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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

    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

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

    PubMed

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

    2008-10-01

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

  3. Hepatitis C virus genotype and HIV coinfection affect cytokine mRNA levels in unstimulated PBMC but do not shift the T1/T2 balance.

    PubMed

    Lee, Silvia; Watson, Mark W; Clark, Ben; Flexman, James P; Cheng, Wendy; French, Martyn A H; Price, Patricia

    2006-08-01

    Rapid progression of hepatitis C virus (HCV) disease in patients with HIV/HCV may reflect different cytokine responses and be influenced by HCV genotype. This is addressed by a study of patients with HIV/HCV coinfection and infection with HCV genotype 2 or 3 (2/3). They are compared with coinfected patients infected with genotype 1 and HCV monoinfected patients matched for HCV genotype. IFN-gamma, IL-10, IL-4 and IL-4delta2 mRNA were quantified by real-time PCR in unstimulated PBMC and after in vitro stimulation with HCV core or nonstructural 3/4A antigen. In unstimulated PBMC, levels of IFN-gamma and IL-4 mRNA were lowest in HIV/HCV genotype 1 patients, intermediate in HIV/HCV genotype 2/3 patients and highest in HCV genotype 2/3 patients. Neither HCV genotype nor HIV affected levels of IL-10 mRNA in unstimulated PBMC or IFN-gamma, IL-4 and IL-10 mRNA in PBMC stimulated with HCV antigens. Levels of IL-4 and IL-4delta2 mRNA correlated in mitogen-stimulated PBMC from all patient groups but both were low in HIV/HCV genotype 1 patients. Serum soluble CD30 levels (a putative marker of a T2 cytokine environment) did not differ between patient groups. The data do not suggest a shift in the T1/T2 balance driven by HIV coinfection or HCV genotype but either may affect IL-4 bioavailability. PMID:16834574

  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. Human papillomavirus genotyping, human papillomavirus mRNA expression, and p16/Ki-67 cytology to detect anal cancer precursors in HIV-infected MSM

    PubMed Central

    Wentzensen, Nicolas; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren; Lorey, Thomas S.; Sahasrabuddhe, Vikrant V.; Lamere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Castle, Philip E.

    2014-01-01

    Objective Anal cancer incidence is high in HIV-infected MSM. Screening for anal intraepithelial lesions and cancers is performed at specialized clinics and relies on high-resolution anoscopy (HRA) and anal cytology. Both approaches have limited reproducibility and sensitivity for detecting anal cancer precursors. We evaluated biomarkers for human papillomavirus (HPV)-related disease in a population of HIV-infected MSM. Methods A cross-sectional screening study with passive follow-up included 363 MSM followed at a HIV/AIDS clinic. All men had anal cytology samples taken and were evaluated using HRA and anal biopsies. Using a composite endpoint of biopsy results and cytology, we compared the performance of HPV16/18 genotyping, HPVE6/E7 mRNA expression, and p16/Ki-67 cytology to detect high-grade anal intraepithelial neoplasias (AINs). Results For all biomarkers analyzed, there was a significant trend of increasing percentage of men testing positive with increasing severity of disease (P< 0.001). HPV DNA testing had the highest sensitivity for anal intraepithelial neoplasia grade 2 and anal intraepithelial neoplasia grade 3 (AIN3), followed by p16/Ki-67, HPVE6/E7 mRNA testing, and HPV16/18 genotyping. The highest Youden's index was observed for HPVE6/E7 mRNA testing, followed by HPV16/18 genotyping, p16/Ki-67 cytology, and HPV DNA testing. Increasing the threshold for positivity of p16/Ki-67 to five or more positive cells led to significantly higher specificity, but unchanged sensitivity for detecting AIN3. Conclusion Molecular features of anal disease categories are similar to those of corresponding cervical lesions. Biomarkers evaluated for cervical cancer screening may be used for primary anal cancer screening or to decide who should require immediate treatment vs. expectant management. PMID:23018436

  6. A comparison of human papillomavirus genotype-specific DNA and E6/E7 mRNA detection to identify anal precancer among HIV-infected men who have sex with men

    PubMed Central

    Castle, Philip E.; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren M.; Lorey, Thomas S.; LaMere, Brandon; Gage, Julia C.; Fetterman, Barbara; Darragh, Teresa M.; Rodriguez, Ana Cecilia; Wentzensen, Nicolas

