    2002-05-10

    Here we describe the isolation of specific 2'F-substituted RNA ligands for the SU glycoprotein, gp120, of HIV-1 strain HXB2. These aptamers bind the target protein with an affinity of the order of 10(-7) M. Binding was specific to SU glycoprotein and directed to a non-neutralizing epitope that was not shared with the related strain, HIV-1(BaL). The structure of one aptamer was defined by a combination of deletion analysis and enzymatic probing studies, revealing a 42 nt minimal element comprising a three-helix junction that retained the binding affinity of the parental sequence. Interestingly, binding to SU glycoprotein was accompanied by structural changes in the aptamer that stabilized the weakest of the 3 helices.

  16. HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.

    PubMed

    Stake, Matthew; Singh, Deepali; Singh, Gatikrushna; Marcela Hernandez, J; Kaddis Maldonado, Rebecca; Parent, Leslie J; Boris-Lawrie, Kathleen

    2015-12-01

    Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.

  17. A small molecule probe induces a conformation in HIV TAR RNA capable of binding drug-like fragments

    PubMed Central

    Davidson, Amy; Begley, Darren W.; Lau, Carmen; Varani, Gabriele

    2011-01-01

    The HIV-1 TAR/Tat interaction is a potentially valuable target for treating HIV infection, but efforts to develop TAR-binding antiviral drugs have not yet yielded a successful candidate for clinical development. In this work, we describe a novel approach towards screening fragments against RNA that uses a chemical probe to target the Tat binding region of TAR. This probe fulfills two critical roles in the screen: by locking the RNA into a conformation capable of binding other fragments, it simultaneously allows the identification of proximal binding fragments by ligand-based NMR. Using this approach, we have discovered six novel TAR-binding fragments, three of which were docked relative to the probe/RNA structure using experimental NMR restraints. The consistent orientations of functional groups in our data-driven docked structures and common electrostatic properties across all fragment leads reveal a surprising level of selectivity by our fragment-sized screening hits. These models further suggest linking strategies for the development of higher affinity lead compounds for the inhibition of the TAR/Tat interaction. PMID:21763501

  18. HIV-1 and two avian retroviral 5' untranslated regions bind orthologous human and chicken RNA binding proteins.

    PubMed

    Stake, Matthew; Singh, Deepali; Singh, Gatikrushna; Marcela Hernandez, J; Kaddis Maldonado, Rebecca; Parent, Leslie J; Boris-Lawrie, Kathleen

    2015-12-01

    Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages. PMID:26584240

  19. HIV-1 TAR element is processed by Dicer to yield a viral micro-RNA involved in chromatin remodeling of the viral LTR

    PubMed Central

    Klase, Zachary; Kale, Prachee; Winograd, Rafael; Gupta, Madhur V; Heydarian, Mohammad; Berro, Reem; McCaffrey, Timothy; Kashanchi, Fatah

    2007-01-01

    Background RNA interference (RNAi) is a regulatory mechanism conserved in higher eukaryotes. The RNAi pathway generates small interfering RNA (siRNA) or micro RNA (miRNA) from either long double stranded stretches of RNA or RNA hairpins, respectively. The siRNA or miRNA then guides an effector complex to a homologous sequence of mRNA and regulates suppression of gene expression through one of several mechanisms. The suppression of gene expression through these mechanisms serves to regulate endogenous gene expression and protect the cell from foreign nucleic acids. There is growing evidence that many viruses have developed in the context of RNAi and express either a suppressor of RNAi or their own viral miRNA. Results In this study we investigated the possibility that the HIV-1 TAR element, a hairpin structure of ~50 nucleotides found at the 5' end of the HIV viral mRNA, is recognized by the RNAi machinery and processed to yield a viral miRNA. We show that the protein Dicer, the enzyme responsible for cleaving miRNA and siRNA from longer RNA sequences, is expressed in CD4+ T-cells. Interestingly, the level of expression of Dicer in monocytes is sub-optimal, suggesting a possible role for RNAi in maintaining latency in T-cells. Using a biotin labeled TAR element we demonstrate that Dicer binds to this structure. We show that recombinant Dicer is capable of cleaving the TAR element in vitro and that TAR derived miRNA is present in HIV-1 infected cell lines and primary T-cell blasts. Finally, we show that a TAR derived miRNA is capable of regulating viral gene expression and may be involved in repressing gene expression through transcriptional silencing. Conclusion HIV-1 TAR element is processed by the Dicer enzyme to create a viral miRNA. This viral miRNA is detectable in infected cells and appears to contribute to viral latency. PMID:17663774

  20. ASSESEMENT OF THE SEVERITY OF IMMUNODEFICIENCY IN PATIENTS OF ASIAN ETHNICITY WITH HIV/HCV CO-INFECTION.

    PubMed

    Begaidarova, R; Asenova, L; Alshynbekova, G; Devdariani, Kh; Dyusembaeva, A; Starikov, Y; Zolotareva, O

    2016-05-01

    The aim of the study was to determine the HIV RNA viral load and CD4+ cell count to assess the severity of immune deficiency in patients of Asian ethnicity with HIV/HCV co-infection by different HCV genotypes. 181 HIV-infected patients of Asian ethnicity were studied from several regions of Kazakhstan, predominantly from Karaganda region, including prisons. The diagnosis of HIV infection was confirmed by immunoblotting after a positive ELISA screening test. The patient data for analysis were extracted from the patients' medical charts. Analysis of peripheral blood and urine, blood biochemistry tests were performed for each patient. The flow cytometry was used to determine the CD3, CD4 and CD8 lymphocytes in the whole blood. Detection and quantification of HCV RNA in clinical samples of serum or plasma was performed by reverse transcription of viral RNA followed by amplification using DNA real-time polymerase chain reaction (RT-qPCR) with hybridization-fluorescence detection of PCR products in real-time. Statistical analysis was performed using STATISTICA software package. Student's t test was used to evaluate the differences between the means. HCV antibodies were detected in 73 patients out of 181. HCV genotyping showed presence of first genotype in 18 (24.7%), genotype 2 in 19 (26.0%) and genotype 3 in 36 (49.3%) patients. The third genotype was the most prevalent according to our study. More severe immunosuppression was observed in patients with HCV genotypes 1 and 3 in comparison with those with HCV genotype 2. HCV infection is a negative risk-factor in the course of HIV infection, accelerating the decrease of CD4+ cells. The greatest risk of progression of HIV infection to AIDS occurs in the presence of HCV genotype 1 and 3 which can be explained by more aggressive course of the disease and a poorer prognosis in comparison with the infection with genotype 2. PMID:27348168

  1. Changes in HIV-1 Subtypes B and C Genital Tract RNA in Women and Men After Initiation of Antiretroviral Therapy

    PubMed Central

    Fiscus, Susan A.; Cu-Uvin, Susan; Eshete, Abel Tilahun; Hughes, Michael D.; Bao, Yajing; Hosseinipour, Mina; Grinsztejn, Beatriz; Badal-Faesen, Sharlaa; Dragavon, Joan; Coombs, Robert W.; Braun, Ken; Moran, Laura; Hakim, James; Flanigan, Timothy; Kumarasamy, N.; Campbell, Thomas B.; Klingman, Karin L.; Nair, Apsara; Walawander, Ann; Smeaton, Laura M.; De Gruttola, Victor; Martinez, Ana I.; Swann, Edith; Barnett, Ronald L.; Brizz, Barbara; Delph, Yvette; Gettinger, Nikki; Mitsuyasu, Ronald T.; Eshleman, Susan; Safren, Steven; Andrade, Adriana; Haas, David W.; Amod, Farida; Berthaud, Vladimir; Bollinger, Robert C.; Bryson, Yvonne; Celentano, David; Chilongozi, David; Cohen, Myron; Collier, Ann C.; Currier, Judith Silverstein; Eron, Joseph; Firnhaber, Cynthia; Flexner, Charles; Gallant, Joel E.; Gulick, Roy M.; Hammer, Scott M.; Hoffman, Irving; Kazembe, Peter; Kumwenda, Johnstone; Kumwenda, Newton; Lama, Javier R.; Lawrence, Jody; Maponga, Chiedza; Martinson, Francis; Mayer, Kenneth; Nielsen, Karin; Pendame, Richard B.; Ramratnam, Bharat; Rooney, James F.; Sanchez, Jorge; Sanne, Ian; Schooley, Robert T.; Snowden, Wendy; Solomon, Suniti; Tabet, Steve; Taha, Taha; Uy, Jonathan; van der Horst, Charles; Wanke, Christine; Gormley, Joan; Marcus, Cheryl J.; Putnam, Beverly; Ntshele, Smanga; Loeliger, Edde; Pappa, Keith A.; Webb, Nancy; Shugarts, David L.; Winters, Mark A.; Descallar, Renard S.; Sharma, Jabin; Poongulali, S.; Cardoso, Sandra Wagner; Faria, Deise Lucia; Berendes, Sima; Burke, Kelly; Kanyama, Cecelia; Kayoyo, Virginia; Samaneka, Wadzanai P.; Chisada, Anthony; Santos, Breno; La Rosa, Alberto; Infante, Rosa; Balfour, Henry H.; Mullan, Beth; Kim, Ge-Youl; Klebert, Michael K.; Mildvan, Donna; Revuelta, Manuel; Jan Geiseler, P.; Santos, Bartolo; Daar, Eric S.; Lopez, Ruben; Frarey, Laurie; Currin, David; Haas, David H.; Bailey, Vicki L.; Tebas, Pablo; Zifchak, Larisa; Sha, Beverly E.; Fritsche, Janice M.

    2013-01-01

    Background. Combination antiretroviral therapy (cART) reduces genital tract human immunodeficiency virus type 1 (HIV-1) load and reduces the risk of sexual transmission, but little is known about the efficacy of cART for decreasing genital tract viral load (GTVL) and differences in sex or HIV-1 subtype. Methods. HIV-1 RNA from blood plasma, seminal plasma, or cervical wicks was quantified at baseline and at weeks 48 and 96 after entry in a randomized clinical trial of 3 cART regimens. Results. One hundred fifty-eight men and 170 women from 7 countries were studied (men: 55% subtype B and 45% subtype C; women: 24% subtype B and 76% subtype C). Despite similar baseline CD4+ cell counts and blood plasma viral loads, women with subtype C had the highest GTVL (median, 5.1 log10 copies/mL) compared to women with subtype B and men with subtype C or B (4.0, 4.0, and 3.8 log10 copies/mL, respectively; P < .001). The proportion of participants with a GTVL below the lower limit of quantification (LLQ) at week 48 (90%) and week 96 (90%) was increased compared to baseline (16%; P < .001 at both times). Women were significantly less likely to have GTVL below the LLQ compared to men (84% vs 94% at week 48, P = .006; 84% vs 97% at week 96, P = .002), despite a more sensitive assay for seminal plasma than for cervical wicks. No difference in GTVL response across the 3 cART regimens was detected. Conclusions. The female genital tract may serve as a reservoir of persistent HIV-1 replication during cART and affect the use of cART to prevent sexual and perinatal transmission of HIV-1. PMID:23532477

  2. HIV-Derived ssRNA Binds to TLR8 to Induce Inflammation-Driven Macrophage Foam Cell Formation

    PubMed Central

    Bernard, Mark A.; Han, Xinbing; Inderbitzin, Sonya; Agbim, Ifunanya; Zhao, Hui; Koziel, Henry; Tachado, Souvenir D.

    2014-01-01

    Even though combined anti-retroviral therapy (cART) dramatically improves patient survival, they remain at a higher risk of being afflicted with non-infectious complications such as cardiovascular disease (CVD). This increased risk is linked to persistent inflammation and chronic immune activation. In this study, we assessed whether this complication is related to HIV-derived ssRNAs inducing in macrophages increases in TNFα release through TLR8 activation leading to foam cell formation. HIV ssRNAs induced foam cell formation in monocyte-derived macrophages (MDMs) in a dose-dependent manner. This response was reduced when either endocytosis or endosomal acidification was inhibited by dynasore or chloroquine, respectively. Using a flow cytometry FRET assay, we demonstrated that ssRNAs bind to TLR8 in HEK cells. In MDMs, ssRNAs triggered a TLR8-mediated inflammatory response that ultimately lead to foam cell formation. Targeted silencing of the TLR8 and MYD88 genes reduced foam cell formation. Furthermore, foam cell formation induced by these ssRNAs was blocked by an anti-TNFα neutralizing antibody. Taken together in MDMs, HIV ssRNAs are internalized; bind TLR8 in the endosome followed by endosomal acidification. TLR8 signaling then triggers TNFα release and ultimately leads to foam cell formation. As this response was inhibited by a blocking anti-TNFα antibody, drug targeting HIV ssRNA-driven TLR8 activation may serve as a potential therapeutic target to reduce chronic immune activation and inflammation leading to CVD in HIV+ patients. PMID:25090652

  3. Effects of co-infection with hepatitis C virus and GB virus C on CD4 cell count and HIV-RNA level among HIV-infected patients treated with highly active antiretroviral therapy.

    PubMed

    Voirin, Nicolas; Trépo, Christian; Estève, Jacques; Chevallier, Philippe; Ritter, Jacques; Fabry, Jacques; Vanhems, Philippe

    2002-07-26

    The effects of co-infection with hepatitis C virus (HCV) and GB virus C (GBV-C) on CD4 cell counts and plasma HIV-RNA levels has been investigated in HIV-infected patients treated with highly active antiretroviral therapy (HAART). Patients co-infected with HCV and GBV-C experienced a CD4 cell increase during 4 years of HAART, whereas the increase stopped after 2 years in the other groups.

  4. H2 genotypes of G4P[6], G5P[7], and G9[23] porcine rotaviruses show super-short RNA electropherotypes.

    PubMed

    Nagai, Makoto; Shimada, Saya; Fujii, Yoshiki; Moriyama, Hiromitsu; Oba, Mami; Katayama, Yukie; Tsuchiaka, Shinobu; Okazaki, Sachiko; Omatsu, Tsutomu; Furuya, Tetsuya; Koyama, Satoshi; Shirai, Junsuke; Katayama, Kazuhiko; Mizutani, Tetsuya

    2015-04-17

    During group A rotavirus (RVA) surveillance of pig farms in Japan, we detected three RVA strains (G4P[6], G5P[7], and G9P[23] genotypes), which showed super-short RNA patterns by polyacrylamide gel electrophoresis, in samples from a healthy eight-day-old pig and two pigs of seven and eight days old with diarrhea from three farms. Reverse transcription PCR and sequencing revealed that the full-length NSP5 gene of these strains contained 952 or 945 nucleotides, which is consistent with their super-short electropherotypes. Due to a lack of whole genome data on Japanese porcine RVAs, we performed whole genomic analyses of the three strains. The genomic segments of these RVA strains showed typical porcine RVA constellations, except for H2 NSP5 genotype, (G4,5,9-P[6,7,23]-I5-R1-C1-M1-A8-N1-T1-E1-H2 representing VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes). In phylogenetic analyses, these porcine RVA strains clustered with porcine and porcine-like human RVA strains and showed a typical porcine RVA backbone, except for the NSP5 gene; however, intra-genotype reassortment events among porcine and porcine-like human RVA strains were observed. The NSP5 gene segments of these strains were clustered within the H2b genotype with super-short human RVA strains. The H2 genotype has to date only been identified in human and lapine RVA strains. Thus, to our knowledge, this report presents the first case of H2 NSP5 genotype showing a super-short RNA pattern in porcine RVA. These data suggest the possibility of interspecies transmission between pigs and humans and imply that super-short porcine RVA strains possessing H2 genotype are circulating among both asymptomatic and diarrheic porcine populations in Japan. PMID:25724331

  5. HIV

    PubMed Central

    Chawla, Sumit; Sahoo, Soumya Swaroop; Jain, Rambilas; Khanna, Pardeep; Mehta, Bharti; Singh, Inderjeet

    2014-01-01

    Getting to zero: zero new HIV infections, zero deaths from AIDS-related illness, zero discrimination is the theme of World AIDS Day 2012. Given the spread of the epidemic today, getting to zero may sound difficult, but significant progress is underway. The total annual loss for the entire country due to HIV is 7% of GDP, which exceeds India’s annual health expenditure in 2004. The additional loss due to loss of labor income and increased medical expenditure as measured by the external transfers, account for 5% of the country’s health expenditure and 0.23% of GDP. Given that the HIV incidence rate is only 0.27% in India, these losses are quite staggering. Despite the remarkable achievements in development of anti-retroviral therapies against HIV and the recent advances in new prevention technologies, the rate of new HIV infections continue to outpace efforts on HIV prevention and control. Thus, the development of a safe and effective vaccine for prevention and control of AIDS remains a global public health priority and the greatest opportunity to eventually end the AIDS pandemic. PMID:24056755

  6. A Short Sequence Motif in the 5′ Leader of the HIV-1 Genome Modulates Extended RNA Dimer Formation and Virus Replication*

    PubMed Central

    van Bel, Nikki; Das, Atze T.; Cornelissen, Marion; Abbink, Truus E. M.; Berkhout, Ben

    2014-01-01

    The 5′ leader of the HIV-1 RNA genome encodes signals that control various steps in the replication cycle, including the dimerization initiation signal (DIS) that triggers RNA dimerization. The DIS folds a hairpin structure with a palindromic sequence in the loop that allows RNA dimerization via intermolecular kissing loop (KL) base pairing. The KL dimer can be stabilized by including the DIS stem nucleotides in the intermolecular base pairing, forming an extended dimer (ED). The role of the ED RNA dimer in HIV-1 replication has hardly been addressed because of technical challenges. We analyzed a set of leader mutants with a stabilized DIS hairpin for in vitro RNA dimerization and virus replication in T cells. In agreement with previous observations, DIS hairpin stability modulated KL and ED dimerization. An unexpected previous finding was that mutation of three nucleotides immediately upstream of the DIS hairpin significantly reduced in vitro ED formation. In this study, we tested such mutants in vivo for the importance of the ED in HIV-1 biology. Mutants with a stabilized DIS hairpin replicated less efficiently than WT HIV-1. This defect was most severe when the upstream sequence motif was altered. Virus evolution experiments with the defective mutants yielded fast replicating HIV-1 variants with second site mutations that (partially) restored the WT hairpin stability. Characterization of the mutant and revertant RNA molecules and the corresponding viruses confirmed the correlation between in vitro ED RNA dimer formation and efficient virus replication, thus indicating that the ED structure is important for HIV-1 replication. PMID:25368321

  7. Inhibition of Rev-mediated HIV-1 expression by an RNA binding protein encoded by the interferon-inducible 9-27 gene

    SciTech Connect

    Constantoulakis, P.; Campbell, M.; Felber, B.K.; Nasioulas, G.; Afonina, E.; Pavlakis, G.N. )

    1993-02-26

    Interferon inhibits expression of human immunodeficiency virus type-1 (HIV-1) through unknown mechanisms. A gene inducible by interferon-[alpha] (IFN-[alpha]) and interferon-[gamma] (IFN-[gamma]) was isolated by screening of a human complementary DNA library for proteins binding to the Rev-responsive element (RRE) of HIV-1. The product of this gene, RBP9-27, was shown to bind RNA in vitro and to inhibit HIV-1 expression after transfection into human cells. RBP9-27 primarily inhibited Rev-dependent posttransscriptional steps of viral gene expression. Thus, RBP9-27 is a cellular factor that antagonizes Rev function. These results suggest an inteferon-induced antiviral mechanism operating through the induction of RNA binding proteins such as RBP9-27. Elucidation of RBP9-27 function may lead to a better understanding of the mechanism of interferon action during HIV-1 infection. 29 refs., 4 figs.

  8. Validation of the Gen-Probe Aptima qualitative HIV-1 RNA assay for diagnosis of human immunodeficiency virus infection in infants.

    PubMed

    Fiscus, Susan A; McMillion, Takesha; Nelson, Julie A E; Miller, William C

    2013-12-01

    The qualitative Roche HIV-1 DNA Amplicor assay has been used for the past 20 years to diagnose HIV infection in infants and young children but is being phased out; hence, alternative assays must be found. The Gen-Probe Aptima qualitative HIV-1 RNA assay is currently the only FDA-cleared HIV-1 nucleic acid assay approved for diagnosis, but data on the use of this assay with infant plasma are limited. We assessed Aptima's performance using control material for reproducibility and limit of detection and 394 plasma samples (0.2 to 0.5 ml) from HIV-exposed infected and uninfected infants and children for analytical sensitivity and specificity. Assays to assess within-run repeatability and between-run reproducibility indicated that the controls with 10,000 (5 of 5), 200 (5 of 5), 100 (16 of 16), 50 (12 of 12), and 25 (20 of 20) HIV-1 RNA copies/ml (cp/ml) were always positive, and negatives were always negative (20 of 20). The limit of detection was 14 cp/ml, as determined by probit analysis. The analytic sensitivity of the assay was 99.5% (189/190 samples; 95% confidence interval [CI], 97.1 to 99.9%) and specificity was 99.5% (199/200 samples; 95% CI, 97.2 to 99.9%). These results suggest that the assay is suitable for early infant diagnosis of HIV-1.