    2012-01-01

    Background Human papillomavirus (HPV) RNA detection is reportedly more specific for the detection of anogenital precancer than HPV DNA but it is unknown whether this is due to detection of RNA or due to HPV genotype restriction. Materials and Methods 363 human immunodeficiency virus (HIV)-positive men who have sex with men had two anal cytology samples taken and were evaluated using high-resolution anoscopy and biopsies of visible lesions. Anal specimens were tested for E6/E7 RNA for 5 carcinogenic HPV genotypes (HPV16, 18, 31, 33, and 45) and tested for the DNA of 13 carcinogenic HPV genotypes. Results DNA testing was more likely to be positive than RNA testing (53% vs. 48%, p = 0.02) for the same 5 HPV genotypes in aggregate. When restricted to 5 HPV genotypes targeted by the RNA test, the sensitivity to detect anal precancer was the same for DNA and RNA (81%) while RNA was more specific than DNA (65% vs. 58%, p = 0.007). By comparison, DNA detection of all 13 carcinogenic HPV genotypes was more sensitive (96% vs. 81%, p = 0.001) but much less specific (65% vs. 33%, p < 0.001) compared to RNA detection of the 5 HPV genotypes. Conclusion After controlling for HPV genotypes, RNA was only slightly more specific than DNA detection for anal precancer. Impact DNA or RNA testing for a subset of the most carcinogenic HPV genotypes may be useful for distinguishing between those HPV-positive men at higher and lower risk of anal precancer and cancer. PMID:23155136

  7. Similar hepatitis C virus RNA kinetics in HIV/hepatitis C virus monoinfected genotype 2 or 3 matched controls during hepatitis C virus combination therapy.

    PubMed

    Karlström, Olle; Sönnerborg, Anders; Weiland, Ola

    2008-04-23

    We prospectively studied early hepatitis C virus kinetics and sustained virological response rates in HIV/HCV coinfected (n = 13) and HCV monoinfected matched controls (n = 26) with HCV genotype 2/3 treated with pegylated interferon (peg-IFN) alpha-2a 135 microg/week plus ribavirin 11 mg/kg daily during 24 weeks. No significant difference in HCV-RNA decay was seen at any time point during the initial 12 weeks of therapy. Sustained virological response was achieved in 9/13 (69%) versus 20/26 (77%) patients (intent-to-treat), respectively. The lower-than-standard peg-IFN dose offered high compliance and reasonable sustained virological response rates. PMID:18427210

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

    PubMed Central

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

    2016-01-01

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

  9. HIV drug resistance interpreted by cumulative versus last genotypes in HIV-infected patients with multiple treatment failures.

    PubMed

    Punyacam, Punthiya; Iemwimangsa, Nareenart; Chantratita, Wasun; Sukasem, Chonlaphat; Sungkanuparph, Somnuek

    2012-04-01

    Genotypic resistance test has been recommended to evaluate HIV drug resistance and guide the effective regimens of antiretroviral therapy (ART) in HIV-infected patients with treatment failure. In patients with multiple treatment failures, drug resistance-associated mutations may disappear due to the loss of selective drug pressure after switching regimens. A cohort study was conducted among HIV-infected patients who had ≥2 genotypic resistance tests during 2003-2011. HIV-1 pol nucleotide sequencing of reverse transcriptase and protease region was carried out using TRUGENE HIV-1 Genotypic Assay. Sequencing data was analyzed using Stanford rule-based interpretation algorithms. Of 54 patients with mean age of 30.1 years, 46.3% were males. HIV-1 subtype A/E was observed in 88.9% of patients. At the latest failure, 55.3% were receiving protease inhibitor-based regimens. Median CD4 and HIV RNA were 167 cells/mm(3) and 22,359 copies/mL. During a median duration of ART of 38.6 months, 72.2%, 22.2%, and 5.6% had 5, 3, and 2 genotype tests, respectively. When compared between using cumulative (CG) and last genotypes (LG), CG interpreted resistance to any drug 59.3% higher than LG did. For NRTI, NNRTI, and PI drug classes, CG interpreted as resistance 42.6%, 27.8%, and 7.4% higher than LG, respectively. The most common drugs that CG interpreted resistance with the higher rate than LG were lamivudine/emtricitabine, nevirapine, efavirenz, etravirine and abacavir. In conclusion, CG interprets HIV drug resistance at a higher rate than LG and may be more accurate to use for selecting the next effective regimen of ART among HIV-infected patients with multiple treatment failures. PMID:22497699

  10. 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.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  13. Reliable Genotypic Tropism Tests for the Major HIV-1 Subtypes

    PubMed Central

    Cashin, Kieran; Gray, Lachlan R.; Harvey, Katherine L.; Perez-Bercoff, Danielle; Lee, Guinevere Q.; Sterjovski, Jasminka; Roche, Michael; Demarest, James F.; Drummond, Fraser; Harrigan, P. Richard; Churchill, Melissa J.; Gorry, Paul R.

    2015-01-01

    Over the past decade antiretroviral drugs have dramatically improved the prognosis for HIV-1 infected individuals, yet achieving better access to vulnerable populations remains a challenge. The principal obstacle to the CCR5-antagonist, maraviroc, from being more widely used in anti-HIV-1 therapy regimens is that the pre-treatment genotypic “tropism tests” to determine virus susceptibility to maraviroc have been developed primarily for HIV-1 subtype B strains, which account for only 10% of infections worldwide. We therefore developed PhenoSeq, a suite of HIV-1 genotypic tropism assays that are highly sensitive and specific for establishing the tropism of HIV-1 subtypes A, B, C, D and circulating recombinant forms of subtypes AE and AG, which together account for 95% of HIV-1 infections worldwide. The PhenoSeq platform will inform the appropriate use of maraviroc and future CCR5 blocking drugs in regions of the world where non-B HIV-1 predominates, which are burdened the most by the HIV-1 pandemic. PMID:25712827

  14. 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

  15. Aptamer-siRNA chimeras for HIV.

    PubMed

    Takahashi, Mayumi; Burnett, John C; Rossi, John J

    2015-01-01

    Since 1980s, HIV/AIDS has escalated into a global pandemic. Although combinatorial antiretroviral therapy (cART) regimens can suppress plasma virus levels to below the detection limit and the survival rate of HIV-1 infected patients has been improving, long-term cART holds the potential to cause a number of chronic diseases. RNA interference (RNAi) is considered as a powerful method for developing new generation of therapeutics. Discovery of small interfering RNAs (siRNAs) shed light on limitations of targets that are "undruggable" with current technologies. However, delivery remains a major hurdle of siRNA-based therapy. Recent progress in technology of engineering nucleic acid enables a targeted delivery of siRNAs using aptamers, which, as often regarded as nucleic acid "antibodies," can recognize/bind to multiple different proteins and small-molecule targets by forming scaffolds for molecular interactions. SELEX technology enabled to isolate highly target specific aptamers from a random sequence oligonucleotide library. A number of aptamers for HIV-1 proteins as well as host proteins that interact with HIV-1 have been developed and some of them have potent viral neutralization ability and inhibition of HIV-1 infectivity. The availability of these aptamers has given an idea of using aptamers for targeting delivery of siRNAs. So far, aptamers against either HIV-1 gp120 or CD4 have been eagerly evaluated as the aptamer portion of the aptamer-siRNA chimeras for the treatment or prevention of HIV-1. In this chapter, we highlight the development and therapeutic potential of aptamer-siRNA chimeras for HIV-1. PMID:25757623

  16. Genital HIV-1 RNA Quantity Predicts Risk of Heterosexual HIV-1 Transmission

    PubMed Central

    Baeten, Jared M.; Kahle, Erin; Lingappa, Jairam R.; Coombs, Robert W.; Delany-Moretlwe, Sinead; Nakku-Joloba, Edith; Mugo, Nelly R.; Wald, Anna; Corey, Lawrence; Donnell, Deborah; Campbell, Mary S.; Mullins, James I.; Celum, Connie

    2011-01-01

    High plasma HIV-1 RNA concentrations are associated with an increased risk of HIV-1 transmission. Although plasma and genital HIV-1 RNA concentrations are correlated, no study has evaluated the relationship between genital HIV-1 RNA and the risk of heterosexual HIV-1 transmission. In a prospective study of 2521 African HIV-1 serodiscordant couples, we assessed genital HIV-1 RNA quantity and HIV-1 transmission risk. HIV-1 transmission linkage was established within the partnership by viral sequence analysis. We tested endocervical samples from 1805 women, including 46 who transmitted HIV-1 to their partner, and semen samples from 716 men, including 32 who transmitted HIV-1 to their partner. Genital and plasma HIV-1 concentrations were correlated: For endocervical swabs, Spearman’s rank correlation coefficient rho was 0.56 (p<0.001), and for semen rho was 0.55 (p<0.001). Each 1 log10 increase in genital HIV-1 RNA was associated with a 2.20-fold (for endocervical swabs, 95% confidence interval 1.60–3.04, p<0.001) and a 1.79-fold (for semen, 95% confidence interval 1.30–2.47, p<0.001) increased risk of HIV-1 transmission. Genital HIV-1 RNA independently predicted HIV-1 transmission risk after adjusting for plasma HIV-1 quantity (hazard ratio 1.67 for endocervical swabs and 1.68 for semen). Seven female-to-male and four male-to-female HIV-1 transmissions (incidence <1% per year) occurred from persons with undetectable genital HIV-1 RNA, but in all eleven plasma HIV-1 RNA was detected. Thus, higher genital HIV-1 RNA concentrations are associated with greater risk of heterosexual HIV-1 transmission, and this effect was independent of plasma HIV-1 concentrations. These data suggest that HIV-1 RNA in genital secretions could be used as a marker of HIV-1 sexual transmission risk. PMID:21471433

  17. 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

  18. MicroRNA-155 Reinforces HIV Latency.

    PubMed

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

    2015-05-29

    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

  19. 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

  20. 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

  1. The Brazilian Network for HIV-1 Genotyping External Quality Control Assurance Programme

    PubMed Central

    2011-01-01

    The Brazilian network for genotyping is composed of 21 laboratories that perform and analyze genotyping tests for all HIV-infected patients within the public system, performing approximately 25,000 tests per year. We assessed the interlaboratory and intralaboratory reproducibility of genotyping systems by creating and implementing a local external quality control evaluation. Plasma samples from HIV-1-infected individuals (with low and intermediate viral loads) or RNA viral constructs with specific mutations were used. This evaluation included analyses of sensitivity and specificity of the tests based on qualitative and quantitative criteria, which scored laboratory performance on a 100-point system. Five evaluations were performed from 2003 to 2008, with 64% of laboratories scoring over 80 points in 2003, 81% doing so in 2005, 56% in 2006, 91% in 2007, and 90% in 2008 (Kruskal-Wallis, p = 0.003). Increased performance was aided by retraining laboratories that had specific deficiencies. The results emphasize the importance of investing in laboratory training and interpretation of DNA sequencing results, especially in developing countries where public (or scarce) resources are used to manage the AIDS epidemic. PMID:21936945