  9. Altered subcellular localization of the NeuN/Rbfox3 RNA splicing factor in HIV-associated neurocognitive disorders (HAND).

    PubMed

    Lucas, Calixto-Hope; Calvez, Mathilde; Babu, Roshni; Brown, Amanda

    2014-01-13

    The anti-NeuN antibody has been widely used for over 15 years to unambiguously identify post-mitotic neurons in the central nervous system of a wide variety of vertebrates including mice, rats and humans. In contrast to its widely reported nuclear localization, we found significantly higher NeuN reactivity in the cytoplasm of neurons in brain sections from HIV-infected individuals with cognitive impairment compared to controls. The protein target of anti-NeuN antisera was recently identified as the neuron-specific RNA splicing factor, Rbfox3, but its significance in diseases affecting the brain has not been previously reported. RNA splicing occurs in the nucleus hence, the altered localization of RbFox3 to the cytoplasm may lead to the downregulation of neuronal gene expression.

  10. Intra-host evolution of multiple genotypes of hepatitis C virus in a chronically infected patient with HIV along a 13-year follow-up period.

    PubMed

    Culasso, A C A; Baré, P; Aloisi, N; Monzani, M C; Corti, M; Campos, R H

    2014-01-20

    The intra-host evolutionary process of hepatitis C virus (HCV) was analyzed by phylogenetic and coalescent methodologies in a patient co-infected with HCV-1a, HCV-2a, HCV-3a and human immunodeficiency virus (HIV) along a 13-year period. Direct sequence analysis of the E2 and NS5A regions showed diverse evolutionary dynamics, in agreement with different relationships between these regions and the host factors. The Bayesian Skyline Plot analyses of the E2 sequences (cloned) yielded different intra-host evolutionary patterns for each genotype: a steady state of a "consensus" sequence for HCV-1a; a pattern of lineage splitting and extinction for HCV-2a; and a two-phase (drift/diversification) process for HCV-3a. Each genotype evolving in the same patient and at the same time presents a different pattern apparently modulated by the immune pressure of the host. This study provides useful information for the management of co-infected patients and provides insights into the mechanisms behind the intra-host evolution of HCV.

  11. 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.

  12. An Intronic G Run within HIV-1 Intron 2 Is Critical for Splicing Regulation of vif mRNA

    PubMed Central

    Widera, Marek; Erkelenz, Steffen; Hillebrand, Frank; Krikoni, Aikaterini; Widera, Darius; Kaisers, Wolfgang; Deenen, René; Gombert, Michael; Dellen, Rafael; Pfeiffer, Tanya; Kaltschmidt, Barbara; Münk, Carsten; Bosch, Valerie; Köhrer, Karl

    2013-01-01

    Within target T lymphocytes, human immunodeficiency virus type I (HIV-1) encounters the retroviral restriction factor APOBEC3G (apolipoprotein B mRNA-editing enzyme, catalytic polypeptide-like 3G; A3G), which is counteracted by the HIV-1 accessory protein Vif. Vif is encoded by intron-containing viral RNAs that are generated by splicing at 3′ splice site (3′ss) A1 but lack splicing at 5′ss D2, which results in the retention of a large downstream intron. Hence, the extents of activation of 3′ss A1 and repression of D2, respectively, determine the levels of vif mRNA and thus the ability to evade A3G-mediated antiviral effects. The use of 3′ss A1 can be enhanced or repressed by splicing regulatory elements that control the recognition of downstream 5′ss D2. Here we show that an intronic G run (GI2-1) represses the use of a second 5′ss, termed D2b, that is embedded within intron 2 and, as determined by RNA deep-sequencing analysis, is normally inefficiently used. Mutations of GI2-1 and activation of D2b led to the generation of transcripts coding for Gp41 and Rev protein isoforms but primarily led to considerable upregulation of vif mRNA expression. We further demonstrate, however, that higher levels of Vif protein are actually detrimental to viral replication in A3G-expressing T cell lines but not in A3G-deficient cells. These observations suggest that an appropriate ratio of Vif-to-A3G protein levels is required for optimal virus replication and that part of Vif level regulation is effected by the novel G run identified here. PMID:23255806

  13. MicroRNA Profile in CD8+ T-Lymphocytes from HIV-Infected Individuals: Relationship with Antiviral Immune Response and Disease Progression

    PubMed Central

    Bargalló, Manel E.; Planet, Evarist; Vilaplana, Elisenda; Escribà, Tuixent; Pérez, Iñaki; Gatell, Josep Maria; García, Felipe; Arnedo, Mireia; Plana M, Montserrat

    2016-01-01

    Background The relationship between host microRNAs (miRNA), viral control and immune response has not yet been elucidated in the field of HIV. The aim of this study was to assess the differential miRNA profile in CD8+ T-cells between HIV-infected individuals who differ in terms of viral replication control and immune response. Methods miRNA profile from resting and CD3/CD28-stimulated CD8+ T-cells from uninfected individuals (HIV-, n = 11), Elite Controllers (EC, n = 15), Viremic Controllers (VC, n = 15), Viremic Progressors (VP, n = 13) and HIV-infected patients on therapy (ART, n = 14) was assessed using Affymetrix miRNA 3.1 arrays. After background correction, quantile normalization and median polish summarization, normalized data were fit to a linear model. The analysis comprised: resting samples between groups; stimulated samples between groups; and stimulated versus resting samples within each group. Enrichment analyses of the putative target genes were perfomed using bioinformatic algorithms. Results A downregulated miRNA pattern was observed when resting samples from all infected groups were compared to HIV-. A miRNA downregulation was also observed when stimulated samples from EC, ART and HIV- groups were compared to VP, being hsa-miR-4492 the most downregulated. Although a preferential miRNA downregulation was observed when stimulated samples were compared to the respective resting samples, VP presented a differential miRNA expression pattern. In fact, hsa-miR-155 and hsa-miR-181a were downregulated in VP whereas in the other groups, either an upregulation or no differences were observed after stimulation, respectively. Overall, functional enrichment analysis revealed that the predicted target genes were involved in signal transduction pathways, metabolic regulation, apoptosis, and immune response. Conclusions Resting CD8+ T-cells do not exhibit a differential miRNA expression between HIV-infected individuals but they do differ from non

  14. Interactions of the HIV-1 Tat and RAP74 proteins with the RNA polymerase II CTD phosphatase FCP1.

    PubMed

    Abbott, Karen L; Archambault, Jacques; Xiao, Hua; Nguyen, Bao D; Roeder, Robert G; Greenblatt, Jack; Omichinski, James G; Legault, Pascale

    2005-03-01

    FCP1, a phosphatase specific for the carboxyl-terminal domain of the largest subunit of RNA polymerase II, is regulated by the HIV-1 Tat protein, CK2, TFIIB, and the large subunit of TFIIF (RAP74). We have characterized the interactions of Tat and RAP74 with the BRCT-containing central domain of FCP1 (FCP1(562)(-)(738)). We demonstrated that FCP1 is required for Tat-mediated transactivation in vitro and that amino acids 562-685 of FCP1 are necessary for Tat interaction in yeast two-hybrid studies. From sequence alignments, we identified a conserved acidic/hydrophobic region in FCP1 adjacent to its highly conserved BRCT domain. In vitro binding studies with purified proteins indicate that HIV-1 Tat interacts with both the acidic/hydrophobic region and the BRCT domain of FCP1, whereas RAP74(436)(-)(517) interacts solely with a portion of the acidic/hydrophobic region containing a conserved LXXLL-like motif. HIV-1 Tat inhibits the binding of RAP74(436)(-)(517) to FCP1. In a companion paper (K. Abbott et al. (2005) Enhanced Binding of RNAPII CTD Phosphatase FCP1 to RAP74 Following CK2 Phosphorylation, Biochemistry 44, 2732-2745, we identified a novel CK2 site adjacent to this conserved LXXLL-like motif. Phosphorylation of FCP1(562)(-)(619) by CK2 at this site increases binding to RAP74(436)(-)(517), but this phosphorylation is inhibited by Tat. Our results provide insights into the mechanisms by which Tat inhibits the FCP1 CTD phosphatase activity and by which FCP1 mediates transcriptional activation by Tat. In addition to increasing our understanding of the role of HIV-1 Tat in transcriptional regulation, this study defines a clear role for regions adjacent to the BRCT domain in promoting important protein-protein interactions. PMID:15723517

  15. Integrated analysis of miRNA and mRNA paired expression profiling of prenatal skeletal muscle development in three genotype pigs

    PubMed Central

    Tang, Zhonglin; Yang, Yalan; Wang, Zishuai; Zhao, Shuanping; Mu, Yulian; Li, Kui

    2015-01-01

    MicroRNAs (miRNAs) play a vital role in muscle development by binding to messenger RNAs (mRNAs). Based on prenatal skeletal muscle at 33, 65 and 90 days post-coitus (dpc) from Landrace, Tongcheng and Wuzhishan pigs, we carried out integrated analysis of miRNA and mRNA expression profiling. We identified 33, 18 and 67 differentially expressed miRNAs and 290, 91 and 502 mRNA targets in Landrace, Tongcheng and Wuzhishan pigs, respectively. Subsequently, 12 mRNAs and 3 miRNAs differentially expressed were validated using quantitative real-time PCR (qPCR), and 5 predicted miRNA targets were confirmed via dual luciferase reporter or western blot assays. We identified a set of miRNAs and mRNA genes differentially expressed in muscle development. Gene ontology (GO) enrichment analysis suggests that the miRNA targets are primarily involved in muscle contraction, muscle development and negative regulation of cell proliferation. Our data indicated that more mRNAs are regulated by miRNAs at earlier stages than at later stages of muscle development. Landrace and Tongcheng pigs also had longer phases of myoblast proliferation than Wuzhishan pigs. This study will be helpful to further explore miRNA-mRNA interactions in myogenesis and aid to uncover the molecular mechanisms of muscle development and phenotype variance in pigs. PMID:26496978

  16. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

    PubMed Central

    Masuda, Takao; Sato, Yoko; Huang, Yu-Lun; Koi, Satoshi; Takahata, Tatsuro; Hasegawa, Atsuhiko; Kawai, Gota; Kannagi, Mari

    2015-01-01

    Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription. PMID:26631448

  17. The biological characteristics of predominant strains of HIV-1 genotype: modeling of HIV-1 infection among men who have sex with men.

    PubMed

    Dai, Di; Shang, Hong; Han, Xiao-Xu; Zhao, Bin; Liu, Jing; Ding, Hai-Bo; Xu, Jun-Jie; Chu, Zhen-Xing

    2015-04-01

    To investigate the molecular subtypes of prevalent HIV-1 strains and characterize the genetics of dominant strains among men who have sex with men. Molecular epidemiology surveys in this study concentrated on the prevalent HIV-1 strains in Liaoning province by year. 229 adult patients infected with HIV-1 and part of a high-risk group of men who have sex with men were recruited. Reverse transcription and nested PCR amplification were performed. Sequencing reactions were conducted and edited, followed by codon-based alignment. NJ phylogenetic tree analyses detected two distinct CRF01_AE phylogenetic clusters, designated clusters 1 and 2. Clusters 1 and 2 accounted for 12.8% and 84.2% of sequences in the pol gene and 17.6% and 73.1% of sequences in the env gene, respectively. Another six samples were distributed on other phylogenetic clusters. Cluster 1 increased significantly from 5.6% to 20.0%, but cluster 2 decreased from 87.5% to 80.0%. Genetic distance analysis indicated that CRF01_AE cluster 1 in Liaoning was homologous to epidemic CRF01_AE strains, but CRF01_AE cluster 2 was different from other scattered strains. Additionally, significant differences were found in tetra-peptide motifs at the tip of V3 loop between cluster 1 and 2; however, differences in coreceptor usage were not detected. This study shows that subtype CRF01_AE strain may be the most prevalent epidemic strain in the men who have sex with men. Genetic characteristics of the subtype CRF01_AE cluster strain in Liaoning showed homology to the prevalent strains of men who have sex with men in other parts of China.

  18. 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

  19. Impact of HIV-1 genetic diversity on plasma HIV-1 RNA Quantification: usefulness of the Agence Nationale de Recherches sur le SIDA second-generation long terminal repeat-based real-time reverse transcriptase polymerase chain reaction test.

    PubMed

    Rouet, François; Chaix, Marie-Laure; Nerrienet, Eric; Ngo-Giang-Huong, Nicole; Plantier, Jean-Christophe; Burgard, Marianne; Peeters, Martine; Damond, Florence; Ekouevi, Didier Koumavi; Msellati, Philippe; Ferradini, Laurent; Rukobo, Sandra; Maréchal, Valérie; Schvachsa, Nilda; Wakrim, Lahcen; Rafalimanana, Christian; Rakotoambinina, Benjamin; Viard, Jean-Paul; Seigneurin, Jean-Marie; Rouzioux, Christine

    2007-08-01

    The high genetic diversity of HIV-1 has a major impact on the quantification of plasma HIV-1 RNA, representing an increasingly difficult challenge. A total of 898 plasma specimens positive for HIV-1 RNA by commercial assays (Amplicor v1.5; Roche Diagnostic Systems, Alameda, CA or Versant v3.0; Bayer Diagnostics, Emeryville, CA) were tested using the Agence Nationale de Recherches sur le SIDA second-generation (G2) real-time reverse transcriptase polymerase chain reaction (RT-PCR) test: 518 samples containing HIV-1 of known subtype, including 88 from 2 subtype panels and 430 harboring B (n = 266) and non-B (n = 164) group M HIV-1 subtypes from patients followed up in 2002 through 2005 at Necker Hospital (Paris, France), and 380 samples from 10 different countries (Argentina, Cambodia, Cameroon, Central African Republic, France, Ivory Coast, Madagascar, Morocco, Thailand, and Zimbabwe). HIV-1 RNA values obtained by G2 real-time PCR were highly correlated with those obtained by the Amplicor v1.5 for B and non-B subtypes (R = 0.892 and 0.892, respectively) and for samples from diverse countries (R = 0.867 and 0.893 for real-time PCR vs. Amplicor v1.5 and real-time PCR vs. Versant v3.0, respectively). Approximately 30% of specimens harboring non-B subtypes were underquantified by at least -0.51 log10 in Amplicor v1.5 versus 5% underquantified in G2 real-time PCR. Discrepant results were also obtained with subtype B samples (14% underquantified by Amplicor v1.5 vs. 7% by G2 real-time PCR). Similar percentages were observed when comparing results obtained with the G2 real-time PCR assay with those obtained using the Versant assay. Addressing HIV-1 diversity, continual monitoring of HIV-1 RNA assays, together with molecular epidemiology studies, is required to improve the accuracy of all HIV RNA assays.

  20. Combined Genotype Analyses of Precursor miRNA196a2 and 499a Variants with Hepatic and Renal Cancer Susceptibility a Preliminary Study.

    PubMed

    Toraih, Eman A; Fawz, Manal S; Elgazzaz, Mona G; Hussein, Mohammad H; Shehata, Rasha H; Daoud, Hisham G

    2016-01-01

    MicroRNAs, a novel class of small noncoding RNAs, are key players in many cellular processes, including cell proliferation, differentiation, invasion and regeneration. Tissue and circulatory microRNAs could serve as useful clinical biomarkers and deregulated expression levels have been observed in various cancers. Gene variants may alter microRNA processing and maturation. Thus, we aimed to investigate the association of MIR196a2 rs11614913 (C/T), MIR499a rs3746444 (A/G) polymorphisms and their combination with cancer susceptibility in an Egyptian population. Sixty five renal cell carcinoma (RCC) and 60 hepatocellular carcinoma (HCC) patients and 150 controls were enrolled in the study. They were genotyped using realtime polymerase chain reaction technology. Both miR196a2*T and miR499a*G were associated with RCC risk, but only miR196a*T was associated with HCC development. Carriage of the homozygote combinations (MIR196a2*TT + MIR499a*AA) and (MIR196a2*CC + MIR499a*GG) was associated with 25 and 48 fold elevation of likelhood to develop RCC, respectively. The miR196a2 SNP was also linked with larger tumor size in RCC and advanced tumor stage in HCC. miR196a2 and miR499a combined genotypes were associated with RCC and HCC. Further functional analysis of SNPs is required to confirm relationships between genotypes and phenotypes. PMID:27509977

  1. Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

    PubMed Central

    Vallejos, Maricarmen; Carvajal, Felipe; Pino, Karla; Navarrete, Camilo; Ferres, Marcela; Huidobro-Toro, Juan Pablo; Sargueil, Bruno; López-Lastra, Marcelo

    2012-01-01

    The 5′untranslated regions (UTR) of the full length mRNA of the HIV-1 proviral clones pNL4.3 and pLAI, harbor an internal ribosomal entry site (IRES). In this study we extend this finding by demonstrating that the mRNA 5′UTRs of natural variants of HIV-1 also exhibit IRES-activity. Cap-independent translational activity was demonstrated using bicistronic mRNAs in HeLa cells and in Xenopus laevis oocytes. The possibility that expression of the downstream cistron in these constructs was due to alternative splicing or to cryptic promoter activity was ruled out. The HIV-1 variants exhibited significant 5′UTR nucleotide diversity with respect to the control sequence recovered from pNL4.3. Interestingly, translational activity from the 5′UTR of some of the HIV-1 variants was enhanced relative to that observed for the 5′UTR of pNL4.3. In an attempt to explain these findings we probed the secondary structure of the variant HIV-1 5′UTRs using enzymatic and chemical approaches. Yet subsequent structural analyses did not reveal significant variations when compared to the pNL4.3-5′UTR. Thus, the increased IRES-activity observed for some of the HIV-1 variants cannot be ascribed to a specific structural modification. A model to explain these findings is proposed. PMID:22496887

  2. Identification and comparative analysis of the microRNA transcriptome in roots of two contrasting tobacco genotypes in response to cadmium stress

    PubMed Central

    He, Xiaoyan; Zheng, Weite; Cao, Fangbin; Wu, Feibo

    2016-01-01

    Tobacco (Nicotiana tabacum L.) is more acclimated to cadmium (Cd) uptake and preferentially enriches Cd in leaves than other crops. MicroRNAs (miRNAs) play crucial roles in regulating expression of various stress response genes in plants. However, genome-wide expression of miRNAs and their target genes in response to Cd stress in tobacco are still unknown. Here, miRNA high-throughput sequencing technology was performed using two contrasting tobacco genotypes Guiyan 1 and Yunyan 2 of Cd-sensitive and tolerance. Comprehensive analysis of miRNA expression profiles in control and Cd treated plants identified 72 known (27 families) and 14 novel differentially expressed miRNAs in the two genotypes. Among them, 28 known (14 families) and 5 novel miRNAs were considered as Cd tolerance associated miRNAs, which mainly involved in cell growth, ion homeostasis, stress defense, antioxidant and hormone signaling. Finally, a hypothetical model of Cd tolerance mechanism in Yunyan 2 was presented. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in Cd tolerance. PMID:27667199

  3. Identification and comparative analysis of the microRNA transcriptome in roots of two contrasting tobacco genotypes in response to cadmium stress

    NASA Astrophysics Data System (ADS)

    He, Xiaoyan; Zheng, Weite; Cao, Fangbin; Wu, Feibo

    2016-09-01

    Tobacco (Nicotiana tabacum L.) is more acclimated to cadmium (Cd) uptake and preferentially enriches Cd in leaves than other crops. MicroRNAs (miRNAs) play crucial roles in regulating expression of various stress response genes in plants. However, genome-wide expression of miRNAs and their target genes in response to Cd stress in tobacco are still unknown. Here, miRNA high-throughput sequencing technology was performed using two contrasting tobacco genotypes Guiyan 1 and Yunyan 2 of Cd-sensitive and tolerance. Comprehensive analysis of miRNA expression profiles in control and Cd treated plants identified 72 known (27 families) and 14 novel differentially expressed miRNAs in the two genotypes. Among them, 28 known (14 families) and 5 novel miRNAs were considered as Cd tolerance associated miRNAs, which mainly involved in cell growth, ion homeostasis, stress defense, antioxidant and hormone signaling. Finally, a hypothetical model of Cd tolerance mechanism in Yunyan 2 was presented. Our findings suggest that some miRNAs and their target genes and pathways may play critical roles in Cd tolerance.