  2. Prevalence of Primary HIV Drug Resistance in Thailand Detected by Short Reverse Transcriptase Genotypic Resistance Assay

    PubMed Central

    Kiertiburanakul, Sasisopin; Pinsai, Subencha; Chantratita, Wasun; Pasomsub, Ekawat; Leechawengwongs, Manoon; Thipmontree, Wilawan; Siriyakorn, Nirada; Sungkanuparph, Somnuek

    2016-01-01

    Background HIV drug resistance (HIVDR) is the major cause of treatment failure after scaling up of antiretroviral therapy (ART). HIVDR testing prior to ART initiation is not routinely performed in resource-limited settings. We aimed to assess the prevalence of primary HIVDR by short reverse transcriptase (RT) genotypic resistance assay and evaluate of the impact of the mutations on the treatment outcomes. Methods A prospective cohort study was conducted in treatment-naïve HIV-infected patients. Fourteen major mutations of codon 99–191 on the RT gene were selected (K103N, V106A/M, V108I, Q151M, Y181C/I, M184V/I, Y188C/L/H, and G190S/A) at a cost of testing of 35 USD. The association between the presence of primary HIVDR and undetectable HIV RNA (<50 copies/mL) after 6 months of ART was determined. Results A total of 265 HIV-infected patients were included, with a median age of 35.2 (range, 16.8–75.2) years; 62.6% were males. The median (interquartile range) CD4 cell count at ART initiation was 216 (77–381) cells/mm3. The overall prevalence of primary HIVDR was 7.9%. The prevalence of each HIVDR mutation were K103N 6.0%, V106I 1.1%, V108I 0.4%, Y181C 2.3%, Y181I 0.7%, Y181V 0.4%, M184V 3.0%, M184I 1.5%, and G190A 2.3%. No associated factor of having primary HIVDR was determined. By multiple stepwise logistic regression, factors associated with undetectable HIV RNA after 6 months of ART were: having M184V/I (odds ratio [OR] 0.11; 95% confidence interval [CI] 0.02–0.62, p = 0.013), condom use (OR 2.38; 95% CI 1.12–5.06, p = 0.024), and adherence per 5% increase (OR 1.16; 95% CI 1.00–1.35, p = 0.044). Conclusions The prevalence of primary HIVDR is approximately 8%; it is associated with detectable HIV RNA at 6 months after ART initiation. Routine “short RT” genotypic resistance assay should be considered in resource-limited settings to maximize treatment outcome. PMID:26828876

  3. 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 resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  4. 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 resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  5. 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 resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  6. 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 resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  7. 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 resistance genotype assay. 866.3950 Section 866.3950 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT... Serological Reagents § 866.3950 In vitro human immunodeficiency virus (HIV) drug resistance genotype assay....

  8. HIV Vpr protein upregulates microRNA-122 expression and stimulates hepatitis C virus replication

    PubMed Central

    Peng, Milin; Xiao, Xinqiang; He, Yan; Jiang, Yongfang; Zhang, Min; Peng, Feng; Tian, Yi; Xu, Yun

    2015-01-01

    Human immunodeficiency virus (HIV)/hepatitis C virus (HCV) co-infection is characterized by higher serum HCV RNA loads compared with HCV mono-infection. However, the relationship between HIV and HCV replication remains to be clarified. HIV Vpr has been shown to play an essential role in HIV replication. In this study, we aimed to explore the role of Vpr in HCV replication and pathogenesis. We therefore used the genotype 2a full-length HCV strain JFH1 infection system and the genotype 1b full-length HCV replicon OR6 cell line to analyse the effects of Vpr on HCV replication. We found that Vpr promoted HCV 5′ UTR activity, HCV RNA replication and HCV protein expression in two HCV infection cell models. Additionally, lymphocyte-produced Vpr significantly induced HCV 5′ UTR activity and HCV replication in hepatocytes. We also found that Vpr upregulated the expression of miR-122 by stimulating its promoter activity. Furthermore, an miR-122 inhibitor suppressed the Vpr-mediated enhancement of both HCV 5′ UTR activity and HCV replication. In summary, our results revealed that the Vpr-upregulated expression of miR-122 is closely related to the stimulation of HCV 5′ UTR activity and HCV replication by Vpr, providing new evidence for how HIV interacts with HCV during HIV/HCV co-infection. PMID:25920531