  4. Inhibition of HIV-1 reverse transcription by triple-helix forming oligonucleotides with viral RNA.

    PubMed Central

    Volkmann, S; Jendis, J; Frauendorf, A; Moelling, K

    1995-01-01

    Reverse transcription of retroviral RNA into double-stranded DNA is catalyzed by reverse transcriptase (RT). A highly conserved polypurine tract (PPT) on the viral RNA serves as primer for plus-strand DNA synthesis and is a possible target for triple-helix formation. Triple-helix formation during reverse transcription involves either single-stranded RNA or an RNA.DNA hybrid. The effect of triple-helix formation on reverse transcription has been analyzed here in vitro using a three-strand-system consisting of an RNA.DNA hybrid and triplex-forming oligonucleotides (TFOs) consisting either of DNA or RNA. Three strand triple-helices inhibit RNase H cleavage of the PPT-RNA.DNA hybrid and initiation of plus-strand DNA synthesis in vitro. Triple-helix formation on a single-stranded RNA target has also been tested in a two-strand-system with TFOs comprising Watson-Crick and Hoogsteen base-pairing sequences, both targeted to the PPT-RNA, on a single strand connected by a linker (T)4. TFOs prevent RNase H cleavage of the PPT-RNA and initiation of plus-strand DNA synthesis in vitro. In cell culture experiments one TFO is an efficient inhibitor of retrovirus replication, leading to a block of p24 synthesis and inhibition of syncytia formation in newly infected cells. Images PMID:7537875

  5. Transcription elongation regulator 1 (TCERG1) regulates competent RNA polymerase II-mediated elongation of HIV-1 transcription and facilitates efficient viral replication

    PubMed Central

    2013-01-01

    Background Control of RNA polymerase II (RNAPII) release from pausing has been proposed as a checkpoint mechanism to ensure optimal RNAPII activity, especially in large, highly regulated genes. HIV-1 gene expression is highly regulated at the level of elongation, which includes transcriptional pausing that is mediated by both viral and cellular factors. Here, we present evidence for a specific role of the elongation-related factor TCERG1 in regulating the extent of HIV-1 elongation and viral replication in vivo. Results We show that TCERG1 depletion diminishes the basal and viral Tat-activated transcription from the HIV-1 LTR. In support of a role for an elongation mechanism in the transcriptional control of HIV-1, we found that TCERG1 modifies the levels of pre-mRNAs generated at distal regions of HIV-1. Most importantly, TCERG1 directly affects the elongation rate of RNAPII transcription in vivo. Furthermore, our data demonstrate that TCERG1 regulates HIV-1 transcription by increasing the rate of RNAPII elongation through the phosphorylation of serine 2 within the carboxyl-terminal domain (CTD) of RNAPII and suggest a mechanism for the involvement of TCERG1 in relieving pausing. Finally, we show that TCERG1 is required for HIV-1 replication. Conclusions Our study reveals that TCERG1 regulates HIV-1 transcriptional elongation by increasing the elongation rate of RNAPII and phosphorylation of Ser 2 within the CTD. Based on our data, we propose a general mechanism for TCERG1 acting on genes that are regulated at the level of elongation by increasing the rate of RNAPII transcription through the phosphorylation of Ser2. In the case of HIV-1, our evidence provides the basis for further investigation of TCERG1 as a potential therapeutic target for the inhibition of HIV-1 replication PMID:24165037

  6. Strength, power, fiber types, and mRNA expression in trained men and women with different ACTN3 R577X genotypes.

    PubMed

    Norman, Barbara; Esbjörnsson, Mona; Rundqvist, Håkan; Osterlund, Ted; von Walden, Ferdinand; Tesch, Per A

    2009-03-01

    Alpha-actinins are structural proteins of the Z-line. Human skeletal muscle expresses two alpha-actinin isoforms, alpha-actinin-2 and alpha-actinin-3, encoded by their respective genes ACTN2 and ACTN3. ACTN2 is expressed in all muscle fiber types, while only type II fibers, and particularly the type IIb fibers, express ACTN3. ACTN3 (R577X) polymorphism results in loss of alpha-actinin-3 and has been suggested to influence skeletal muscle function. The X allele is less common in elite sprint and power athletes than in the general population and has been suggested to be detrimental for performance requiring high power. The present study investigated the association of ACTN3 genotype with muscle power during 30-s Wingate cycling in 120 moderately to well-trained men and women and with knee extensor strength and fatigability in a subset of 21 men performing isokinetic exercise. Muscle biopsies were obtained from the vastus lateralis muscle to determine fiber-type composition and ACTN2 and ACTN3 mRNA levels. Peak and mean power and the torque-velocity relationship and fatigability output showed no difference across ACTN3 genotypes. Thus this study suggests that R577X polymorphism in ACTN3 is not associated with differences in power output, fatigability, or force-velocity characteristics in moderately trained individuals. However, repeated exercise bouts prompted an increase in peak torque in RR but not in XX genotypes, suggesting that ACTN3 genotype may modulate responsiveness to training. Our data further suggest that alpha-actinins do not play a significant role in determining muscle fiber-type composition. Finally, we show that ACTN2 expression is affected by the content of alpha-actinin-3, which implies that alpha-actinin-2 may compensate for the lack of alpha-actinin-3 and hence counteract the phenotypic consequences of the deficiency.

  7. RNA.

    ERIC Educational Resources Information Center

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  8. HIV-1 Viral RNA Dynamics at the Plasma Membrane May Provide Insight into Viral Assembly | Poster

    Cancer.gov

    Many aspects of how infectious viruses assemble in cells have yet to be completely deciphered. However, as reported in a recent Journal of Virology paper, researchers may be one step closer to understanding how HIV-1, the virus that causes AIDS, assembles and replicates.

  9. The interaction of HIV-1 Tat(32-72) with its target RNA: a fluorescence and nuclear magnetic resonance study.

    PubMed

    Metzger, A U; Bayer, P; Willbold, D; Hoffmann, S; Frank, R W; Goody, R S; Rösch, P

    1997-12-01

    We performed intrinsic peptide fluorescence experiments to characterize the interaction between variants of the HIV-1 Tat(32-72) peptide BP1 and TAR RNA. Kd values for wild-type BP1 and cysteine-modified BP1 were found to be in the range of 60 to 70 nM for both peptides, indicating that free sulfhydryl groups of the cysteines within the peptide are not required for high affinity TAR binding. Thus, the mutant peptide BP1 (C34S, C37W) (BP1SW) was used to further investigate peptide RNA interaction by fluorescence studies. Titration of BP1SW with TAR resulted in a dissociation constant (Kd = 9 nM) nearly an order of magnitude lower than that of the wild-type peptide. The change of the BP1SW fluorescence intensity on TAR binding was used to investigate the kinetics of this interaction by stopped-flow experiments. The results can be explained in terms of a two-step binding model, with a rapid diffusion-limited initial formation of a tight, but unspecific peptide RNA complex, followed by a relatively slow structural rearrangement step (k approximately 60 s-1) in order to form the specific BP1SW-TAR complex. Comparison of heteronuclear two-dimensional NMR spectra of BP1SW and BP1SW bound to TAR shows that only resonances from amino acid residues of the core and basic sequence regions are shifted on TAR binding.

  10. RNA-directed remodeling of the HIV-1 protein Rev orchestrates assembly of the Rev–Rev response element complex

    PubMed Central

    Jayaraman, Bhargavi; Crosby, David C; Homer, Christina; Ribeiro, Isabel; Mavor, David; Frankel, Alan D

    2014-01-01

    The HIV-1 protein Rev controls a critical step in viral replication by mediating the nuclear export of unspliced and singly-spliced viral mRNAs. Multiple Rev subunits assemble on the Rev Response Element (RRE), a structured region present in these RNAs, and direct their export through the Crm1 pathway. Rev-RRE assembly occurs via several Rev oligomerization and RNA-binding steps, but how these steps are coordinated to form an export–competent complex is unclear. Here, we report the first crystal structure of a Rev dimer-RRE complex, revealing a dramatic rearrangement of the Rev-dimer upon RRE binding through re-packing of its hydrophobic protein–protein interface. Rev-RNA recognition relies on sequence-specific contacts at the well-characterized IIB site and local RNA architecture at the second site. The structure supports a model in which the RRE utilizes the inherent plasticity of Rev subunit interfaces to guide the formation of a functional complex. DOI: http://dx.doi.org/10.7554/eLife.04120.001 PMID:25486594

  11. Human Immunodeficiency Virus Replication and Genotypic Resistance in Blood and Lymph Nodes after a Year of Potent Antiretroviral Therapy

    PubMed Central

    Günthard, Huldrych F.; Wong, Joseph K.; Ignacio, Caroline C.; Guatelli, John C.; Riggs, Nanette L.; Havlir, Diane V.; Richman, Douglas D.

    1998-01-01

    Potent antiretroviral therapy can reduce human immunodeficiency virus (HIV) in plasma to levels below the limit of detection for up to 2 years, but the extent to which viral replication is suppressed is unknown. To search for ongoing viral replication in 10 patients on combination antiretroviral therapy for up to 1 year, the emergence of genotypic drug resistance across different compartments was studied and correlated with plasma viral RNA levels. In addition, lymph node (LN) mononuclear cells were assayed for the presence of multiply spliced RNA. Population sequencing of HIV-1 pol was done on plasma RNA, peripheral blood mononuclear cell (PBMC) RNA, PBMC DNA, LN RNA, LN DNA, and RNA from virus isolated from PBMCs or LNs. A special effort was made to obtain sequences from patients with undetectable plasma RNA, emphasizing the rapidly emerging lamivudine-associated M184V mutation. Furthermore, concordance of drug resistance mutations across compartments was investigated. No evidence for viral replication was found in patients with plasma HIV RNA levels of <20 copies/ml. In contrast, evolving genotypic drug resistance or the presence of multiply spliced RNA provided evidence for low-level replication in subjects with plasma HIV RNA levels between 20 and 400 copies/ml. All patients failing therapy showed multiple drug resistance mutations in different compartments, and multiply spliced RNA was present upon examination. Concordance of nucleotide sequences from different tissue compartments obtained concurrently from individual patients was high: 98% in the protease and 94% in the reverse transcriptase regions. These findings argue that HIV replication differs significantly between patients on potent antiretroviral therapy with low but detectable viral loads and those with undetectable viral loads. PMID:9499103

  12. Secondary metabolism pathway polymorphisms and plasma efavirenz concentrations in HIV-infected adults with CYP2B6 slow metabolizer genotypes

    PubMed Central

    Haas, David W.; Kwara, Awewura; Richardson, Danielle M.; Baker, Paxton; Papageorgiou, Ioannis; Acosta, Edward P.; Morse, Gene D.; Court, Michael H.

    2014-01-01

    Objectives Efavirenz is widely prescribed for HIV-1 infection, and CYP2B6 polymorphisms 516G→T and 983T→C define efavirenz slow metabolizer genotypes. To identify genetic predictors of higher plasma efavirenz concentrations beyond these two common functional alleles, we characterized associations with mid-dosing interval efavirenz concentrations in 84 HIV-infected adults, all carrying two copies of these major loss-of-function CYP2B6 alleles. Methods Study participants had been randomized to efavirenz-containing regimens in prospective clinical trials and had available plasma efavirenz assay data. Analyses focused on secondary metabolism pathway polymorphisms CYP2A6 -48T→G (rs28399433), UGT2B7 735A→G (rs28365062) and UGT2B7 802T→C (rs7439366). Exploratory analyses also considered 196 polymorphisms and 8 copy number variants in 41 drug metabolism/transport genes. Mid-dosing interval efavirenz concentrations at steady-state were obtained ≥8 h but <19 h post-dose. Linear regression was used to test for associations between polymorphisms and log-transformed efavirenz concentrations. Results Increased efavirenz concentrations were associated with CYP2A6 -48T→G in all subjects (P = 3.8 × 10−4) and in Black subjects (P = 0.027) and White subjects (P = 0.0011) analysed separately; and with UGT2B7 735 G/G homozygosity in all subjects (P = 0.006) and in Black subjects (P = 0.046) and White subjects (P = 0.062) analysed separately. In a multivariable model, CYP2A6 -48T→G and UGT2B7 735 G/G homozygosity remained significant (P < 0.05 for each). No additional polymorphisms or copy number variants were significantly associated with efavirenz concentrations. Conclusions Among individuals with a CYP2B6 slow metabolizer genotype, CYP2A6 and possibly UGT2B7 polymorphisms contribute to even higher efavirenz concentrations. PMID:24729586

  13. The IL-6 system in HIV-1-infection and in HAART-related fat redistribution syndromes.

    PubMed

    Saumoy, Maria; López-Dupla, Miguel; Veloso, Sergi; Alonso-Villaverde, Carlos; Domingo, Pere; Broch, Montserrat; Miranda, Merce; Coll, Blai; Saurí, Amadeu; Vendrell, Joan; Richart, Cristóbal; Vidal, Francesc

    2008-04-23

    We determined the IL-6 -174 G>C single nucleotide polymorphism, IL-6 mRNA expression in subcutaneous adipose tissue (SAT) and IL-6 plasma levels in HIV-1-infected patients with and without lipodystrophy and uninfected controls. HIV-1-infected patients had a greater prevalence of the IL-6 -174 C/C genotype and the C allele, higher SAT IL-6 mRNA expression and plasma IL-6 levels than controls. The IL-6 -174 G>C genotype distribution and allele frequencies, SAT IL-6 mRNA expression and IL-6 plasma levels were non-significantly different between HIV-1-infected patients with and without lipodystrophy.

  14. A study of financial incentives to reduce plasma HIV RNA among patients in care.

    PubMed

    Farber, Steven; Tate, Janet; Frank, Cyndi; Ardito, David; Kozal, Michael; Justice, Amy C; Scott Braithwaite, R

    2013-09-01

    The role of financial incentives in HIV care is not well studied. We conducted a single-site study of monetary incentives for viral load suppression, using each patient as his own control. The incentive size ($100/quarter) was designed to be cost-neutral, offsetting estimated downstream costs averted through reduced HIV transmission. Feasibility outcomes were clinic workflow, patient acceptability, and patient comprehension. Although the study was not powered for effectiveness, we also analyzed viral load suppression. Of 80 eligible patients, 77 consented, and 69 had 12 month follow-up. Feasibility outcomes showed minimal impact on patient workflow, near-unanimous patient acceptability, and satisfactory patient comprehension. Among individuals with detectable viral loads pre-intervention, the proportion of undetectable viral load tests increased from 57 to 69 % before versus after the intervention. It is feasible to use financial incentives to reward ART adherence, and to specify the incentive by requiring cost-neutrality and targeting biological outcomes.

  15. Acceptability and Accuracy of Cervical Cancer Screening Using a Self-Collected Tampon for HPV Messenger-RNA Testing among HIV-Infected Women in South Africa

    PubMed Central

    Adamson, Paul C.; Huchko, Megan J.; Moss, Alison M.; Kinkel, Hans F.; Medina-Marino, Andrew

    2015-01-01

    Background HIV increases women’s risk for high-risk human papillomavirus (hrHPV) infection and invasive cervical cancer. South Africa has a high HIV prevalence but low cervical cancer screening coverage. Self-collection of cervical specimens and hrHPV testing, including hrHPV messenger-RNA (mRNA) testing, are methods aimed at increasing screening rates. However, data are limited on the acceptability and accuracy of tampon-based self-collection for hrHPV mRNA testing in HIV-infected women. Methods We recruited 325 HIV-infected women seeking care at a government HIV clinic in Pretoria, South Africa. A clinician performed a pelvic examination and obtained an endocervical specimen. Study participants performed self-collection using a tampon. Both clinician- and self-collected specimens were tested for hrHPV mRNA. Acceptability of both collection methods was assessed, the prevalence of hrHPV mRNA in our study population was estimated, test positivity of the two collection methods were compared, and test agreement was assessed by calculating the κ-statistic, sensitivity, and specificity. Results Over 90% of women reported no difficulties self-collecting specimens and 82% were willing to perform the tampon-collection at home. Based on clinician-collection specimens, the prevalence of hrHPV mRNA in our study population was 36.7% (95% CI: 31.4%– 42.0%). There was no difference in test positivity between clinician-collection, 36.7%, and tampon-collection, 43.5% (p-value = 0.08). Using clinician-collection as the reference test, the sensitivity and specificity for hrHPV mRNA of tampon-collection were 77.4% (95% CI: 69.8–85.0%) and 77.8% (95% CI: 71.9–83.6%), respectively. Conclusions Tampon-based self-collection is acceptable to women and has similar hrHPV mRNA positivity rates as clinician-collection, but has reduced sensitivity and specificity compared to clinician-collection. The hrHPV mRNA prevalence in our study population is high, but similar to other high

  16. HIV-1 Tat-associated RNA polymerase C-terminal domain kinase, CDK2, phosphorylates CDK7 and stimulates Tat-mediated transcription.

    PubMed Central

    Nekhai, Sergei; Zhou, Meisheng; Fernandez, Anne; Lane, William S; Lamb, Ned J C; Brady, John; Kumar, Ajit

    2002-01-01

    HIV-1 gene expression is regulated by a viral transactivator protein (Tat) which induces transcriptional elongation of HIV-1 long tandem repeat (LTR). This induction requires hyperphosphorylation of the C-terminal domain (CTD) repeats of RNA polymerase II (Pol II). To achieve CTD hyperphosphorylation, Tat stimulates CTD kinases associated with general transcription factors of the promoter complex, specifically TFIIH-associated CDK7 and positive transcription factor b-associated CDK9 (cyclin-dependent kinase 9). Other studies indicate that Tat may bind an additional CTD kinase that regulates the target-specific phosphorylation of RNA Pol II CTD. We previously reported that Tat-associated T-cell-derived kinase (TTK), purified from human primary T-cells, stimulates Tat-dependent transcription of HIV-1 LTR in vivo [Nekhai, Shukla, Fernandez, Kumar and Lamb (2000) Virology 266, 246-256]. In the work presented here, we characterized the components of TTK by biochemical fractionation and the function of TTK in transcription assays in vitro. TTK uniquely co-purified with CDK2 and not with either CDK9 or CDK7. Tat induced the TTK-associated CDK2 kinase to phosphorylate CTD, specifically at Ser-2 residues. The TTK fraction restored Tat-mediated transcription activation of HIV-1 LTR in a HeLa nuclear extract immunodepleted of CDK9, but not in the HeLa nuclear extract double-depleted of CDK9 and CDK7. Direct microinjection of the TTK fraction augmented Tat transactivation of HIV-1 LTR in human primary HS68 fibroblasts. The results argue that TTK-associated CDK2 may function to maintain target-specific phosphorylation of RNA Pol II that is essential for Tat transactivation of HIV-1 promoter. They are also consistent with the observed cell-cycle-specific induction of viral gene transactivation. PMID:12049628

  17. Complement-mediated, antibody-dependent enhancement of HIV-1 infection in vitro is characterized by increased protein and RNA syntheses and infectious virus release.

    PubMed

    Robinson, W E; Montefiori, D C; Gillespie, D H; Mitchell, W M

    1989-01-01

    Antibody-dependent enhancement (ADE) of human immunodeficiency virus type 1 (HIV-1) infection in vitro has been described recently and was shown to occur by two mechanisms: either participation of the alternative pathway of complement or to involve an Fc receptor-mediated, complement-independent mechanism. Complement-mediated ADE results in an accelerated cytopathic effect in target cells that can abrogate the protective properties of neutralizing antibodies. This study characterizes the surface antigens of MT-2 cells using flow cytometric analysis and shows that these cells express high levels of both CD4 and complement receptor type 2 (CR2) while several CD4+ cell lines that do not demonstrate complement-mediated ADE lack high levels of complement receptors. Further, utilizing MT-2 cell cultures, it is demonstrated that complement-mediated ADE of HIV-1 infection is conferred by the sera from more than 80% of HIV-1 antibody-positive individuals (N = 85). Complement-mediated ADE of HIV-1 infection causes an acceleration of several parameters indicative of HIV-1 infection in vitro including increased HIV-1 antigen synthesis as detected by indirect immunofluorescence, RNA accumulation as measured by a solution hybridization protocol, reverse transcriptase release, and progeny virus production. PMID:2465404

  18. Recognition of HIV-TAR RNA using neomycin-benzimidazole conjugates.

    PubMed

    Ranjan, Nihar; Kumar, Sunil; Watkins, Derrick; Wang, Deyun; Appella, Daniel H; Arya, Dev P

    2013-10-15

    Synthesis of a novel class of compounds and their biophysical studies with TAR-RNA are presented. The synthesis of these compounds was achieved by conjugating neomycin, an aminoglycoside, with benzimidazoles modeled from a B-DNA minor groove binder, Hoechst 33258. The neomycin-benzimidazole conjugates have varying linkers that connect the benzimidazole and neomycin units. The linkers of varying length (5-23 atoms) in these conjugates contain one to three triazole units. The UV thermal denaturation experiments showed that the conjugates resulted in greater stabilization of the TAR-RNA than either neomycin or benzimidazole used in the synthesis of conjugates. These results were corroborated by the FID displacement and tat-TAR inhibition assays. The binding of ligands to the TAR-RNA is affected by the length and composition of the linker. Our results show that increasing the number of triazole groups and the linker length in these compounds have diminishing effect on the binding to TAR-RNA. Compounds that have shorter linker length and fewer triazole units in the linker displayed increased affinity towards the TAR RNA.

  19. Impact of HIV infection on sustained virological response to treatment against hepatitis C virus with pegylated interferon plus ribavirin.

    PubMed

    Monje-Agudo, P; Castro-Iglesias, A; Rivero-Juárez, A; Martínez-Marcos, F; Ortega-González, E; Real, L M; Pernas, B; Merchante, N; Cid, P; Macías, J; Merino, M D; Rivero, A; Mena, A; Neukam, K; Pineda, J A

    2015-10-01

    It is commonly accepted that human immunodeficiency (HIV) coinfection negatively impacts on the rates of sustained virological response (SVR) to therapy with pegylated interferon plus ribavirin (PR). However, this hypothesis is derived from comparing different studies. The aim of this study was to determine the impact of HIV coinfection on SVR to PR in one single population. In a multicentric, prospective study conducted between 2000 and 2013, all previously naïve hepatitis C virus (HCV)-infected patients who started PR in five Spanish hospitals were analyzed. SVR was evaluated 24 weeks after the scheduled end of therapy. Of the 1046 patients included in this study, 413 (39%) were coinfected with HIV. Three hundred and forty-one (54%) HCV-monoinfected versus 174 (42%) HIV/HCV-coinfected patients achieved SVR (p < 0.001). The corresponding figures for undetectable HCV RNA at treatment week 4 were 86/181 (47%) versus 59/197 (30%), p < 0.001. SVR was observed in 149 (69%) HCV genotype 2/3-monoinfected subjects versus 91 (68%) HIV/HCV genotype 2/3-coinfected subjects (p = 0.785). In the HCV genotype 1/4-infected population, 188 (46%) monoinfected patients versus 82 (30%) with HIV coinfection (p < 0.001) achieved SVR. In this subgroup, absence of HIV coinfection was independently associated with higher SVR [adjusted odds ratio (95% confidence interval): 2.127 (1.135-3.988); p = 0.019] in a multivariate analysis adjusted for age, sex, baseline HCV RNA load, IL28B genotype, fibrosis stage, and type of pegylated interferon. HIV coinfection impacts on the rates of SVR to PR only in HCV genotype 1/4-infected patients, while it has no effect on SVR in the HCV genotype 2/3-infected subpopulation.

  20. Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones.

    PubMed

    Pascale, Lise; Azoulay, Stéphane; Di Giorgio, Audrey; Zenacker, Laura; Gaysinski, Marc; Clayette, Pascal; Patino, Nadia

    2013-06-01

    RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous 'polyamide amino acids' (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy-entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

  1. Thermodynamic studies of a series of homologous HIV-1 TAR RNA ligands reveal that loose binders are stronger Tat competitors than tight ones

    PubMed Central

    Pascale, Lise; Azoulay, Stéphane; Di Giorgio, Audrey; Zenacker, Laura; Gaysinski, Marc; Clayette, Pascal; Patino, Nadia

    2013-01-01

    RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous ‘polyamide amino acids’ (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy–entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets. PMID:23605042

  2. Quantitative Effect of Suboptimal Codon Usage on Translational Efficiency of mRNA Encoding HIV-1 gag in Intact T Cells

    PubMed Central

    Ngumbela, Kholiswa C.; Ryan, Kieran P.; Sivamurthy, Rohini; Brockman, Mark A.; Gandhi, Rajesh T.; Bhardwaj, Nina; Kavanagh, Daniel G.

    2008-01-01

    Background The sequences of wild-isolate strains of Human Immunodeficiency Virus-1 (HIV-1) are characterized by low GC content and suboptimal codon usage. Codon optimization of DNA vectors can enhance protein expression both by enhancing translational efficiency, and by altering RNA stability and export. Although gag codon optimization is widely used in DNA vectors and experimental vaccines, the actual effect of altered codon usage on gag translational efficiency has not been quantified. Methodology and Principal Findings To quantify translational efficiency of gag mRNA in live T cells, we transfected Jurkat cells with increasing doses of capped, polyadenylated synthetic mRNA corresponding to wildtype or codon-optimized gag sequences, measured Gag production by quantitative ELISA and flow cytometry, and estimated the translational efficiency of each transcript as pg of Gag antigen produced per µg of input mRNA. We found that codon optimization yielded a small increase in gag translational efficiency (approximately 1.6 fold). In contrast when cells were transfected with DNA vectors requiring nuclear transcription and processing of gag mRNA, codon optimization resulted in a very large enhancement of Gag production. Conclusions We conclude that suboptimal codon usage by HIV-1 results in only a slight loss of gag translational efficiency per se, with the vast majority of enhancement in protein expression from DNA vectors due to altered processing and export of nuclear RNA. PMID:18523584

  3. Lipid Profiles and APOE4 Allele Impact Midlife Cognitive Decline in HIV-Infected Men on Antiretroviral Therapy

    PubMed Central

    Mukerji, Shibani S.; Locascio, Joseph J.; Misra, Vikas; Lorenz, David R.; Holman, Alex; Dutta, Anupriya; Penugonda, Sudhir; Wolinsky, Steven M.; Gabuzda, Dana

    2016-01-01

    Background. Dyslipidemia and apolipoprotein E4 (APOE ϵ4) allele are risk factors for age-related cognitive decline, but how these risks are modified by human immunodeficiency virus (HIV) infection is unclear. Methods. In a longitudinal nested study from the Multicenter AIDS Cohort Study, 273 HIV type 1–infected (HIV+) men aged 50–65 years with baseline HIV RNA <400 copies/mL and on continuous antiretroviral therapy (ART) in ≥95% of follow-up visits were matched by sociodemographic variables to 516 HIV-uninfected (HIV–) controls. The association between lipid markers (total cholesterol, low-density lipoprotein cholesterol [LDL-C], high-density lipoprotein cholesterol [HDL-C], and triglycerides), APOE genotype, and cognitive decline in HIV infection was examined using mixed-effects models. Results. The median baseline age of participants was 51, 81% were white, and 89% had education >12 years. HIV+ men had similar baseline total cholesterol and LDL-C, but lower HDL-C and higher triglycerides than controls (P < .001). Higher total cholesterol and LDL-C were associated with faster rates of cognitive decline (P < .01), whereas higher HDL-C attenuated decline (P = .02) in HIV+ men. In HIV+ men with elevated cholesterol, statin use was associated with a slower estimated rate of decline (P = .02). APOE ϵ4 genotype accelerated cognitive decline in HIV+ but not HIV– men (P = .01), with trajectories diverging from HIV– ε4 carriers after age 50. Total cholesterol levels did not modify the association of ϵ4 genotype with decline (P = .9). Conclusions. Elevated cholesterol and APOE ϵ4 genotype are independent risk factors for cognitive decline in ART-adherent HIV+ men aged >50 years. Treatment of dyslipidemia may be an effective strategy to reduce cognitive decline in older HIV+ individuals. PMID:27448678

  4. Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers

    PubMed Central

    Hendrix, Jelle; Baumgärtel, Viola; Schrimpf, Waldemar; Ivanchenko, Sergey; Digman, Michelle A.; Gratton, Enrico; Kräusslich, Hans-Georg; Müller, Barbara

    2015-01-01

    Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly. PMID:26283800

  5. Large-Scale SNP Discovery through RNA Sequencing and SNP Genotyping by Targeted Enrichment Sequencing in Cassava (Manihot esculenta Crantz)

    PubMed Central

    Pootakham, Wirulda; Shearman, Jeremy R.; Ruang-areerate, Panthita; Sonthirod, Chutima; Sangsrakru, Duangjai; Jomchai, Nukoon; Yoocha, Thippawan; Triwitayakorn, Kanokporn; Tragoonrung, Somvong; Tangphatsornruang, Sithichoke

    2014-01-01

    Cassava (Manihot esculenta Crantz) is one of the most important crop species being the main source of dietary energy in several countries. Marker-assisted selection has become an essential tool in plant breeding. Single nucleotide polymorphism (SNP) discovery via transcriptome sequencing is an attractive strategy for genome complexity reduction in organisms with large genomes. We sequenced the transcriptome of 16 cassava accessions using the Illumina HiSeq platform and identified 675,559 EST-derived SNP markers. A subset of those markers was subsequently genotyped by capture-based targeted enrichment sequencing in 100 F1 progeny segregating for starch viscosity phenotypes. A total of 2,110 non-redundant SNP markers were used to construct a genetic map. This map encompasses 1,785 cM and consists of 19 linkage groups. A major quantitative trait locus (QTL) controlling starch pasting properties was identified and shown to coincide with the QTL previously reported for this trait. With a high-density SNP-based linkage map presented here, we also uncovered a novel QTL associated with starch pasting time on LG 10. PMID:25551642

  6. Large-scale SNP discovery through RNA sequencing and SNP genotyping by targeted enrichment sequencing in cassava (Manihot esculenta Crantz).

    PubMed

    Pootakham, Wirulda; Shearman, Jeremy R; Ruang-Areerate, Panthita; Sonthirod, Chutima; Sangsrakru, Duangjai; Jomchai, Nukoon; Yoocha, Thippawan; Triwitayakorn, Kanokporn; Tragoonrung, Somvong; Tangphatsornruang, Sithichoke

    2014-01-01

    Cassava (Manihot esculenta Crantz) is one of the most important crop species being the main source of dietary energy in several countries. Marker-assisted selection has become an essential tool in plant breeding. Single nucleotide polymorphism (SNP) discovery via transcriptome sequencing is an attractive strategy for genome complexity reduction in organisms with large genomes. We sequenced the transcriptome of 16 cassava accessions using the Illumina HiSeq platform and identified 675,559 EST-derived SNP markers. A subset of those markers was subsequently genotyped by capture-based targeted enrichment sequencing in 100 F1 progeny segregating for starch viscosity phenotypes. A total of 2,110 non-redundant SNP markers were used to construct a genetic map. This map encompasses 1,785 cM and consists of 19 linkage groups. A major quantitative trait locus (QTL) controlling starch pasting properties was identified and shown to coincide with the QTL previously reported for this trait. With a high-density SNP-based linkage map presented here, we also uncovered a novel QTL associated with starch pasting time on LG 10.

  7. Baseline characteristics of HIV & hepatitis B virus (HIV/HBV) co-infected patients from Kolkata, India

    PubMed Central

    Sarkar, Jayeeta; Saha, Debraj; Bandyopadhyay, Bhaswati; Saha, Bibhuti; Kedia, Deepika; Guha Mazumder, D.N.; Chakravarty, Runu; Guha, Subhasish Kamal

    2016-01-01

    Background & objectives: Hepatitis B virus (HBV) and HIV co-infection has variable prevalence worldwide. In comparison to HBV mono-infection, the course of chronic HBV infection is accelerated in HIV/HBV co-infected patients. The present study was carried out to analyse the baseline characteristics (clinical, biochemical, serological and virological) of treatment naïve HIV/HBV co-infected and HIV mono-infected patients. Methods: Between July 2011 and January 2013, a total number of 1331 HIV-seropositive treatment naïve individuals, enrolled in the ART Centre of Calcutta School of Tropical Medicine, Kolkata, India, were screened for hepatitis B surface antigen (HBsAg). A total of 1253 HIV mono-infected and 78 HIV/HBV co-infected patients were characterized. The co-infected patients were evaluated for HBeAg and anti-HBe antibody by ELISA. HIV RNA was quantified for all co-infected patients. HBV DNA was detected and quantified by real time-PCR amplification followed by HBV genotype determination. Results: HIV/HBV co-infected patients had proportionately more advanced HIV disease (WHO clinical stage 3 and 4) than HIV mono-infected individuals (37.1 vs. 19.9%). The co-infected patients had significantly higher serum bilirubin, alanine aminotransferase (ALT), alkaline phosphatase and ALT/platelet ratio index (APRI). CD4 count was non-significantly lower in co-infected patients. Majority (61.5%) were HBeAg positive with higher HIV RNA (P<0.05), HBV DNA (P<0.001) and APRI (P<0.05) compared to those who were HBeAg negative. HBV/D was the predominant genotype (73.2%) and D2 (43.7%) was the commonest subgenotype. Interpretation & conclusions: HIV/HBV co-infected patients had significantly higher serum bilirubin, ALT, alkaline phosphatase and lower platelet count. HBeAg positive co-infected patients had higher HIV RNA and HBV DNA compared to HBeAg negative co-infected patients. Prior to initiation of antiretroviral treatment (ART) all patients should be screened for HBsAg to

  8. MicroRNA-21 dysregulates the expression of MEF2C in neurons in monkey and human SIV/HIV neurological disease.

    PubMed

    Yelamanchili, S V; Chaudhuri, A Datta; Chen, L-N; Xiong, Huangui; Fox, H S

    2010-01-01

    MicroRNAs (miRNAs) play important roles in regulating a plethora of physiological and pathophysiogical processes including neurodegeneration. In both HIV associated dementia in humans and its monkey model SIV encephalitis we find miR-21, a miRNA largely known for its link to oncogenesis, to be significantly upregulated in the brain. In situ hybridization of the diseased brain sections revealed induction of miR-21 in neurons. MiR-21 can be induced in neurons by prolonged N-methyl-D-aspartic acid receptor stimulation, an excitotoxic process active in HIV and other neurodegenerative diseases. Introduction of miR-21 into human neurons leads to pathological functional defects. Furthermore, we show that miR-21 specifically targets the mRNA of myocyte enhancer factor 2C (MEF2C), a transcription factor crucial for neuronal function, and reduces its expression. MEF2C is dramatically downregulated in neurons of HIV-associated dementia patients as well as monkeys with SIVE. Together, this study elucidates a novel role for miR-21 in the brain, not only as a potential signature of neurological disease but also as a crucial effector of HIV induced neuronal dysfunction and neurodegeneration.

  9. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry.

    PubMed

    Taylor, Ethan Will; Ruzicka, Jan A; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3' end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

  10. Cellular Selenoprotein mRNA Tethering via Antisense Interactions with Ebola and HIV-1 mRNAs May Impact Host Selenium Biochemistry

    PubMed Central

    Taylor, Ethan Will; Ruzicka, Jan A.; Premadasa, Lakmini; Zhao, Lijun

    2016-01-01

    Regulation of protein expression by non-coding RNAs typically involves effects on mRNA degradation and/or ribosomal translation. The possibility of virus-host mRNA-mRNA antisense tethering interactions (ATI) as a gain-of-function strategy, via the capture of functional RNA motifs, has not been hitherto considered. We present evidence that ATIs may be exploited by certain RNA viruses in order to tether the mRNAs of host selenoproteins, potentially exploiting the proximity of a captured host selenocysteine insertion sequence (SECIS) element to enable the expression of virally-encoded selenoprotein modules, via translation of in-frame UGA stop codons as selenocysteine. Computational analysis predicts thermodynamically stable ATIs between several widely expressed mammalian selenoprotein mRNAs (e.g., isoforms of thioredoxin reductase) and specific Ebola virus mRNAs, and HIV-1 mRNA, which we demonstrate via DNA gel shift assays. The probable functional significance of these ATIs is further supported by the observation that, in both viruses, they are located in close proximity to highly conserved in-frame UGA stop codons at the 3′ end of open reading frames that encode essential viral proteins (the HIV-1 nef protein and the Ebola nucleoprotein). Significantly, in HIV/AIDS patients, an inverse correlation between serum selenium and mortality has been repeatedly documented, and clinical benefits of selenium in the context of multi-micronutrient supplementation have been demonstrated in several well-controlled clinical trials. Hence, in the light of our findings, the possibility of a similar role for selenium in Ebola pathogenesis and treatment merits serious investigation. PMID:26369818

  11. Impact of genotypic drug resistance mutations on clinical and immunological outcomes in HIV-infected adults on HAART in West Africa

    PubMed Central

    Seyler, Catherine; Adjé-Touré, Christiane; Messou, Eugène; Dakoury-Dogbo, Nicole; Rouet, François; Gabillard, Delphine; Nolan, Monica; Toure, Siaka; Anglaret, Xavier

    2007-01-01

    Background The impact of HIV-1 drug resistance mutations in African adults on HAART has never been reported. Methods In 2004 in Abidjan, 106 adults on HAART had plasma viral load (VL) measurements. Patients with detectable VL had resistance genotypic tests. Patients were followed-up until 2006. Main outcomes were serious morbidity and immunological failure (CD4 count < 200/mm3). Results At study entry, the median previous time on HAART was 37 months and the median CD4 count 266/mm3; 58% of patients had undetectable VL, 20% detectable VL with no major resistance mutations, and 22% detectable VL with ≥1 major mutations. At study termination, 20% of patients had <200 CD4/mm3. Factors associated with immunological failure were a low baseline CD4 count (p=0.007) and ≥1 resistance mutations at baseline (p=0.04). Compared with patients with undetectable VL, those with detectable VL without mutations and those with ≥1 mutations had adjusted hazard ratios of immunological failure of 2.56 (95%CI 0.76–8.54) and 4.32 (1.38–13.57), respectively. In patients with undetectable VL and detectable VL without and with mutations, the median change in CD4 count between study entry and termination was +129/mm3, +51/mm3 and +3/mm3, respectively. One patient died. The 18-months probability of remaining free of morbidity was 0.79 in patients with undetectable VL and 0.69 in those with resistance mutations (p=0.19). Conclusion In this setting with restricted access to second-line HAART regimens, patients with major resistance mutations had higher rates of immunological failure, but most of them maintained stable CD4 count and stayed alive during 20 months. PMID:17502726

  12. Comparison of SIV and HIV-1 genomic RNA structures reveals impact of sequence evolution on conserved and non-conserved structural motifs.

    PubMed

    Pollom, Elizabeth; Dang, Kristen K; Potter, E Lake; Gorelick, Robert J; Burch, Christina L; Weeks, Kevin M; Swanstrom, Ronald

    2013-01-01

    RNA secondary structure plays a central role in the replication and metabolism of all RNA viruses, including retroviruses like HIV-1. However, structures with known function represent only a fraction of the secondary structure reported for HIV-1(NL4-3). One tool to assess the importance of RNA structures is to examine their conservation over evolutionary time. To this end, we used SHAPE to model the secondary structure of a second primate lentiviral genome, SIVmac239, which shares only 50% sequence identity at the nucleotide level with HIV-1NL4-3. Only about half of the paired nucleotides are paired in both genomic RNAs and, across the genome, just 71 base pairs form with the same pairing partner in both genomes. On average the RNA secondary structure is thus evolving at a much faster rate than the sequence. Structure at the Gag-Pro-Pol frameshift site is maintained but in a significantly altered form, while the impact of selection for maintaining a protein binding interaction can be seen in the conservation of pairing partners in the small RRE stems where Rev binds. Structures that are conserved between SIVmac239 and HIV-1(NL4-3) also occur at the 5' polyadenylation sequence, in the plus strand primer sites, PPT and cPPT, and in the stem-loop structure that includes the first splice acceptor site. The two genomes are adenosine-rich and cytidine-poor. The structured regions are enriched in guanosines, while unpaired regions are enriched in adenosines, and functionaly important structures have stronger base pairing than nonconserved structures. We conclude that much of the secondary structure is the result of fortuitous pairing in a metastable state that reforms during sequence evolution. However, secondary structure elements with important function are stabilized by higher guanosine content that allows regions of structure to persist as sequence evolution proceeds, and, within the confines of selective pressure, allows structures to evolve. PMID:23593004

  13. Structural domains within the HIV-1 mRNA and the ribosomal protein S25 influence cap-independent translation initiation.