  9. Quantitation of HIV-1 RNA in dried blood and plasma spots.

    PubMed

    Reigadas, S; Schrive, M H; Aurillac-Lavignolle, V; Fleury, H J

    2009-10-01

    Dried blood spots (DBSs) and dried plasma spots (DPSs) are an attractive method for serological and molecular diagnosis of HIV infection. Recently, Youngpairoj et al. [Youngpairoj, A.S., Masciotra, S., Garrido, C., Zahonero, N., de Mendoza, C., Garcia-Lerma, J.G., 2008. HIV-1 drug resistance genotyping from dried blood spots stored for 1 year at 4 degrees C. J. Antimicrob. Chemother. 61, 1217-1220] showed that HIV-1 can be genotyped efficiently from DBS specimens stored at 4 degrees C for 1 year. The viral load obtained from DBS and DPS samples was compared with that obtained from plasma samples. A total of 86 samples was prepared from people infected with HIV subtype B or non-B and spotted on 903 filter papers stored with desiccant at 4 degrees C. RNA was extracted using the QIAamp Viral RNA mini kit (Qiagen, Courtaboeuf, France). RNA from DBS or DPS samples was quantified in accordance with the Agence Nationale de Recherche sur le SIDA (ANRS, AC11, Paris, France) assay for HIV-1 quantitation. When the mean viral load of plasma samples and DPS samples or plasma samples and DBS samples were compared, there were no significant differences. The overall data showed that although the sensitivity threshold of the assays was different, there was a correlation between the three specimen types and that DBS and DPS samples can be routinely used for viral load quantification particularly in resource-limited settings. PMID:19523984

  10. 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

  11. 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

  12. Different effects of the TAR structure on HIV-1 and HIV-2 genomic RNA translation

    PubMed Central

    Soto-Rifo, Ricardo; Limousin, Taran; Rubilar, Paulina S.; Ricci, Emiliano P.; Décimo, Didier; Moncorgé, Olivier; Trabaud, Mary-Anne; André, Patrice; Cimarelli, Andrea; Ohlmann, Théophile

    2012-01-01

    The 5′-untranslated region (5′-UTR) of the genomic RNA of human immunodeficiency viruses type-1 (HIV-1) and type-2 (HIV-2) is composed of highly structured RNA motifs essential for viral replication that are expected to interfere with Gag and Gag-Pol translation. Here, we have analyzed and compared the properties by which the viral 5′-UTR drives translation from the genomic RNA of both human immunodeficiency viruses. Our results showed that translation from the HIV-2 gRNA was very poor compared to that of HIV-1. This was rather due to the intrinsic structural motifs in their respective 5′-UTR without involvement of any viral protein. Further investigation pointed to a different role of TAR RNA, which was much inhibitory for HIV-2 translation. Altogether, these data highlight important structural and functional differences between these two human pathogens. PMID:22121214

  13. In situ hybridization for detection of HIV RNA.

    PubMed

    Fox, C H; Cottler-Fox, M

    2001-05-01

    In HIV studies, in situ hybridization can be used for identifying virion RNA, mRNA being produced for virion packaging, and proviral DNA in the cytoplasm or integrated in the nucleus. This unit focuses primarily on identifying virion RNA, because this is the most sensitive means by which in situ hybridization can be employed to detect HIV expression. In situ hybridization, as developed for HIV RNA detection, involves several protocols: (1) preparation of a radioactive or nonradioactive RNA probe; (2) in situ hybridization of probe to cells and paraffin sections of tissue; (3) detection of radiolabeled probe by emulsion autoradiography; (4) development, staining, and mounting of slides; and finally (5) examination of slides by bright-field, dark-field, specular reflectance, or laser-scanning confocal microscopy. The protocols presented in this unit describe a setup involving up to 150 slides. PMID:18432712

  14. Ribozyme-mediated inhibition of HIV 1 suggests nucleolar trafficking of HIV-1 RNA

    PubMed Central

    Michienzi, Alessandro; Cagnon, Laurence; Bahner, Ingrid; Rossi, John J.

    2000-01-01

    The HIV regulatory proteins Tat and Rev have a nucleolar localization property in human cells. However, no functional role has been attributed to this localization. Recently it has been demonstrated that expression of Rev induces nucleolar relocalization of some protein factors involved in Rev export. Because the function of Rev is to bind HIV RNA and facilitate transport of singly spliced and unspliced RNA to the cytoplasm, it is likely that the nucleolus plays a critical role in HIV-1 RNA export. As a test for trafficking of HIV-1 RNAs into the nucleolus, a hammerhead ribozyme that specifically cleaves HIV-1 RNA was inserted into the body of the U16 small nucleolar RNA, resulting in accumulation of the ribozyme within the nucleoli of human cells. HeLa CD4+ and T cells expressing this nucleolar localized ribozyme exhibit dramatically suppressed HIV-1 replication. The results presented here suggest a trafficking of HIV-1 RNA through the nucleoli of human cells, thus posing a different paradigm for lentiviral RNA processing. PMID:10922055