    PubMed

    Carvajal, Felipe; Vallejos, Maricarmen; Walters, Beth; Contreras, Nataly; Hertz, Marla I; Olivares, Eduardo; Cáceres, Carlos J; Pino, Karla; Letelier, Alejandro; Thompson, Sunnie R; López-Lastra, Marcelo

    2016-07-01

    The 5' leader of the HIV-1 genomic RNA is a multifunctional region that folds into secondary/tertiary structures that regulate multiple processes during viral replication including translation initiation. In this work, we examine the internal ribosome entry site (IRES) located in the 5' leader that drives translation initiation of the viral Gag protein under conditions that hinder cap-dependent translation initiation. We show that activity of the HIV-1 IRES relies on ribosomal protein S25 (eS25). Additionally, a mechanistic and mutational analysis revealed that the HIV-1 IRES is modular in nature and that once the 40S ribosomal subunit is recruited to the IRES, translation initiates without the need of ribosome scanning. These findings elucidate a mechanism of initiation by the HIV-1 IRES whereby a number of highly structured sites present within the HIV-1 5' leader leads to the recruitment of the 40S subunit directly at the site of initiation of protein synthesis. PMID:27191820

  14. RNA helicase MOV10 functions as a co-factor of HIV-1 Rev to facilitate Rev/RRE-dependent nuclear export of viral mRNAs.

    PubMed

    Huang, Feng; Zhang, Junsong; Zhang, Yijun; Geng, Guannan; Liang, Juanran; Li, Yingniang; Chen, Jingliang; Liu, Chao; Zhang, Hui

    2015-12-01

    Human immunodeficiency virus type 1 (HIV-1) exploits multiple host factors during its replication. The REV/RRE-dependent nuclear export of unspliced/partially spliced viral transcripts needs the assistance of host proteins. Recent studies have shown that MOV10 overexpression inhibited HIV-1 replication at various steps. However, the endogenous MOV10 was required in certain step(s) of HIV-1 replication. In this report, we found that MOV10 potently enhances the nuclear export of viral mRNAs and subsequently increases the expression of Gag protein and other late products through affecting the Rev/RRE axis. The co-immunoprecipitation analysis indicated that MOV10 interacts with Rev in an RNA-independent manner. The DEAG-box of MOV10 was required for the enhancement of Rev/RRE-dependent nuclear export and the DEAG-box mutant showed a dominant-negative activity. Our data propose that HIV-1 utilizes the anti-viral factor MOV10 to function as a co-factor of Rev and demonstrate the complicated effects of MOV10 on HIV-1 life cycle.

  15. mRNA transfection of a novel TAL effector nuclease (TALEN) facilitates efficient knockout of HIV co-receptor CCR5.

    PubMed

    Mock, Ulrike; Machowicz, Rafał; Hauber, Ilona; Horn, Stefan; Abramowski, Pierre; Berdien, Belinda; Hauber, Joachim; Fehse, Boris

    2015-06-23

    Homozygosity for a natural deletion variant of the HIV-coreceptor molecule CCR5, CCR5Δ32, confers resistance toward HIV infection. Allogeneic stem cell transplantation from a CCR5Δ32-homozygous donor has resulted in the first cure from HIV ('Berlin patient'). Based thereon, genetic disruption of CCR5 using designer nucleases was proposed as a promising HIV gene-therapy approach. Here we introduce a novel TAL-effector nuclease, CCR5-Uco-TALEN that can be efficiently delivered into T cells by mRNA electroporation, a gentle and truly transient gene-transfer technique. CCR5-Uco-TALEN mediated high-rate CCR5 knockout (>90% in PM1 and >50% in primary T cells) combined with low off-target activity, as assessed by flow cytometry, next-generation sequencing and a newly devised, very convenient gene-editing frequency digital-PCR (GEF-dPCR). GEF-dPCR facilitates simultaneous detection of wild-type and gene-edited alleles with remarkable sensitivity and accuracy as shown for the CCR5 on-target and CCR2 off-target loci. CCR5-edited cells were protected from infection with HIV-derived lentiviral vectors, but also with the wild-type CCR5-tropic HIV-1BaL strain. Long-term exposure to HIV-1BaL resulted in almost complete suppression of viral replication and selection of CCR5-gene edited T cells. In conclusion, we have developed a novel TALEN for the targeted, high-efficiency knockout of CCR5 and a useful dPCR-based gene-editing detection method.

  16. Next-Generation Sequencing of Small RNAs from HIV-Infected Cells Identifies Phased microRNA Expression Patterns and Candidate Novel microRNAs Differentially Expressed upon Infection

    PubMed Central

    Chang, Stewart T.; Thomas, Matthew J.; Sova, Pavel; Green, Richard R.; Palermo, Robert E.; Katze, Michael G.

    2013-01-01

    ABSTRACT HIV infection of CD4+ T cells induces a range of host transcriptional changes in mRNAs as well as microRNAs that may coordinate changes in mRNAs. To survey these dynamic changes, we applied next-generation sequencing, analyzing the small RNA fraction of HIV-infected cells at 5, 12, and 24 h postinfection (RNA-Seq). These time points afforded a view of the transcriptomic changes occurring both before and during viral replication. In the resulting small RNA-Seq data set, we detected a phased pattern of microRNA expression. Largely distinct sets of microRNAs were found to be suppressed at 5 and 12 h postinfection, and both sets of changes rebounded later in infection. A larger set of microRNA changes was observed at 24 h postinfection. When integrated with mRNA expression data, the small RNA-Seq data indicated a role for microRNAs in transcriptional regulation, T cell activation, and cell cycle during HIV infection. As a unique benefit of next-generation sequencing, we also detected candidate novel host microRNAs differentially expressed during infection, including one whose downregulation at 24 h postinfection may allow full replication of HIV to proceed. Collectively, our data provide a uniquely comprehensive view of the changes in host microRNAs induced by HIV during cellular infection. PMID:23386435

  17. Lysine directed cross-linking of viral DNA-RNA:DNA hybrid substrate to the isolated RNase H domain of HIV-1 reverse transcriptase.

    PubMed

    Guaitiao, Juan P; Zúñiga, Roberto A; Roth, Monica J; Leon, Oscar

    2004-02-10

    An isolated ribonuclease H domain of HIV-1 reverse transcriptase is capable of specifically removing the tRNA primer within an oligonucleotide mimic. The determinants for substrate specificity are located in a region within the terminal octanucleotide of the acceptor stem of the tRNA. Recognition of the substrate by HIV-1 RNase H was analyzed by the introduction of a cross-linking reagent directed toward lysines on the thymine residue complementary to the scissile bond, facing the major groove of the DNA-RNA:DNA substrate. Cross-linking of the modified substrate to RNase H required the presence of Mn(2+). The Mn(2+) titration of cross-linking paralleled the Mn(2+) requirement for activity. Modified substrate quenched with glycine prior to binding of substrate was efficiently cleaved, whereas the RNA within the cross-linked product was intact. Tryptic digestion of the isolated RNase H-nucleic acid covalent complex revealed a main cross-linked peptide whose N-terminal peptide sequence is VVTLTDTTNQ, indicating that the cross-linked lysine corresponds to Lys476. Cross-linking to K476 was confirmed by analysis of K476C RNase H. Mutation of K476C disrupted the chemical cross-linking while maintaining activity. On the basis of the size of the cross-linker arm, the results indicate that K476 is in closer proximity to the tRNA mimic substrate within the isolated RNase H domain than observed for the RNase H-resistant polypurine tract (PPT) substrate within the HIV-1 RT.

  18. HIV Drug Resistance Mutations in Proviral DNA from a Community Treatment Program

    PubMed Central

    Derache, Anne; Shin, Hyoung-Shik; Balamane, Maya; White, Elizabeth; Israelski, Dennis; Klausner, Jeffrey D.; Freeman, Alexandra H.; Katzenstein, David

    2015-01-01

    Background Drug resistance mutations archived in resting memory CD4+ cells may persist despite suppression of HIV RNA to <50 copies/ml. We sequenced pol gene from proviral DNA among viremic and suppressed patients to identify drug resistance mutations. Methods The Peninsula AIDS Research Cohort study enrolled and followed over 2 years 120 HIV infected patients from San Mateo and San Francisco Counties. HIV-1 pol genotyping by bulk sequencing was performed on 38 DNA and RNA from viremic patients and DNA only among 82 suppressed patients at baseline. Antiretroviral susceptibility was predicted by HIVDB.stanford.edu. Results Among 120 subjects, 81% were on antiretroviral therapy and had been treated for a median time of 7 years. Thirty-two viremic patients showed concordant RNA and DNA genotypes (84%); the discordant profiles were mainly observed in patients with low-level viremia. Among suppressed patients, 21 had drug resistance mutations in proviral DNA (26%) with potential resistance to one, two or three ARV classes in 16, 4 and 1 samples respectively. Conclusions The high level of genotype concordance between DNA and RNA in viremic patients suggested that DNA genotyping might be used to assess drug resistance in resource-limited settings, and further investigation of extracted DNA from dried blood spots is needed. Drug resistance mutations in proviral DNA in 26% of subjects with less than 50 copies/ml pose a risk for the transmission of drug resistant virus with virologic failure, treatment interruption or decreased adherence. PMID:25635815

  19. Deconvoluting the Composition of Low-Frequency Hepatitis C Viral Quasispecies: Comparison of Genotypes and NS3 Resistance-Associated Variants between HCV/HIV Coinfected Hemophiliacs and HCV Monoinfected Patients in Japan

    PubMed Central

    Ogishi, Masato; Yotsuyanagi, Hiroshi; Tsutsumi, Takeya; Gatanaga, Hiroyuki; Ode, Hirotaka; Sugiura, Wataru; Moriya, Kyoji; Oka, Shinichi; Kimura, Satoshi; Koike, Kazuhiko

    2015-01-01

    Pre-existing low-frequency resistance-associated variants (RAVs) may jeopardize successful sustained virological responses (SVR) to HCV treatment with direct-acting antivirals (DAAs). However, the potential impact of low-frequency (∼0.1%) mutations, concatenated mutations (haplotypes), and their association with genotypes (Gts) on the treatment outcome has not yet been elucidated, most probably owing to the difficulty in detecting pre-existing minor haplotypes with sufficient length and accuracy. Herein, we characterize a methodological framework based on Illumina MiSeq next-generation sequencing (NGS) coupled with bioinformatics of quasispecies reconstruction (QSR) to realize highly accurate variant calling and genotype-haplotype detection. The core-to-NS3 protease coding sequences in 10 HCV monoinfected patients, 5 of whom had a history of blood transfusion, and 11 HCV/HIV coinfected patients with hemophilia, were studied. Simulation experiments showed that, for minor variants constituting more than 1%, our framework achieved a positive predictive value (PPV) of 100% and sensitivities of 91.7–100% for genotyping and 80.6% for RAV screening. Genotyping analysis indicated the prevalence of dominant Gt1a infection in coinfected patients (6/11 vs 0/10, p = 0.01). For clinical samples, minor genotype overlapping infection was prevalent in HCV/HIV coinfected hemophiliacs (10/11) and patients who experienced whole-blood transfusion (4/5) but none in patients without exposure to blood (0/5). As for RAV screening, the Q80K/R and S122K/R variants were particularly prevalent among minor RAVs observed, detected in 12/21 and 6/21 cases, respectively. Q80K was detected only in coinfected patients, whereas Q80R was predominantly detected in monoinfected patients (1/11 vs 7/10, p < 0.01). Multivariate interdependence analysis revealed the previously unrecognized prevalence of Gt1b-Q80K, in HCV/HIV coinfected hemophiliacs [Odds ratio = 13.4 (3.48–51.9), p < 0.01]. Our study

  20. Aminoglycoside binding to the HIV-1 RNA dimerization initiation site: thermodynamics and effect on the kissing-loop to duplex conversion.

    PubMed

    Bernacchi, Serena; Freisz, Séverine; Maechling, Clarisse; Spiess, Bernard; Marquet, Roland; Dumas, Philippe; Ennifar, Eric

    2007-01-01

    Owing to a striking, and most likely fortuitous, structural and sequence similarity with the bacterial 16 S ribosomal A site, the RNA kissing-loop complex formed by the HIV-1 genomic RNA dimerization initiation site (DIS) specifically binds 4,5-disubstituted 2-deoxystreptamine (2-DOS) aminoglycoside antibiotics. We used chemical probing, molecular modeling, isothermal titration calorimetry (ITC) and UV melting to investigate aminoglycoside binding to the DIS loop-loop complex. We showed that apramycin, an aminoglycoside containing a bicyclic moiety, also binds the DIS, but in a different way than 4,5-disubstituted 2-DOS aminoglycosides. The determination of thermodynamic parameters for various aminoglycosides revealed the role of the different rings in the drug-RNA interaction. Surprisingly, we found that the affinity of lividomycin and neomycin for the DIS (K(d) approximately 30 nM) is significantly higher than that obtained in the same experimental conditions for their natural target, the bacterial A site (K(d) approximately 1.6 microM). In good agreement with their respective affinity, aminoglycoside increase the melting temperature of the loop-loop interaction and also block the conversion from kissing-loop complex to extended duplex. Taken together, our data might be useful for selecting new molecules with improved specificity and affinity toward the HIV-1 DIS RNA.

  1. HIV-1 splicing is controlled by local RNA structure and binding of splicing regulatory proteins at the major 5' splice site.

    PubMed

    Mueller, Nancy; Berkhout, Ben; Das, Atze T

    2015-07-01

    The 5' leader region of the human immunodeficiency virus 1 (HIV-1) RNA genome contains the major 5' splice site (ss) that is used in the production of the many spliced viral RNAs. This splice-donor (SD) region can fold into a stable stem-loop structure and the thermodynamic stability of this RNA hairpin influences splicing efficiency. In addition, splicing may be modulated by binding of splicing regulatory (SR) proteins, in particular SF2/ASF (SRSF1), SC35 (SRSF2), SRp40 (SRSF5) and SRp55 (SRSF6), to sequence elements in the SD region. The role of RNA structure and SR protein binding in splicing control was previously studied by functional analysis of mutant SD sequences. The interpretation of these studies was complicated by the fact that most mutations simultaneously affect both structure and sequence elements. We therefore tried to disentangle the contribution of these two variables by designing more precise SD region mutants with a single effect on either the sequence or the structure. The current analysis indicates that HIV-1 splicing at the major 5'ss is modulated by both the stability of the local RNA structure and the binding of splicing regulatory proteins. PMID:25779589

  2. Genotypic Characterization of Bradyrhizobium Strains Nodulating Small Senegalese Legumes by 16S-23S rRNA Intergenic Gene Spacers and Amplified Fragment Length Polymorphism Fingerprint Analyses

    PubMed Central

    Doignon-Bourcier, Florence; Willems, Anne; Coopman, Renata; Laguerre, Gisele; Gillis, Monique; de Lajudie, Philippe

    2000-01-01

    We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations. PMID:10966419

  3. Genotypic characterization of Bradyrhizobium strains nodulating small Senegalese legumes by 16S-23S rRNA intergenic gene spacers and amplified fragment length polymorphism fingerprint analyses.

    PubMed

    Doignon-Bourcier, F; Willems, A; Coopman, R; Laguerre, G; Gillis, M; de Lajudie, P

    2000-09-01

    We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.

  4. Factors associated with hepatitis C virus RNA levels in early chronic infection: The InC3 Study

    PubMed Central

    Hajarizadeh, Behzad; Grady, Bart; Page, Kimberly; Kim, Arthur Y.; McGovern, Barbara H.; Cox, Andrea L.; Rice, Thomas M.; Sacks-Davis, Rachel; Bruneau, Julie; Morris, Meghan; Amin, Janaki; Schinkel, Janke; Applegate, Tanya; Maher, Lisa; Hellard, Margaret; Lloyd, Andrew R.; Prins, Maria; Geskus, Ronald B; Dore, Gregory J.; Grebely, Jason

    2014-01-01

    Improved understanding of natural history of hepatitis C virus (HCV) RNA levels in chronic infection provides enhanced insights into immunopathogenesis of HCV and has implications for the clinical management of chronic HCV infection. This study assessed factors associated with HCV RNA levels during early chronic infection in a population with well-defined early chronic HCV infection. Data were from an international collaboration of nine prospective cohorts studying acute HCV infection (InC3 Study). Individuals with persistent HCV and detectable HCV RNA during early chronic infection (one year [±4 months] post-infection) were included. Distribution of HCV RNA levels during early chronic infection were compared by selected host and virological factors. A total of 308 individuals were included. Median HCV RNA levels was significantly higher among males (vs. females; 5.15 vs. 4.74 log IU/mL; P<0.01) and among individuals with HIV co-infection (vs. no HIV; 5.89 vs. 4.86; P=0.02). In adjusted logistic regression, male sex (vs. female, adjusted odds ratio [AOR]: 1.93; 95%CI: 1.01, 3.69), interferon lambda 4 (IFNL4) rs12979860 CC genotype (vs. TT/CT; AOR: 2.48; 95%CI: 1.42, 4.35), HIV co-infection (vs. no HIV; AOR: 3.27; 95%CI: 1.35, 7.93), and HCV genotype G2 (vs. G3; AOR: 5.40; 95%CI: 1.63, 17.84) were independently associated with high HCV RNA levels (>5.6 log IU/mL=400,000 IU/mL). In conclusion, this study demonstrated that IFNL4 rs12979860 CC genotype, male sex, HIV co-infection, and HCV genotype G2 are associated with high HCV RNA levels in early chronic infection. These factors exert their role as early as one year following infection. PMID:25580520

  5. Comparison of Sanger and next generation sequencing performance for genotyping Cryptosporidium isolates at the 18S rRNA and actin loci.

    PubMed

    Paparini, Andrea; Gofton, Alexander; Yang, Rongchang; White, Nicole; Bunce, Michael; Ryan, Una M

    2015-01-01

    Cryptosporidium is an important enteric pathogen that infects a wide range of humans and animals. Rapid and reliable detection and characterisation methods are essential for understanding the transmission dynamics of the parasite. Sanger sequencing, and high-throughput sequencing (HTS) on an Ion Torrent platform, were compared with each other for their sensitivity and accuracy in detecting and characterising 25 Cryptosporidium-positive human and animal faecal samples. Ion Torrent reads (n = 123,857) were obtained at both 18S rRNA and actin loci for 21 of the 25 samples. Of these, one isolate at the actin locus (Cattle 05) and three at the 18S rRNA locus (HTS 10, HTS 11 and HTS 12), suffered PCR drop-out (i.e. PCR failures) when using fusion-tagged PCR. Sanger sequences were obtained for both loci for 23 of the 25 samples and showed good agreement with Ion Torrent-based genotyping. Two samples both from pythons (SK 02 and SK 05) produced mixed 18S and actin chromatograms by Sanger sequencing but were clearly identified by Ion Torrent sequencing as C. muris. One isolate (SK 03) was typed as C. muris by Sanger sequencing but was identified as a mixed C. muris and C. tyzzeri infection by HTS. 18S rRNA Type B sequences were identified in 4/6 C. parvum isolates when deep sequenced but were undetected in Sanger sequencing. Sanger was cheaper than Ion Torrent when sequencing a small numbers of samples, but when larger numbers of samples are considered (n = 60), the costs were comparative. Fusion-tagged amplicon based approaches are a powerful way of approaching mixtures, the only draw-back being the loss of PCR efficiency on low-template samples when using primers coupled to MID tags and adaptors. Taken together these data show that HTS has excellent potential for revealing the "true" composition of species/types in a Cryptosporidium infection, but that HTS workflows need to be carefully developed to ensure sensitivity, accuracy and contamination are

  6. A 1.3-Å resolution crystal structure of the HIV-1 trans-activation response region RNA stem reveals a metal ion-dependent bulge conformation

    PubMed Central

    Ippolito, Joseph A.; Steitz, Thomas A.