  15. 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

  16. 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

  17. 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

  18. NF45 and NF90 Bind HIV-1 RNA and Modulate HIV Gene Expression

    PubMed Central

    Li, Yan; Belshan, Michael

    2016-01-01

    A previous proteomic screen in our laboratory identified nuclear factor 45 (NF45) and nuclear factor 90 (NF90) as potential cellular factors involved in human immunodeficiency virus type 1 (HIV-1) replication. Both are RNA binding proteins that regulate gene expression; and NF90 has been shown to regulate the expression of cyclin T1 which is required for Tat-dependent trans-activation of viral gene expression. In this study the roles of NF45 and NF90 in HIV replication were investigated through overexpression studies. Ectopic expression of either factor potentiated HIV infection, gene expression, and virus production. Deletion of the RNA binding domains of NF45 and NF90 diminished the enhancement of HIV infection and gene expression. Both proteins were found to interact with the HIV RNA. RNA decay assays demonstrated that NF90, but not NF45, increased the half-life of the HIV RNA. Overall, these studies indicate that both NF45 and NF90 potentiate HIV infection through their RNA binding domains. PMID:26891316

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

    PubMed Central

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

    2014-01-01

    Background Both 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 ones risk for neurocognitive impairment. Both HIV and stimulant literature 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 was examined longitudinally over a 10 year period. Methods 952 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-HAART data was 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. Results No 4-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 3-way interactions were found that involved genotype and HIV status. All immunologic-related genes found to interact with HIV status affected neurocognitive functioning in the expected direction; however, only 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. Conclusion The findings support the role of immunologic-related genetic differences in CCL2 and CCL3 in neurocognitive functioning among HIV+ individuals; however their impact is minor. Consistent with findings from another cohort, DA-related genetic differences do not appear to impact the longitudinal neurocognitive functioning of HIV

  20. RNA interference directed to CDK2 inhibits HIV-1 transcription.

    PubMed

    Ammosova, Tatyana; Berro, Reem; Kashanchi, Fatah; Nekhai, Sergei

    2005-10-25

    We previously reported that cell cycle-dependent kinase 2 (CDK2) is required for human immunodeficiency virus-1 (HIV-1) Tat-dependent transcription in vitro. In the present study, CDK2-specific RNA interference in cultured HEK293T cells inhibited CDK2 expression and Tat-induced HIV-1 transcription from non-integrated HIV-1 promoter but not basal HIV-1 transcription or transcription from CMV or beta-actin promoters. Also, CDK2-specific RNA interference inhibited Tat-induced transcription from the integrated HIV-1 promoter in HeLa-CD4-LTR-beta-gal cells and potently blocked TNFalpha-induced HIV-1 viral replication in OM10.1 cells. CDK2-specific RNA interference did not have an effect on cell cycle progression, but it augmented TNFalpha-induced apoptosis of OM10.1 cells. Our results indicate that CDK2 participates in Tat-mediated HIV-1 transcription and may serve as a potential therapeutic target. PMID:16085226

  1. HIV RNA--should you get a quantitative HIV test today?

    PubMed

    Schouten, J T

    1995-01-01

    The theory that in early HIV infection the virus remains hidden within cells or lymph nodes waiting for a signal to replicate and then causes the disease to progress, has been disputed. Current testing methods have shown that HIV can be detected in the blood of almost all HIV-infected patients throughout the course of the disease. Blood tests that allow detection of HIV include the branched DNA test (bDNA) and the quantitative polymerase chain reaction (QPCR). Both of these tests measure HIV RNA; Food and Drug Administration (FDA) approval of both test methods is expected within a year. Upon approval, people may obtain an HIV RNA test through their physician, and insurance companies may pick up the cost of the test. Analysis of HIV RNA has been shown to be the best predictor of progression to AIDS, even better than the CD4 count. Long-term non-progressors with no immune system deterioration were found to have very low to non-detectable levels of plasma HIV. These tests may potentially be used for guidance in antiviral therapies and for monitoring the risk of maternal-fetal transmission of HIV. Information on obtaining either of these tests and costs for each are provided. PMID:11362716

  2. Defective HIV-1 proviruses produce novel protein-coding RNA species in HIV-infected patients on combination antiretroviral therapy.

    PubMed

    Imamichi, Hiromi; Dewar, Robin L; Adelsberger, Joseph W; Rehm, Catherine A; O'Doherty, Una; Paxinos, Ellen E; Fauci, Anthony S; Lane, H Clifford

    2016-08-01

    Despite years of plasma HIV-RNA levels <40 copies per milliliter during combination antiretroviral therapy (cART), the majority of HIV-infected patients exhibit persistent seropositivity to HIV-1 and evidence of immune activation. These patients also show persistence of proviruses of HIV-1 in circulating peripheral blood mononuclear cells. Many of these proviruses have been characterized as defective and thus thought to contribute little to HIV-1 pathogenesis. By combining 5'LTR-to-3'LTR single-genome amplification and direct amplicon sequencing, we have identified the presence of "defective" proviruses capable of transcribing novel unspliced HIV-RNA (usHIV-RNA) species in patients at all stages of HIV-1 infection. Although these novel usHIV-RNA transcripts had exon structures that were different from those of the known spliced HIV-RNA variants, they maintained translationally competent ORFs, involving elements of gag, pol, env, rev, and nef to encode a series of novel HIV-1 chimeric proteins. These novel usHIV-RNAs were detected in five of five patients, including four of four patients with prolonged viral suppression of HIV-RNA levels <40 copies per milliliter for more than 6 y. Our findings suggest that the persistent defective proviruses of HIV-1 are not "silent," but rather may contribute to HIV-1 pathogenesis by stimulating host-defense pathways that target foreign nucleic acids and proteins. PMID:27432972

  3. Evaluation of two commercial kits for the detection of genotypic drug resistance on a panel of HIV type 1 subtypes A through J.

    PubMed

    Fontaine, E; Riva, C; Peeters, M; Schmit, J C; Delaporte, E; Van Laethem, K; Van Vaerenbergh, K; Snoeck, J; Van Wijngaerden, E; De Clercq, E; Van Ranst, M; Vandamme, A M

    2001-11-01

    We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes. PMID:11694832

  4. 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

  5. HIV genotyping among female sex workers in the State of Santa Catarina.

    PubMed

    Schuelter-Trevisol, Fabiana; da Silva, Marcos Vinicius; Oliveira, Cristina M; Rodrigues, Rosângela

    2007-01-01

    The objective of this study was to investigate the frequency of HIV infection among female sex workers in the port area of Imbituba (State of Santa Catarina), and to identify the viral subtype and its susceptibility to antiretroviral medications. Ninety women were interviewed between December 2003 and February 2004. Six (6.7%) were HIV-positive. Genotyping for HIV, performed on four samples, detected subtype C in three of them, which is predominant in Africa and Asia, and subtype B in one of them, which is prevalent in Brazil, USA and Europe. The results suggest that the Port of Imbituba may be one of the gateways for HIV-1 subtype C to enter Brazil, and for its dissemination to the rest of the country and the Mercosul area, along the highway BR-101. This points towards the need for preventive work to reduce the introduction and dissemination of HIV subtype C in Brazil. PMID:17653456

  6. Genotypic Changes in Human Immunodeficiency Virus Type 1 Associated with Loss of Suppression of Plasma Viral RNA Levels in Subjects Treated with Ritonavir (Norvir) Monotherapy

    PubMed Central

    Eastman, P. Scott; Mittler, John; Kelso, Reed; Gee, Chris; Boyer, Eric; Kolberg, Janice; Urdea, Mickey; Leonard, John M.; Norbeck, Daniel W.; Mo, Hongmei; Markowitz, Martin

    1998-01-01

    Ten subjects received 600 to 1,200 mg of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor ritonavir per day. Following 2 weeks of therapy, plasma HIV RNA levels decreased by a mean of 1.57 (range, 0.89 to 1.96) log units. With continued therapy, HIV RNA levels began to rise in eight subjects. The initial rise in plasma RNA levels was temporally associated with the development and quantitative increase in the V82 resistance mutation. Doubling times of the V82A mutant virus were estimated to be 2.4 to 4.8 days. An L63P/A mutation was commonly present at baseline even in subjects with a durable virologic response. The concomitant acquisition of an L63P/A mutation with the V82A/F mutation at the time when plasma RNA levels rebounded suggests a role for the L63P/A mutation in improving the fitness of the V82A/F mutation. Subsequent additional genotypic changes at codons 54 and 84 were often associated with further increases in plasma RNA levels. Ongoing viral replication in the presence of drugs resulted in the appearance of additional genotypic changes, including the L90M saquinavir resistance mutation, and decreased phenotypic susceptibility. The relative fitness of the protease V82A ritonavir resistance mutation and reverse transcriptase T215Y/F zidovudine resistance mutation following drug withdrawal were estimated to be 96 to 98% that of the wild type. Durability of the virologic response was associated with plasma RNA levels at the nadir. A virologic response beyond 60 days was not observed unless plasma HIV RNA levels were suppressed below 2,000 copies/ml, consistent with estimates from V82A doubling times for selection of a single resistance mutation to dominate the replicating population. PMID:9573287

  7. Quantification of Total and 2-LTR (Long terminal repeat) HIV DNA, HIV RNA and Herpesvirus DNA in PBMCs

    PubMed Central

    Massanella, Marta; Gianella, Sara; Lada, Steven M.; Richman, Douglas D.; Strain, Matthew C.

    2016-01-01

    Almost all individuals infected with human immunodeficiency virus (HIV) are also infected with cytomegalovirus (CMV) and Epstein Barr virus (EBV). The aims of our studies have included characterizing and measuring the latent HIV reservoir and understanding the association between asymptomatic replication of CMV (and other herpesvirus, including EBV) and this HIV reservoir (Gianella et al., 2014). This protocol was designed to simultaneously co-extract DNA and RNA from the same peripheral blood mononuclear cell (PBMC) aliquot and quantify HIV, CMV and EBV DNA, as well as HIV RNA using droplet digital PCR (ddPCR). For collection and processing of male genital secretions and quantification of HIV RNA and DNA from seven human herpesviruses from seminal plasma, refer to protocol “Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma” (Vargas-Meneses et al., 2015).

  8. 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.