    1998-01-01

    The crystal structure of an HIV-1 trans-activation response region (TAR) RNA fragment containing the binding site for the trans-activation protein Tat has been determined to 1.3-Å resolution. In this crystal structure, the characteristic UCU bulge of TAR adopts a conformation that is stabilized by three divalent calcium ions and differs from those determined previously by solution NMR. One metal ion, crucial to the loop conformation, binds directly to three phosphates in the loop region. The structure emphasizes the influence of metal ion binding on RNA structure and, given the abundance of divalent metal ion in the cell, raises the question of whether metal ions play a role in the conformation of TAR RNA and the interaction of TAR with Tat and cyclin T in vivo. PMID:9707559

  7. Low-level Viremia Early in HIV Infection

    PubMed Central

    Chen, Iris; Cummings, Vanessa; Fogel, Jessica M.; Marzinke, Mark A.; Clarke, William; Connor, Matthew B.; Griffith, Sam; Buchbinder, Susan; Shoptaw, Steven; del Rio, Carlos; Magnus, Manya; Mannheimer, Sharon; Wheeler, Darrell P.; Mayer, Kenneth H.; Koblin, Beryl A.; Eshleman, Susan H.

    2014-01-01

    HIV RNA levels are usually high early in HIV infection. In the HPTN 061 study, men were tested for HIV infection every six months; six (21.4%) of 28 men who acquired HIV infection during the study had low or undetectable HIV RNA at the time of HIV diagnosis. Antiretroviral drugs were not detected at the time of HIV diagnosis. False-negative HIV test results were obtained for two men using multiple assays. Antiretroviral drug resistance mutations were detected in HIV from one man. Additional studies are needed to identify factors associated with low HIV RNA levels during early HIV infection. PMID:25140905

  8. Detection of HIV-1 minority variants containing the K103N drug-resistance mutation using a simple method to amplify RNA targets (SMART).

    PubMed

    Morabito, Kenneth; Kantor, Rami; Tai, Warren; Schreier, Leeann; Tripathi, Anubhav

    2013-05-01

    The simple method for amplifying RNA targets (SMART) was used to detect K103N, a common HIV-1 reverse transcriptase drug-resistance mutation. Novel amplifiable SMART probes served as reporter molecules for RNA sequences that are captured and separated on a microfluidic platform under zero-flow conditions. Assays were performed both off chip and in a microchip reservoir using a modified version of real-time nucleic acid sequence-based amplification, without the noncyclic phase, and 65°C preheat. A total of 6000 copies/mL of the synthetic sequences were detected within 180 minutes of amplification. Although the sensitivity of research platforms is higher, SMART has the potential to offer comparable sensitivity and speed to commercially available viral load and HIV detection kits. Furthermore, SMART uses an inexpensive, practical, and more accurate isothermal exponential amplification technique. The use of molecular beacons resulted in relatively fast real-time detection (<180 minutes); however, they were also shown to hinder the amplification process when compared with end point detection. Finally, SMART probes were used for modeling of K103N concentrations within an unknown sample. Only 1% of the SMART probes was detected within the wild-type population (6 × 10(8) copies/mL). These results establish the groundwork for point-of-care drug resistance and viral load monitoring in clinical samples, which can revolutionize HIV patient care globally.

  9. A peptide nucleic acid-aminosugar conjugate targeting transactivation response element of HIV-1 RNA genome shows a high bioavailability in human cells and strongly inhibits tat-mediated transactivation of HIV-1 transcription.

    PubMed

    Das, Indrajit; Désiré, Jérôme; Manvar, Dinesh; Baussanne, Isabelle; Pandey, Virendra N; Décout, Jean-Luc

    2012-07-12

    The 6-aminoglucosamine ring of the aminoglycoside antibiotic neomycin B (ring II) was conjugated to a 16-mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. For this purpose, we prepared the aminoglucosamine monomer 15 and attached it to the protected PNA prior to its cleavage from the solid support. We found that the resulting PNA-aminoglucosamine conjugate is stable under acidic conditions, efficiently taken up by the human cells and fairly distributed in both cytosol and nucleus without endosomal entrapment because cotreatment with endosome-disrupting agent had no effect on its cellular distribution. The conjugate displayed very high target specificity in vitro and strongly inhibited Tat mediated transactivation of HIV-1 LTR transcription in a cell culture system. The unique properties of this new class of PNA conjugate suggest it to be a potential candidate for therapeutic application.

  10. A Peptide Nucleic Acid-Aminosugar Conjugate Targeting Transactivation Response Element of HIV-1 RNA Genome Shows a High Bioavailability in Human Cells and Strongly Inhibits Tat-mediated Transactivation of HIV-1 Transcription

    PubMed Central

    Das, Indrajit; Désiré, Jérôme; Manvar, Dinesh; Baussanne, Isabelle; Pandey, Virendra N.; Décout, Jean-Luc

    2012-01-01

    The 6-aminoglucosamine ring of the aminoglycoside antibiotic neomycin B (ring II) was conjugated to a 16 mer peptide nucleic acid (PNA) targeting HIV-1 TAR RNA. For this purpose we prepared the aminoglucosamine monomer 15 and attached it to the protected PNA prior to its cleavage from the solid support. We found that the resulting PNA-aminoglucosamine conjugate is stable under acidic condition, efficiently taken up by the human cells and fairly distributed in both cytosol and nucleus without endosomal entrapment since co-treatment with endosome-disrupting agent had no effect on its cellular distribution. The conjugate displayed very high target specificity in vitro and strongly inhibited Tat mediated transactivation of HIV-1 LTR transcription in cell culture system. The unique properties of this new class of PNA conjugate suggest it to be a potential candidate for therapeutic application. PMID:22698070

  11. Negative Elongation Factor Is Required for the Maintenance of Proviral Latency but Does Not Induce Promoter-Proximal Pausing of RNA Polymerase II on the HIV Long Terminal Repeat

    PubMed Central

    Jadlowsky, Julie K.; Wong, Julian Y.; Graham, Amy C.; Dobrowolski, Curtis; Devor, Renee L.; Adams, Mark D.; Fujinaga, Koh

    2014-01-01

    The role of the negative elongation factor (NELF) in maintaining HIV latency was investigated following small hairpin RNA (shRNA) knockdown of the NELF-E subunit, a condition that induced high levels of proviral transcription in latently infected Jurkat T cells. Chromatin immunoprecipitation (ChIP) assays showed that latent proviruses accumulate RNA polymerase II (RNAP II) on the 5′ long terminal repeat (LTR) but not on the 3′ LTR. NELF colocalizes with RNAP II, and its level increases following proviral induction. RNAP II pause sites on the HIV provirus were mapped to high resolution by ChIP with high-throughput sequencing (ChIP-Seq). Like cellular promoters, RNAP II accumulates at around position +30, but HIV also shows additional pausing at +90, which is immediately downstream of a transactivation response (TAR) element and other distal sites on the HIV LTR. Following NELF-E knockdown or tumor necrosis factor alpha (TNF-α) stimulation, promoter-proximal RNAP II levels increase up to 3-fold, and there is a dramatic increase in RNAP II levels within the HIV genome. These data support a kinetic model for proviral transcription based on continuous replacement of paused RNAP II during both latency and productive transcription. In contrast to most cellular genes, HIV is highly activated by the combined effects of NELF-E depletion and activation of initiation by TNF-α, suggesting that opportunities exist to selectively activate latent HIV proviruses. PMID:24636995

  12. Genotypes of ITS region of rRNA in Microcystis (Cyanobacteria) populations in Erhai Lake (China) and their correlation with eutrophication level.

    PubMed

    Song, Gaofei; Jiang, Yongguang; Yu, Gongliang; Li, Renhui

    2015-10-01

    Previous studies on spatiotemporal changes of Microcystis genotypes have shown that the existence and succession of dominant genotypes always occur in eutrophicated freshwater bodies. However, few studies have focused on the correlation between genotype composition and eutrophication level. In the present study, clone libraries of the internal transcribed spacer (ITS) of rrn operon were sequenced from Microcystis populations in Erhai Lake, a subtropical plateau lake in the preliminary eutrophication stage. The genotype composition of the Microcystis populations was highly variable at spatiotemporal scales, and 473 ITS genotypes were identified from the 800 ITS sequences obtained. However, no significantly dominant ITS genotypes existed in the lake. Comparison of Erhai Lake with four major lakes in China, namely, Taihu, Chaohu, Gucheng, and Shijiu Lakes, showed that the Microcystis ITS genotypes and genetic diversity were negatively correlated with eutrophication level. Extensive comparison of the Microcystis ITS genotypes from waters worldwide revealed that 440 ITS genotypes were unique to Erhai Lake, and no obvious phylogenetic correlations can be detected among the dominant genotypes from different water bodies. The high genetic diversity of the Microcystis populations in Erhai Lake may have resulted from the effect of the early stage of eutrophication.

  13. When to Monitor CD4 Cell Count and HIV RNA to Reduce Mortality and AIDS-Defining Illness in Virologically Suppressed HIV-Positive Persons on Antiretroviral Therapy in High-Income Countries: A Prospective Observational Study

    PubMed Central

    Caniglia, Ellen C.; Sabin, Caroline; Robins, James M.; Logan, Roger; Cain, Lauren E.; Abgrall, Sophie; Mugavero, Michael J.; Hernandez-Diaz, Sonia; Meyer, Laurence; Seng, Remonie; Drozd, Daniel R.; Seage, George R.; Bonnet, Fabrice; Dabis, Francois; Moore, Richard R.; Reiss, Peter; van Sighem, Ard; Mathews, William C.; del Amo, Julia; Moreno, Santiago; Deeks, Steven G.; Muga, Roberto; Boswell, Stephen L.; Ferrer, Elena; Eron, Joseph J.; Napravnik, Sonia; Jose, Sophie; Phillips, Andrew; Olson, Ashley; Justice, Amy C.; Tate, Janet P.; Bucher, Heiner C.; Egger, Matthias; Touloumi, Giota; Sterne, Jonathan A.; Costagliola, Dominique; Saag, Michael; Hernán, Miguel A.

    2016-01-01

    Objective: To illustrate an approach to compare CD4 cell count and HIV-RNA monitoring strategies in HIV-positive individuals on antiretroviral therapy (ART). Design: Prospective studies of HIV-positive individuals in Europe and the USA in the HIV-CAUSAL Collaboration and The Center for AIDS Research Network of Integrated Clinical Systems. Methods: Antiretroviral-naive individuals who initiated ART and became virologically suppressed within 12 months were followed from the date of suppression. We compared 3 CD4 cell count and HIV-RNA monitoring strategies: once every (1) 3 ± 1 months, (2) 6 ± 1 months, and (3) 9–12 ± 1 months. We used inverse-probability weighted models to compare these strategies with respect to clinical, immunologic, and virologic outcomes. Results: In 39,029 eligible individuals, there were 265 deaths and 690 AIDS-defining illnesses or deaths. Compared with the 3-month strategy, the mortality hazard ratios (95% CIs) were 0.86 (0.42 to 1.78) for the 6 months and 0.82 (0.46 to 1.47) for the 9–12 month strategy. The respective 18-month risk ratios (95% CIs) of virologic failure (RNA >200) were 0.74 (0.46 to 1.19) and 2.35 (1.56 to 3.54) and 18-month mean CD4 differences (95% CIs) were −5.3 (−18.6 to 7.9) and −31.7 (−52.0 to −11.3). The estimates for the 2-year risk of AIDS-defining illness or death were similar across strategies. Conclusions: Our findings suggest that monitoring frequency of virologically suppressed individuals can be decreased from every 3 months to every 6, 9, or 12 months with respect to clinical outcomes. Because effects of different monitoring strategies could take years to materialize, longer follow-up is needed to fully evaluate this question. PMID:26895294

  14. Quantitation of HIV-1 RNA viral load using nucleic acid sequence based amplification methodology and comparison with other surrogate markers for disease progression.

    PubMed

    Sitnik, R; Pinho, J R

    1998-01-01

    In this study, HIV-1 viral blood quantitation determined by Nucleic Acid Sequence Based Amplification (NASBA) was compared with other surrogate disease progression markers (antigen p24, CD4/CD8 cell counts and beta-2 microglobulin) in 540 patients followed up at São Paulo, SP, Brazil. HIV-1 RNA detection was statistically associated with the presence of antigen p24, but the viral RNA was also detected in 68% of the antigen p24 negative samples, confirming that NASBA is much more sensitive than the determination of antigen p24. Regarding other surrogate markers, no statistically significant association with the detection of viral RNA was found. The reproducibility of this viral load assay was assessed by 14 runs of the same sample, using different reagents batches. Viral load values in this sample ranged from 5.83 to 6.27 log (CV = 36%), less than the range (0.5 log) established to the determination of significant viral load changes. PMID:9698880

  15. NMR structure of a kissing complex formed between the TAR RNA element of HIV-1 and a LNA-modified aptamer

    PubMed Central

    Lebars, Isabelle; Richard, Tristan; Di Primo, Carmelo; Toulmé, Jean-Jacques

    2007-01-01

    The trans-activating responsive (TAR) RNA element located in the 5′ untranslated region of the HIV-1 genome is a 57-nt imperfect stem-loop essential for the viral replication. TAR regulates transcription by interacting with both viral and cellular proteins. RNA hairpin aptamers specific for TAR were previously identified by in vitro selection [Ducongé,F. and Toulmé,J.J. (1999) In vitro selection identifies key determinants for loop-loop interactions: RNA aptamers selective for the TAR RNA element of HIV-1. RNA, 5, 1605–1614]. These aptamers display a 5′-GUCCCAGA-3′ consensus apical loop, partially complementary to the TAR one, leading to the formation of a TAR–aptamer kissing complex. The conserved GA combination (underlined in the consensus sequence) has been shown to be crucial for the formation of a highly stable complex. To improve the nuclease resistance of the aptamer and to increase its affinity for TAR, locked nucleic acid (LNA) nucleotides were introduced in the aptamer apical loop. LNA are nucleic acids analogues that contain a 2′-O,4′-C methylene linkage and that raise the thermostablity of duplexes. We solved the NMR solution structure of the TAR–LNA-modified aptamer kissing complex. Structural analysis revealed the formation of a non-canonical G•A pair leading to increased stacking at the stem-loop junction. Our data also showed that the introduction of LNA residues provides an enhanced stability while maintaining a normal Watson–Crick base pairing with a loop–loop conformation close to an A-type. PMID:17768146

  16. HIV-1 Tat protein enhances the intracellular growth of Leishmania amazonensis via the ds-RNA induced protein PKR

    PubMed Central

    Vivarini, Áislan de Carvalho; Santos Pereira, Renata de Meirelles; Barreto-de-Souza, Victor; Temerozo, Jairo Ramos; Soares, Deivid C.; Saraiva, Elvira M.; Saliba, Alessandra Mattos; Bou-Habib, Dumith Chequer; Lopes, Ulisses Gazos

    2015-01-01

    HIV-1 co-infection with human parasitic diseases is a growing public health problem worldwide. Leishmania parasites infect and replicate inside macrophages, thereby subverting host signaling pathways, including the response mediated by PKR. The HIV-1 Tat protein interacts with PKR and plays a pivotal role in HIV-1 replication. This study shows that Tat increases both the expression and activation of PKR in Leishmania-infected macrophages. Importantly, the positive effect of Tat addition on parasite growth was dependent on PKR signaling, as demonstrated in PKR-deficient macrophages or macrophages treated with the PKR inhibitor. The effect of HIV-1 Tat on parasite growth was prevented when the supernatant of HIV-1-infected macrophages was treated with neutralizing anti-HIV-1 Tat prior to Leishmania infection. The addition of HIV-1 Tat to Leishmania-infected macrophages led to inhibition of iNOS expression, modulation of NF-kB activation and enhancement of IL-10 expression. Accordingly, the expression of a Tat construct containing mutations in the basic region (49–57aa), which is responsible for the interaction with PKR, favored neither parasite growth nor IL-10 expression in infected macrophages. In summary, we show that Tat enhances Leishmania growth through PKR signaling. PMID:26608746

  17. Valacyclovir Decreases Plasma HIV-1 RNA in HSV-2 Seronegative Individuals: A Randomized Placebo-Controlled Crossover Trial

    PubMed Central

    Vanpouille, Christophe; Lisco, Andrea; Grivel, Jean-Charles; Bassit, Leda C.; Kauffman, Robert C.; Sanchez, Jorge; Schinazi, Raymond F.; Lederman, Michael M.; Rodriguez, Benigno; Margolis, Leonid

    2015-01-01

    Background. Acyclovir (ACV), a highly specific anti-herpetic drug, acts as a DNA chain terminator for several human herpesviruses (HHVs), including HHV-2 (HSV-2), a common human immunodeficiency virus (HIV)-1 co-pathogen. Several trials demonstrated that HSV-2 suppressive therapy using ACV or its prodrug valacyclovir (valACV) reduced plasma HIV-1 viral load (VL) in HIV-1/HSV-2 coinfected persons, and this was proposed to be due to a decrease in generalized immune activation. Recently, however, we found that ACV directly suppresses HIV-1 ex vivo in tissues free of HSV-2 but endogenously coinfected with other HHVs. Here, we asked whether valACV suppresses VL in HIV-1 infected HSV-2-seronegative persons. Methods. Eighteen HIV-1 infected HSV-2-seronegative individuals were randomly assigned in a double blind placebo-controlled, crossover trial. Eligible participants had CD4 cell counts of ≥500 cells/µL and were not taking antiretroviral therapy. Subjects in group A received 12 weeks of valACV 500 mg given twice daily by mouth followed by 2 weeks of a no treatment washout and then 12 weeks of placebo; subjects in group B received 12 weeks of placebo followed by 2 weeks of no treatment washout and then 12 weeks of valACV 500 mg twice daily. Results. HIV-1 VL in plasma of patients treated with valACV 500 mg twice daily for 12 weeks was reduced on average by 0.37 log10 copies/mL. Conclusions. These data indicate that the effects of valACV on HIV-1 replication are not related to the suppression of HSV-2-mediated inflammation and are consistent with a direct effect of ACV on HIV-1 replication. PMID:25740794

  18. Production of HIV Particles Is Regulated by Altering Sub-Cellular Localization and Dynamics of Rev Induced by Double-Strand RNA Binding Protein

    PubMed Central

    Urcuqui-Inchima, Silvio; Patiño, Claudia; Zapata, Ximena; García, María Patricia; Arteaga, José; Chamot, Christophe; Kumar, Ajit; Hernandez-Verdun, Danièle

    2011-01-01

    Human immunodeficiency virus (HIV)-1 encoded Rev is essential for export from the nucleus to the cytoplasm, of unspliced and singly spliced transcripts coding for structural and nonstructural viral proteins. This process is spatially and temporally coordinated resulting from the interactions between cellular and viral proteins. Here we examined the effects of the sub-cellular localization and dynamics of Rev on the efficiency of nucleocytoplasmic transport of HIV-1 Gag transcripts and virus particle production. Using confocal microscopy and fluorescence recovery after bleaching (FRAP), we report that NF90ctv, a cellular protein involved in Rev function, alters both the sub-cellular localization and dynamics of Rev in vivo, which drastically affects the accumulation of the viral protein p24. The CRM1–dependent nuclear export of Gag mRNA linked to the Rev Response Element (RRE) is dependent on specific domains of the NF90ctv protein. Taken together, our results demonstrate that the appropriate intracellular localization and dynamics of Rev could regulate Gag assembly and HIV-1 replication. PMID:21364984

  19. Sex-associated Differences in Pre-Antiretroviral Therapy Plasma HIV-1 RNA in Diverse Areas of the World Vary by CD4 Cell Count

    PubMed Central

    Grinsztejn, Beatriz; Smeaton, Laura; Barnett, Ronald; Klingman, Karin; Hakim, James; Flanigan, Timothy; Kumarasamy, N; Campbell, Thomas; Currier, Judith

    2011-01-01

    Background Sex differences in the natural history of HIV infection may vary between resource-rich and resource-limited settings. Objective Baseline characteristics from a randomized clinical trial of treatment naïve subjects conducted at sites in Africa, Asia, the Caribbean, and North and South America were analyzed to determine if there were significant differences by sex. Results Of the 1571 participants, 740 (47.1%) were women. Women had higher mean screening CD4 cell counts (average 15 cells higher, (p<0.001), lower mean hemoglobin and creatinine clearance, a lower mean baseline HIV-1 viral load (4.85 log10 vs. 5.05 log10 copies/mL (P<0.001)) and were less likely to have a prior AIDS diagnosis than men. The sex difference in viral load difference was related to CD4 cell count, however it was independent of country and persisted within the strata with CD4 < 200 cells/mm3. Conclusion Women in resource limited settings have lower levels of plasma HIV-1 RNA and appear to present for enrollment into a clinical trials at an earlier stage of disease than men. The biologic basis for lower viral in women compared to men remains unexplained. It will be important to determine if the sex differences observed at baseline impact clinical outcomes once the PEARLS clinical trial is completed. PMID:22024521

  20. HIV-1 Nucleocapsid Protein Switches the Pathway of TAR RNA/DNA Annealing from Loop-Loop “Kissing” to “Zipper”

    PubMed Central

    Vo, My-Nuong; Barany, George; Rouzina, Ioulia; Musier-Forsyth, Karin

    2009-01-01

    Summary The chaperone activity of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein (NC) facilitates multiple nucleic acid rearrangements that are critical for reverse transcription of the single-stranded RNA genome into double-stranded DNA. Annealing of the trans-activation response element (TAR) RNA hairpin to a complementary TAR DNA hairpin is an essential step in the minus-strand transfer step of reverse transcription. Previously, we used truncated 27-nucleotide (nt) mini-TAR RNA and DNA constructs to investigate this annealing reaction pathway in the presence and absence of HIV-1 NC. In this work, full-length 59-nt TAR RNA and TAR DNA constructs were used to systematically study TAR hairpin annealing kinetics. In the absence of NC, full-length TAR hairpin annealing is ∼10-fold slower than mini-TAR annealing. Similar to mini-TAR annealing, the reaction pathway for TAR in the absence of NC involves the fast formation of an unstable “kissing” loop intermediate, followed by a slower conversion to an extended duplex. NC facilitates the annealing of TAR by ∼105-fold by stabilizing the bimolecular intermediate (∼104-fold) and promoting the subsequent exchange reaction (∼10-fold). In contrast to the mini-TAR annealing pathway, wherein NC-mediated annealing can initiate through both loop-loop kissing and a distinct “zipper” pathway involving nucleation at the 3′/5′ terminal ends, full-length TAR hairpin annealing switches predominantly to the zipper pathway in the presence of saturated NC. PMID:19154737

  1. Interferon-induced HERC5 is evolving under positive selection and inhibits HIV-1 particle production by a novel mechanism targeting Rev/RRE-dependent RNA nuclear export

    PubMed Central

    2014-01-01

    Background Type I interferon (IFN) inhibits virus replication by activating multiple antiviral mechanisms and pathways. It has long been recognized that type I IFNs can potently block HIV-1 replication in vitro; as such, HIV-1 has been used as a system to identify and characterize IFN-induced antiviral proteins responsible for this block. IFN-induced HERC5 contains an amino-terminal Regulator of Chromosome Condensation 1 (RCC1)-like domain and a carboxyl-terminal Homologous to the E6-AP Carboxyl Terminus (HECT) domain. HERC5 is the main cellular E3 ligase that conjugates the IFN-induced protein ISG15 to proteins. This E3 ligase activity was previously shown to inhibit the replication of evolutionarily diverse viruses, including HIV-1. The contribution of the RCC1-like domain to the antiviral activity of HERC5 was previously unknown. Results In this study, we showed that HERC5 inhibits HIV-1 particle production by a second distinct mechanism that targets the nuclear export of Rev/RRE-dependent RNA. Unexpectedly, the E3 ligase activity of HERC5 was not required for this inhibition. Instead, this activity required the amino-terminal RCC1-like domain of HERC5. Inhibition correlated with a reduction in intracellular RanGTP protein levels and/or the ability of RanGTP to interact with RanBP1. Inhibition also correlated with altered subcellular localization of HIV-1 Rev. In addition, we demonstrated that positive evolutionary selection is operating on HERC5. We identified a region in the RCC1-like domain that exhibits an exceptionally high probability of having evolved under positive selection and showed that this region is required for HERC5-mediated inhibition of nuclear export. Conclusions We have identified a second distinct mechanism by which HERC5 inhibits HIV-1 replication and demonstrate that HERC5 is evolving under strong positive selection. Together, our findings contribute to a growing body of evidence suggesting that HERC5 is a novel host restriction factor

  2. A protein ballet around the viral genome orchestrated by HIV-1 reverse transcriptase leads to an architectural switch: from nucleocapsid-condensed RNA to Vpr-bridged DNA

    PubMed Central

    Lyonnais, Sébastien; Gorelick, Robert J.; Heniche-Boukhalfa, Fatima; Bouaziz, Serge; Parissi, Vincent; Mouscadet, Jean-François; Restle, Tobias; Gatell, Jose Maria; Le Cam, Eric; Mirambeau, Gilles

    2012-01-01

    Summary HIV-1 reverse transcription is achieved in the newly infected cell before viral DNA (vDNA) nuclear import. Reverse transcriptase (RT) has previously been shown to function as a molecular motor, dismantling the nucleocapsid complex that binds the viral genome as soon as plus-strand DNA synthesis initiates. We first propose a detailed model of this dismantling in close relationship with the sequential conversion from RNA to double-stranded (ds) DNA, focusing on the nucleocapsid protein (NCp7). The HIV-1 DNA-containing preintegration complex (PIC) resulting from completion of reverse transcription is translocated through the nuclear pore. The PIC nucleoprotein architecture is poorly understood but contains at least two HIV-1 proteins initially from the virion core, namely Integrase (IN) and the viral protein r (Vpr). We next present a set of electron micrographs supporting that Vpr behaves as a DNA architectural protein, initiating multiple DNA bridges over more than 500 base pairs (bp). These complexes are shown to interact with NCp7 bound to single-stranded nucleic acid regions that are thought to maintain IN binding during dsDNA synthesis, concurrently with nucleocapsid complex dismantling. This unexpected binding of Vpr conveniently leads to a compacted but filamentous folding of the vDNA that should favor its nuclear import. Finally, nucleocapsid-like aggregates engaged in dsDNA synthesis appear to efficiently bind to F-actin filaments, a property that may be involved in targeting complexes to the nuclear envelope. More generally, this article highlights unique possibilities offered by in vitro reconstitution approaches combined with macromolecular imaging to gain insights into the mechanisms that alter the nucleoprotein architecture of the HIV-1 genome, ultimately enabling its insertion into the nuclear chromatin. PMID:23017337

  3. The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3

    PubMed Central

    Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal GP; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

    2015-01-01

    RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. PMID:26637431

  4. The Extent of mRNA Editing Is Limited in Chicken Liver and Adipose, but Impacted by Tissular Context, Genotype, Age, and Feeding as Exemplified with a Conserved Edited Site in COG3.

    PubMed

    Roux, Pierre-François; Frésard, Laure; Boutin, Morgane; Leroux, Sophie; Klopp, Christophe; Djari, Anis; Esquerré, Diane; Martin, Pascal G P; Zerjal, Tatiana; Gourichon, David; Pitel, Frédérique; Lagarrigue, Sandrine

    2016-02-01

    RNA editing is a posttranscriptional process leading to differences between genomic DNA and transcript sequences, potentially enhancing transcriptome diversity. With recent advances in high-throughput sequencing, many efforts have been made to describe mRNA editing at the transcriptome scale, especially in mammals, yielding contradictory conclusions regarding the extent of this phenomenon. We show, by detailed description of the 25 studies focusing so far on mRNA editing at the whole-transcriptome scale, that systematic sequencing artifacts are considered in most studies whereas biological replication is often neglected and multi-alignment not properly evaluated, which ultimately impairs the legitimacy of results. We recently developed a rigorous strategy to identify mRNA editing using mRNA and genomic DNA sequencing, taking into account sequencing and mapping artifacts, and biological replicates. We applied this method to screen for mRNA editing in liver and white adipose tissue from eight chickens and confirm the small extent of mRNA recoding in this species. Among the 25 unique edited sites identified, three events were previously described in mammals, attesting that this phenomenon is conserved throughout evolution. Deeper investigations on five sites revealed the impact of tissular context, genotype, age, feeding conditions, and sex on mRNA editing levels. More specifically, this analysis highlighted that the editing level at the site located on COG3 was strongly regulated by four of these factors. By comprehensively characterizing the mRNA editing landscape in chickens, our results highlight how this phenomenon is limited and suggest regulation of editing levels by various genetic and environmental factors. PMID:26637431

  5. Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide.

    PubMed

    Wei, Baojun; Wei, Yuxiang; Zhang, Kuo; Wang, Jing; Xu, Ruihuan; Zhan, Sien; Lin, Guigao; Wang, Wei; Liu, Min; Wang, Lunan; Zhang, Rui; Li, Jinming

    2009-05-01

    RNA-based therapeutic strategies are used widely due to their highly specific mode of action. However, the major obstacle in any RNA-based therapy is cellular delivery and stability in the cells. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for drug delivery. In this study, we utilized the heterobifunctional crosslinker, sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (sulfo-SMPB), to conjugate the human immunodeficiency virus-1 (HIV-1) Tat peptide and MS2 VLPs; the antisense RNA against the 5'-untranslated region (UTR) and the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) was packaged into these particles by using a two-plasmid coexpression system. The MS2 VLPs conjugated with the Tat peptide were then transferred into Huh-7 cells containing an HCV reporter system. The packaged antisense RNA showed an inhibitory effect on the translation of HCV. This paper describes our initial results with this system using the Tat peptide. PMID:18823738

  6. A truncated hnRNP A1 isoform, lacking the RGG-box RNA binding domain, can efficiently regulate HIV-1 splicing and replication.

    PubMed

    Jean-Philippe, Jacques; Paz, Sean; Lu, Michael L; Caputi, Massimo

    2014-01-01

    Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is one of the most abundant RNA binding proteins. hnRNP A1 is localized prevalently in the nucleus but it can relocate to the cytoplasm in response to specific stimuli shuttling between nuclear and cytoplasmic compartments. The cellular localization of this protein is regulated by a short C-terminus motif (M9) and other less defined sequences. The RNA binding specificity of this protein is dependent on multiple RNA binding domains (RBDs), which regulate its role in RNA processing and expression. hnRNP A1 plays multiple roles in gene expression by regulating the biogenesis and translation of messengers RNAs, the processing of miRNAs, affecting transcription and controlling telomere maintenance. The multiple functions of this protein correlate with diverse roles in genetic disease, cancer and the replication of viral pathogens. Utilizing a tagged hnRNP A1 deletion library we have shown that the three hnRNP A1 RBDs contribute to the prevalent nuclear distribution of the protein. Our data also indicate that a truncated form of the protein, lacking one of the RBDs, the RGG-box, can regulate splicing of a splicing reporter minigene and down-regulate replication of the HIV-1 virus with efficiency comparable to the wild-type protein. This functional hnRNP A1 deletion mutant is similar to a predicted hnRNP A1 isoform, which had not been previously experimentally characterized. PMID:24530421

  7. Early Infection HIV-1 Envelope V1-V2 Genotypes Do Not Enhance Binding or Replication in Cells Expressing High Levels of α4β7 Integrin

    PubMed Central

    Etemad, Behzad; Gonzalez, Oscar A.; McDonough, Sean; Pena-Cruz, Victor; Sagar, Manish

    2013-01-01

    It has been postulated that HIV-1 envelope properties, such as shorter and less glycosylated V1-V2 loops commonly observed among non-subtype B early – transmitted viruses, promote utilization of the gut homing integrin α4β7. This property potentially confers an advantage to some HIV-1 variants early after acquisition. We found that replication competent recombinant viruses incorporating HIV-1 subtype A compact and less glycosylated early versus chronic phase V1-V2 loops demonstrated no significant difference in binding to α4β7 high CD8+ T cells or replication in α4β7 high CD4+ T cells. Integrin α4β7 usage does not select for shorter less glycosylated envelopes during transmission. PMID:23797693

  8. Early infection HIV-1 envelope V1-V2 genotypes do not enhance binding or replication in cells expressing high levels of α4β7 integrin.

    PubMed

    Etemad, Behzad; Gonzalez, Oscar A; McDonough, Sean; Pena-Cruz, Victor; Sagar, Manish

    2013-11-01

    It has been postulated that HIV-1 envelope properties, such as shorter and less-glycosylated V1-V2 loops commonly observed among non-subtype B early-transmitted viruses, promote utilization of the gut homing integrin α4β7. This property potentially confers an advantage to some HIV-1 variants early after acquisition. We found that replication-competent recombinant viruses incorporating HIV-1 subtype A compact and less-glycosylated early versus chronic phase V1-V2 loops demonstrated no significant difference in binding to α4β7 high CD8⁺ T cells or replication in α4β7 high CD4⁺ T cells. Integrin α4β7 usage does not select for shorter less-glycosylated envelopes during transmission. PMID:23797693

  9. Early Initiation of Combination Antiretroviral Therapy in HIV-1–Infected Newborns Can Achieve Sustained Virologic Suppression With Low Frequency of CD4+ T Cells Carrying HIV in Peripheral Blood

    PubMed Central

    Bitnun, Ari; Samson, Lindy; Chun, Tae-Wook; Kakkar, Fatima; Brophy, Jason; Murray, Danielle; Justement, Shawn; Soudeyns, Hugo; Ostrowski, Mario; Mujib, Shariq; Harrigan, P. Richard; Kim, John; Sandstrom, Paul; Read, Stanley E.

    2014-01-01

    Background. A human immunodeficiency virus type 1 (HIV-1)–infected infant started on combination antiretroviral therapy (cART) at 30 hours of life was recently reported to have no detectable plasma viremia after discontinuing cART. The current study investigated the impact of early cART initiation on measures of HIV-1 reservoir size in HIV-1–infected children with sustained virologic suppression. Methods. Children born to HIV-1–infected mothers and started on cART within 72 hours of birth at 3 Canadian centers were assessed. HIV serology, HIV-1–specific cell-mediated immune responses, plasma viremia, cell-associated HIV-1 DNA and RNA, presence of replication-competent HIV-1, and HLA genotype were determined for HIV-1–infected children with sustained virologic suppression. Results. Of 136 cART-treated children, 12 were vertically infected (8.8%). In the 4 who achieved sustained virologic suppression, HIV serology, HIV-1–specific cell-mediated immune responses (Gag, Nef), and ultrasensitive viral load were negative. HIV-1 DNA was not detected in enriched CD4+ T cells of the 4 children (<2.6 copies/106 CD4+ T cells), whereas HIV-1 RNA was detected (19.5–130 copies/1.5 µg RNA). No virion-associated HIV-1 RNA was detected following mitogenic stimulation of peripheral blood CD4+ T cells (5.4–8.0 million CD4+ T cells) in these 4 children, but replication competent virus was detected by quantitative co-culture involving a higher number of cells in 1 of 2 children tested (0.1 infectious units/106 CD4+ T cells). Conclusions. In perinatally HIV-1–infected newborns, initiation of cART within 72 hours of birth may significantly reduce the size of the HIV-1 reservoirs. Cessation of cART may be necessary to determine whether functional HIV cure can be achieved in such children. PMID:24917662

  10. Longer duration of homelessness is associated with a lower likelihood of non-detectable plasma HIV-1 RNA viral load among people who use illicit drugs in a Canadian setting.

    PubMed

    Loh, Jane; Kennedy, Mary Clare; Wood, Evan; Kerr, Thomas; Marshall, Brandon; Parashar, Surita; Montaner, Julio; Milloy, M-J

    2016-11-01

    Homelessness is common among people who use drugs (PWUD) and, for those living with HIV/AIDS, an important contributor to sub-optimal HIV treatment outcomes. This study aims to investigate the relationship between the duration of homelessness and the likelihood of plasma HIV-1 RNA viral load (VL) non-detectability among a cohort of HIV-positive PWUD. We used data from the ACCESS study, a long-running prospective cohort study of HIV-positive PWUD linked to comprehensive HIV clinical records including systematic plasma HIV-1 RNA VL monitoring. We estimated the longitudinal relationship between the duration of homelessness and the likelihood of exhibiting a non-detectable VL (i.e., <500 copies/mL plasma) using generalized linear mixed-effects modelling. Between May 1996 and June 2014, 922 highly active antiretroviral therapy-exposed participants were recruited and contributed 8188 observations. Of these, 4800 (59%) were characterized by non-detectable VL. Participants reported they were homeless in 910 (11%) interviews (median: six months, interquartile range: 6-12 months). A longer duration of homelessness was associated with lower odds of VL non-detectability (adjusted odds ratio = 0.71 per six-month period of homelessness, 95% confidence interval: 0.60-0.83) after adjustment for age, ancestry, drug use patterns, engagement in addiction treatment, and other potential confounders. Longer durations of episodes of homelessness in this cohort of HIV-positive illicit drug users were associated with a lower likelihood of plasma VL non-detectability. Our findings suggest that interventions that seek to promptly house homeless individuals, such as Housing First approaches, might assist in maximizing the clinical and public health benefits of antiretroviral therapy among people living with HIV/AIDS. PMID:27248328

  11. Mutations of Conserved Residues in the Major Homology Region Arrest Assembling HIV-1 Gag as a Membrane-Targeted Intermediate Containing Genomic RNA and Cellular Proteins

    PubMed Central

    Tanaka, Motoko; Robinson, Bridget A.; Chutiraka, Kasana; Geary, Clair D.; Reed, Jonathan C.

    2015-01-01

    ABSTRACT The major homology region (MHR) is a highly conserved motif that is found within the Gag protein of all orthoretroviruses and some retrotransposons. While it is widely accepted that the MHR is critical for assembly of HIV-1 and other retroviruses, how the MHR functions and why it is so highly conserved are not understood. Moreover, consensus is lacking on when HIV-1 MHR residues function during assembly. Here, we first addressed previous conflicting reports by confirming that MHR deletion, like conserved MHR residue substitution, leads to a dramatic reduction in particle production in human and nonhuman primate cells expressing HIV-1 proviruses. Next, we used biochemical analyses and immunoelectron microscopy to demonstrate that conserved residues in the MHR are required after assembling Gag has associated with genomic RNA, recruited critical host factors involved in assembly, and targeted to the plasma membrane. The exact point of inhibition at the plasma membrane differed depending on the specific mutation, with one MHR mutant arrested as a membrane-associated intermediate that is stable upon high-salt treatment and other MHR mutants arrested as labile, membrane-associated intermediates. Finally, we observed the same assembly-defective phenotypes when the MHR deletion or conserved MHR residue substitutions were engineered into Gag from a subtype B, lab-adapted provirus or Gag from a subtype C primary isolate that was codon optimized. Together, our data support a model in which MHR residues act just after membrane targeting, with some MHR residues promoting stability and another promoting multimerization of the membrane-targeted assembling Gag oligomer. IMPORTANCE The retroviral Gag protein exhibits extensive amino acid sequence variation overall; however, one region of Gag, termed the major homology region, is conserved among all retroviruses and even some yeast retrotransposons, although the reason for this conservation remains poorly understood. Highly

  12. Ledipasvir/sofosbuvir fixed-dose combination for treatment of hepatitis C virus genotype 4 infection.

    PubMed

    Nehra, V; Tan, E M; Rizza, S A; Temesgen, Z

    2016-02-01

    Hepatitis C virus (HCV) genotype 4 accounts for 8-13% of all chronic HCV infections worldwide. Patients with HCV genotype 4 have been reported to have poor treatment responses to PEGylated interferon and ribavirin regimens. Recently a single tablet, fixed-dose combination of sofosbuvir, an RNA-directed RNA polymerase (NS5B) inhibitor, and ledipasvir, a nonstructural protein 5A (NS5A) inhibitor, has been approved for treatment of chronic HCV infection. Two studies using the fixed-dose combination in chronic HCV genotype 4 for 12 weeks reported sustained virologic response rates at 12 weeks (SVR12) of 93-95%. Data also support the use of ledipasvir/sofosbuvir in chronic HCV genotype 4 and HIV co-infection. Administered as a single once-daily oral regimen, this ribavirin- and interferon-free regimen is well tolerated, with low potential for adverse effects and represents a significant advancement in the treatment of chronic HCV genotype 4 infection.

  13. Cellular HIV type 1 DNA levels are equivalent among drug-sensitive and drug-resistant strains in newly diagnosed and antiretroviral naive patients.

    PubMed

    Antoniadou, Zoi-Anna; Hezka, Johana; Kousiappa, Ioanna; Mamais, Ioannis; Skoura, Lemonia; Pilalas, Dimitris; Metallidis, Simeon; Nicolaidis, Pavlos; Malisiovas, Nicolaos; Kostrikis, Leondios G

    2014-03-01

    The emergence of resistance against current antiretroviral drugs to human immunodeficiency virus type 1 (HIV-1) is an increasingly important concern to the continuous success of antiretroviral therapy to HIV-1-infected patients. In the past decade, a number of studies reported that the prevalence of transmitted drug resistance among newly diagnosed patients has reached an overall 9% prevalence worldwide. Also, a number of studies using longitudinal HIV-1 patient study cohorts demonstrated that the cellular HIV-1 DNA level in peripheral blood mononuclear cells (PBMCs) has a prognostic value for the progression of HIV-1 disease independently of plasma HIV-1 RNA load and CD4 count. Using a previously established molecular-beacon-based real-time PCR methodology, cellular HIV-1 DNA levels were quantified in newly diagnosed and antiretroviral-naive patients in Northern Greece recruited between 2009 and 2010 using a predefined enrolling strategy, in an effort to investigate whether there is any relationship between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. As part of the same study, DNA sequences encoding the env (C2-C5 region of gp120) were also amplified from PBMC-extracted DNA in order to determine the genotypic coreceptor tropism and genetic subtype. Cellular HIV-1 DNA levels had a median of 3.309 log10 HIV-1 copies per 10(6) PBMCs and demonstrated no correlation between cellular HIV-1 DNA levels and HIV-1 transmitted drug resistance. An absence of association between cellular HIV-1 DNA levels with plasma viral HIV-1 RNA load and CD4 levels was also found reconfirming the previously published study. Genotypic analysis of coreceptor tropism indicated that 96% of samples, independently of the presence or not of genotypic drug resistance, were CCR5-tropic. Overall, the findings reconfirmed the previously proposed proposition that transmitted drug resistance does not have an impact on disease progression in HIV-1-infected individuals. Also, CCR5

  14. Association of complement receptor 2 polymorphisms with innate resistance to HIV-1 infection.

    PubMed

    Herrero, R; Real, L M; Rivero-Juárez, A; Pineda, J A; Camacho, Á; Macías, J; Laplana, M; Konieczny, P; Márquez, F J; Souto, J C; Soria, J M; Saulle, I; Lo Caputo, S; Biasin, M; Rivero, A; Fibla, J; Caruz, A

    2015-03-01

    HIV-1 induces activation of complement through the classical and lectin pathways. However, the virus incorporates several membrane-bound or soluble regulators of complement activation (RCA) that inactivate complement. HIV-1 can also use the complement receptors (CRs) for complement-mediated antibody-dependent enhancement of infection (Ć-ADE). We hypothesize that hypofunctional polymorphisms in RCA or CRs may protect from HIV-1 infection. For this purpose, 139 SNPs located in 19 RCA and CRs genes were genotyped in a population of 201 Spanish HIV-1-exposed seronegative individuals (HESN) and 250 HIV-1-infected patients. Two SNPs were associated with infection susceptibility, rs1567190 in CR2 (odds ratio (OR) = 2.27, P = 1 × 10(-4)) and rs2842704 in C4BPA (OR = 2.11, P = 2 × 10(-4)). To replicate this finding, we analyzed a cohort of Italian, sexually HESN individuals. Although not significant (P = 0.25, OR = 1.57), similar genotypic proportions were obtained for the CR2 marker rs1567190. The results of the two association analyses were combined through a random effect meta-analysis, with a significant P-value of 2.6 x 10(-5) (OR = 2.07). Furthermore, we found that the protective CR2 genotype is correlated with lower levels CR2 mRNA as well as differences in the ratio of the long and short CR2 isoforms.

  15. [HIV lipodystrophy].

    PubMed

    Snopková, S; Matýsková, M; Povolná, K; Polák, P; Husa, P

    2010-12-01

    Combined antiretroviral therapy results in extraordinary decrease of morbidity and mortality of HIV-infected patients and in an essential change of the HIV/AIDS disease prognosis. However, long-term intake of antiretroviral medicaments is related to occurrence of metabolic and morphological abnormalities, of which some have been combined into a new syndrome--the so called HIV lipodystrophy. The HIV lipodystrophy syndrome covers metabolic and morphological changes. Metabolic changes include dyslipidaemia with hypercholesterolaemia and/or hypertriglyceridaemia, insulin resistance with hyperinsulinaemia and hyperlaktataemia. Morphological changes have the nature of lipoatrophia (loss of subcutaneous fat--on the cheeks, on extremities, on buttocks and marked prominence of surface veins) or lipohypertrophia (growth of fat tissue--on the chest, in the dorsocervical area, lipomatosis of visceral tissues and organs, fat accumulation in the abdominal area). Several HIV lipodystrophy features are very similar to the metabolic syndrome of the general population. That is why this new syndrome represents a prospective risk of premature atherosclerosis and increase of the cardiovascular risk in young HIV positive individuals. The article mentions major presented studies dealing with the relation of antiretroviral treatment and the cardiovascular risk. The conclusions of the studies are not unequivocal--this is, among others, given by the reason that their length is short from the viewpoint of atherogenesis. The major risk of subclinical atherosclerosis acceleration seems to be related to the deep immunodeficiency and low number of CD4+ lymphocytes and florid, uncontrolled HIV infection with a high number of HIV-1 RNA copies actually circulating in the plasma. The question, whether metabolic and morphological changes related to HIV and cART carry a similar atherogenic potential as in the general population, remains open for future. PMID:21261108

  16. Documented prevalence of HIV type 1 antiretroviral transmitted drug resistance in Ireland from 2004 to 2008.

    PubMed

    De Gascun, Cillian F; Waters, Allison; Regan, Ciara; O'Halloran, Jane; Farrell, Gillian; Coughlan, Suzie; Bergin, Colm; Powderly, William G; Hall, William W

    2012-03-01

    HIV-1-infected individuals with transmitted HIV drug resistance (TDR) begin antiretroviral therapy (ART) with a lower genetic barrier to resistance and a higher risk of both virological failure and of developing further resistance. TDR surveillance informs HIV-1 public health strategies and first line ART. TDR has not been studied nationally in an Irish population. This study includes all new HIV diagnoses from January 2004 to September 2008 from the National Virus Reference Laboratory, University College Dublin. HIV-1 protease and reverse transcriptase sequences were generated, and resistance mutations identified using the Siemens TRUGENE HIV-1 Genotyping System. Subtypes were determined using web-based genotyping tools. The study comprised 1579 patients. There were 305 new diagnoses in 2004 (173 male; 132 female), 298 in 2005 (175M; 123F), 321 in 2006 (197M; 124F), 297 in 2007 (184M; 113F), and 358 (235M; 123F) in 2008. HIV-1 RNA was sequenced from 158/305 patients in 2004, 199/298 in 2005, 225/321 in 2006, 203/297 in 2007, and 275/358 in 2008. The overall TDR rate was 6.3%, peaking in 2006 at 10.4% and declining to 5.3% in 2008. The majority of TDR was seen in Irish born individuals with HIV-1 subtype B infection. The TDR rate in Ireland is comparatively low. Thus, a health technology assessment is required to ascertain the most cost effective use of genotypic antiretroviral resistance testing (GART) in the future: the current approach of performing baseline GART on all new diagnoses, or perhaps a more targeted approach that focuses on patients commencing nonnucleoside reverse transcriptase inhibitor (NNRTI)-based ART.

  17. Killer Cell Immunoglobulin-Like Receptor Alleles Alter HIV Disease in Children

    PubMed Central

    Singh, Kumud K.; Qin, Min; Brummel, Sean S.; Angelidou, Konstantia; Trout, Rodney N.; Fenton, Terence; Spector, Stephen A.

    2016-01-01

    Background HLA class I molecules are ligands for killer cell immunoglobin like receptors (KIR) that control the antiviral response of natural killer (NK) cells. However, the effects of KIR and HLA (KIR/HLA) alleles on HIV disease of children have not been studied. Methods 993 antiretroviral naïve children with symptomatic HIV infection from PACTG protocols P152 and P300 were genotyped for KIR and HLA alleles using the Luminex platform. Linear regression was used to test the association between genotypes and baseline pre-ART HIV RNA, CD4+ lymphocyte count, and cognitive score, adjusting for age, race/ethnicity and study. The interaction between genetic markers and age was investigated. To account for multiple testing the false discovery rate (FDR) was controlled at 0.05. Results Children with the KIR2DS4*ALL FULL LENGTH (KIR2DS4*AFL) allele had higher CD4+ lymphocyte counts. Among children ≤2 years of age, the KIR2DS4*AFL was associated with lower plasma HIV RNA and higher cognitive index scores. KIR Cent2DS3/5_1 had lower CD4+ lymphocyte counts in children ≤2 years of age, while the presence of Tel1, Tel2DS4_2, Tel2DS4_4, Tel8, Tel2DS4_6 had higher CD4+ lymphocyte counts in all children. Presence of Cent2, Cent4 and Cent8 was associated with increased HIV RNA load in children ≤2 years. Presence of KIR3DL1+Bw4 was associated with higher CD4+ lymphocyte counts in all children. Among children >2 years old, KIR3DS1+Bw4-80I was associated with higher plasma HIV RNA, and Bw6/Bw6 was associated with lower plasma HIV RNA compared to children with KIR3DS1+Bw4-80I. Conclusions Presented data show for the first time that specific KIR alleles independently or combined with HLA ligands are associated with HIV RNA and CD4+ lymphocyte counts in infected, antiretroviral naive children; and many of these effect estimates appear to be age dependent. These data support a role for specific KIR alleles in HIV pathogenesis in children. PMID:26983081

  18. The Cleavage and Polyadenylation Specificity Factor 6 (CPSF6) Subunit of the Capsid-recruited Pre-messenger RNA Cleavage Factor I (CFIm) Complex Mediates HIV-1 Integration into Genes.

    PubMed

    Rasheedi, Sheeba; Shun, Ming-Chieh; Serrao, Erik; Sowd, Gregory A; Qian, Juan; Hao, Caili; Dasgupta, Twishasri; Engelman, Alan N; Skowronski, Jacek

    2016-05-27

    HIV-1 favors integration into active genes and gene-enriched regions of host cell chromosomes, thus maximizing the probability of provirus expression immediately after integration. This requires cleavage and polyadenylation specificity factor 6 (CPSF6), a cellular protein involved in pre-mRNA 3' end processing that binds HIV-1 capsid and connects HIV-1 preintegration complexes to intranuclear trafficking pathways that link integration to transcriptionally active chromatin. CPSF6 together with CPSF5 and CPSF7 are known subunits of the cleavage factor I (CFIm) 3' end processing complex; however, CPSF6 could participate in additional protein complexes. The molecular mechanisms underpinning the role of CPSF6 in HIV-1 infection remain to be defined. Here, we show that a majority of cellular CPSF6 is incorporated into the CFIm complex. HIV-1 capsid recruits CFIm in a CPSF6-dependent manner, which suggests that the CFIm complex mediates the known effects of CPSF6 in HIV-1 infection. To dissect the roles of CPSF6 and other CFIm complex subunits in HIV-1 infection, we analyzed virologic and integration site targeting properties of a CPSF6 variant with mutations that prevent its incorporation into CFIm We show, somewhat surprisingly, that CPSF6 incorporation into CFIm is not required for its ability to direct preferential HIV-1 integration into genes. The CPSF5 and CPSF7 subunits appear to have only a minor, if any, role in this process even though they appear to facilitate CPSF6 binding to capsid. Thus, CPSF6 alone controls the key molecular interactions that specify HIV-1 preintegration complex trafficking to active chromatin. PMID:26994143

  19. [Molecular characterization of complex recombinant HIV-1 CRF06_cpx subtype detected in Turkey].

    PubMed

    Sayan, Murat; Kaptan, Figen; Ormen, Bahar; Türker, Nesrin

    2014-01-01

    A major proportion of the global HIV infections is caused by group M of HIV-1 genotype and to date approximately nine subtypes (A, B, C, D, F, G, H, J, K) and 50 circulating recombinant forms (CRFs) have been recognized. Recombinants between different HIV-1 group M subtypes are designated as CRF. The extension 'cpx', for complex, is given if the CRF consists of contributions from three or more different subtypes but the composition of the subtype is not given. The objective of this study was to present, for the first time an HIV-1 positive married couple infected with CRF06_cpx subtype in Izmir, Turkey. A 39-year-old male patient who admitted to hospital with the complaints of oral candidiasis and zona, was found to be anti-HIV positive. CD4+ T lymphocyte count was 21 cells/mm3 and plasma HIV-1 RNA level was 56.380 copies/ml. He reported unprotected heterosexual contact with multiple partners including African women during his stay in Saudi Arabia between 1996 and 2002. After his diagnosis, his 37-year-old wife was screened for HIV infection and she was also found anti-HIV positive, with CD4+ T cell count of 122 cells/mm3. However, her results of basal plasma HIV-1 RNA could not be obtained because of an internal control error. HIV-1 strains were analysed for subtyping, recombination and drug resistance mutations with pol gene region sequencing. HIV-1 sequences were subtyped as CRF06_cpx after phylogenetic analysis using neighbor-joining method. According to the recombination analysis, HIV-1 pol gene regions consisted of group M subtype G, A, D, and B in the male patient and G K, A, F, and D in the female patient. While L10I + L33F mutation associated with protease inhibitor (PI) resistance was detected in both of the patients, K219N mutation associated with nucleoside reverse transcriptase inhibitor (NRTI) resistance was detected only in the male patient. In conclusion, HIV-1 molecular epidemiology studies are important tools for tracking transmission patterns and

  20. Analysis of Immunological, Viral, Genetic, and Environmental Factors That Might Be Associated with Decreased Susceptibility to HIV Infection in Serodiscordant Couples in Florianópolis, Southern Brazil.

    PubMed

    Santos, Íris M; da Rosa, Elis A; Gräf, Tiago; Ferreira, Luiz G E; Petry, Andrea; Cavalheiro, Fernanda; Reiche, Edna M; Zanetti, Carlos R; Pinto, Aguinaldo R

    2015-11-01

    Individuals who have been exposed to human immunodeficiency virus (HIV) and have not been infected might possess natural resistance mechanisms. An understanding of the sociodemographic and immunological conditions that influence resistance to HIV is a challenge, and very little is known about the role of intrinsic antiviral factors that restrict HIV infection. The aim of this study was to analyze potential factors responsible for resistance to HIV infection in serodiscordant couples by comparing HIV-exposed seronegative individuals (HESN) to HIV-seropositive individuals treated with antiretroviral therapy (HIV-ART) along with healthy controls (HC). The results revealed one HLA-B*27 and two HLA-B*57 individuals among the HESN; a CCR5Δ32 heterozygous deletion was observed in one serodiscordant couple, while the homozygous genotype for this variant was not observed. There were no differences in the basal mRNA expression of APOBEC3G, CFLAR, TRIM5α, LEDGF/p75, BST-2, or SAMHD1 in CD4(+) T lymphocyte- and monocyte-enriched populations among the three groups, and lower HBD-3 concentrations were observed in saliva from HIV-ART compared to HESN and HC. The most prevalent HIV-1 subtype was C or C-containing recombinant forms. Six HIV-ART individuals and one HIV-ART individual were infected with the R5 HIV and X4 HIV strains, respectively. The ability to control infection or delay disease progression is probably defined by a balance between viral and host factors, and further evaluation should be performed in larger cohorts. Our data suggest that susceptibility to HIV infection varies among individuals and strengthens the multifactorial characteristics underlying the resistance mechanisms in HIV.

  1. Analysis of Immunological, Viral, Genetic, and Environmental Factors That Might Be Associated with Decreased Susceptibility to HIV Infection in Serodiscordant Couples in Florianópolis, Southern Brazil

    PubMed Central

    Santos, Íris M.; da Rosa, Elis A.; Gräf, Tiago; Ferreira, Luiz G. E.; Petry, Andrea; Cavalheiro, Fernanda; Reiche, Edna M.; Zanetti, Carlos R.

    2015-01-01

    Abstract Individuals who have been exposed to human immunodeficiency virus (HIV) and have not been infected might possess natural resistance mechanisms. An understanding of the sociodemographic and immunological conditions that influence resistance to HIV is a challenge, and very little is known about the role of intrinsic antiviral factors that restrict HIV infection. The aim of this study was to analyze potential factors responsible for resistance to HIV infection in serodiscordant couples by comparing HIV-exposed seronegative individuals (HESN) to HIV-seropositive individuals treated with antiretroviral therapy (HIV-ART) along with healthy controls (HC). The results revealed one HLA-B*27 and two HLA-B*57 individuals among the HESN; a CCR5Δ32 heterozygous deletion was observed in one serodiscordant couple, while the homozygous genotype for this variant was not observed. There were no differences in the basal mRNA expression of APOBEC3G, CFLAR, TRIM5α, LEDGF/p75, BST-2, or SAMHD1 in CD4+ T lymphocyte- and monocyte-enriched populations among the three groups, and lower HBD-3 concentrations were observed in saliva from HIV-ART compared to HESN and HC. The most prevalent HIV-1 subtype was C or C-containing recombinant forms. Six HIV-ART individuals and one HIV-ART individual were infected with the R5 HIV and X4 HIV strains, respectively. The ability to control infection or delay disease progression is probably defined by a balance between viral and host factors, and further evaluation should be performed in larger cohorts. Our data suggest that susceptibility to HIV infection varies among individuals and strengthens the multifactorial characteristics underlying the resistance mechanisms in HIV. PMID:26389741

  2. Importance of an Early HIV Antibody Differentiation Immunoassay for Detection of Dual Infection with HIV-1 and HIV-2

    PubMed Central

    Zbinden, Andrea; Dürig, Roland; Shah, Cyril; Böni, Jürg; Schüpbach, Jörg

    2016-01-01

    Background HIV-2 is primarily endemic in West Africa and India, however, in time of global migration, a possible HIV-2 infection or co-infection with HIV-1 should be recognized right at the time of HIV diagnosis, in order to enable optimized antiretroviral treatment. Laboratory HIV testing consists of a combined HIV1/2/O antibody + antigen screening test and subsequent confirmation and type differentiation by a serological test formatted as a multi-line or multi-spot assay. CDC has proposed a revised alternative HIV diagnostic strategy which, in case of a reactive result in a combined HIV1/2/O antibody + antigen screening test, comprises an HIV-1 nucleic acid test (NAT) for HIV confirmation instead of an antibody differentiation immunoassay (ADI). Only a negative NAT must be further investigated by an ADI, thus saving expenses for ADI in most instances. We have investigated this alternative strategy with respect to its recognition of dual HIV-1 and HIV-2 infection. Methods and Results Anonymized data of HIV notifications of patients newly diagnosed with HIV in Switzerland between 2007 and 2014 were analysed retrospectively. In a total of 4'679 notifications, we found 35 HIV-2 infections, 9 (25.7%) of which were dually infected with HIV-1. In 7 of the 9 dual HIV-1 and HIV-2 infections, HIV-1 RNA testing at the time of HIV diagnosis was positive with concentrations from 102 to 94'300 copies/mL plasma. HIV-1 RNA data were not available for the other two cases. Conclusions The alternative CDC strategy would have missed the concomitant HIV-2 infection in at least 7, but probably even more, of the 9 dually infected patients, as the detectable HIV-1 RNA would have precluded a supplemental ADI. Early ADI is mandatory for diagnosis of dual HIV-1/HIV-2 infection and guidance of appropriate therapy. PMID:27310138

  3. Nitrate Starvation Induced Changes in Root System Architecture, Carbon:Nitrogen Metabolism, and miRNA Expression in Nitrogen-Responsive Wheat Genotypes.

    PubMed

    Sinha, Subodh Kumar; Rani, Manju; Bansal, Niketa; Gayatri; Venkatesh, K; Mandal, P K

    2015-11-01

    Improvement of nutrient use efficiency in cereal crops is highly essential not only to reduce the cost of cultivation but also to save the environmental pollution, reduce energy consumption for production of these chemical fertilizers, improve soil health, and ultimately help in mitigating climate change. In the present investigation, we have studied the morphological (with special emphasis on root system architecture) and biochemical responses (in terms of assay of the key enzymes involved in N assimilation) of two N-responsive wheat genotypes, at the seedling stage, under nitrate-optimum and nitrate-starved conditions grown in hydroponics. Expression profile of a few known wheat micro RNAs (miRNAs) was also studied in the root tissue. Total root size, primary root length, and first- and second-order lateral root numbers responded significantly under nitrate-starved condition. Morphological parameters in terms of root and shoot length and fresh and dry weight of roots and shoots have also been observed to be significant between N-optimum and N-starved condition for each genotypes. Nitrate reductase (NR), glutamine synthatase (GS), and glutamate dehydrogenase (GDH) activity significantly decreased under N-starved condition. Glutamine oxogl