  9. 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

  10. Cost–effectiveness of CYP2B6 genotyping to optimize efavirenz dosing in HIV clinical practice

    PubMed Central

    Schackman, Bruce R; Haas, David W; Park, Sanghee S; Li, X Cynthia; Freedberg, Kenneth A

    2016-01-01

    Aims To assess the cost–effectiveness of CYP2B6 genotyping to guide efavirenz dosing for initial HIV therapy in the USA. Methods We used the Cost–Effectiveness of Preventing AIDS Complications (CEPAC) microsimulation model to project quality-adjusted life expectancy and lifetime costs (2014 US dollars) for efavirenz-based HIV therapy with or without CYP2B6 genotyping. We assumed that with genotyping 60% of patients would be eligible to receive lower doses. Results Current care without CYP2B6 genotyping has an incremental cost–effectiveness ratio >$100,000/QALY compared with genotype-guided dosing, even if lower dosing reduces efficacy. When we assumed generic efavirenz availability, conclusions were similar unless lower dosing reduces efficacy by 6% or more. Conclusion CYP2B6 genotyping can inform efavirenz dosing and decrease HIV therapy cost. PMID:26607811

  11. 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

  12. 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

  13. 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

  14. 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

  15. Evaluation of the Genotypic Prediction of HIV-1 Coreceptor Use versus a Phenotypic Assay and Correlation with the Virological Response to Maraviroc: the ANRS GenoTropism Study▿

    PubMed Central

    Recordon-Pinson, Patricia; Soulié, Cathia; Flandre, Philippe; Descamps, Diane; Lazrek, Mouna; Charpentier, Charlotte; Montes, Brigitte; Trabaud, Mary-Anne; Cottalorda, Jacqueline; Schneider, Véronique; Morand-Joubert, Laurence; Tamalet, Catherine; Desbois, Delphine; Macé, Muriel; Ferré, Virginie; Vabret, Astrid; Ruffault, Annick; Pallier, Coralie; Raymond, Stéphanie; Izopet, Jacques; Reynes, Jacques; Marcelin, Anne-Geneviève; Masquelier, Bernard

    2010-01-01

    Genotypic algorithms for prediction of HIV-1 coreceptor usage need to be evaluated in a clinical setting. We aimed at studying (i) the correlation of genotypic prediction of coreceptor use in comparison with a phenotypic assay and (ii) the relationship between genotypic prediction of coreceptor use at baseline and the virological response (VR) to a therapy including maraviroc (MVC). Antiretroviral-experienced patients were included in the MVC Expanded Access Program if they had an R5 screening result with Trofile (Monogram Biosciences). V3 loop sequences were determined at screening, and coreceptor use was predicted using 13 genotypic algorithms or combinations of algorithms. Genotypic predictions were compared to Trofile; dual or mixed (D/M) variants were considered as X4 variants. Both genotypic and phenotypic results were obtained for 189 patients at screening, with 54 isolates scored as X4 or D/M and 135 scored as R5 with Trofile. The highest sensitivity (59.3%) for detection of X4 was obtained with the Geno2pheno algorithm, with a false-positive rate set up at 10% (Geno2pheno10). In the 112 patients receiving MVC, a plasma viral RNA load of <50 copies/ml was obtained in 68% of cases at month 6. In multivariate analysis, the prediction of the X4 genotype at baseline with the Geno2pheno10 algorithm including baseline viral load and CD4 nadir was independently associated with a worse VR at months 1 and 3. The baseline weighted genotypic sensitivity score was associated with VR at month 6. There were strong arguments in favor of using genotypic coreceptor use assays for determining which patients would respond to CCR5 antagonist. PMID:20530226

  16. 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

  17. 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

  18. HIV-1 RNAs are Not Part of the Argonaute 2 Associated RNA Interference Pathway in Macrophages

    PubMed Central

    Kishore, Shivendra; Jaskiewicz, Lukasz; Hall, Jonathan; Günthard, Huldrych F.; Beerenwinkel, Niko; Metzner, Karin J.

    2015-01-01

    Background MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte derived macrophages (MDM). Methods The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs. Results and Conclusion PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages. PMID:26226348

  19. HIV-1 Modulates the tRNA Pool to Improve Translation Efficiency

    PubMed Central

    van Weringh, Anna; Ragonnet-Cronin, Manon; Pranckeviciene, Erinija; Pavon-Eternod, Mariana; Kleiman, Lawrence; Xia, Xuhua

    2011-01-01

    Despite its poorly adapted codon usage, HIV-1 replicates and is expressed extremely well in human host cells. HIV-1 has recently been shown to package non-lysyl transfer RNAs (tRNAs) in addition to the tRNALys needed for priming reverse transcription and integration of the HIV-1 genome. By comparing the codon usage of HIV-1 genes with that of its human host, we found that tRNAs decoding codons that are highly used by HIV-1 but avoided by its host are overrepresented in HIV-1 virions. In particular, tRNAs decoding A-ending codons, required for the expression of HIV's A-rich genome, are highly enriched. Because the affinity of Gag-Pol for all tRNAs is nonspecific, HIV packaging is most likely passive and reflects the tRNA pool at the time of viral particle formation. Codon usage of HIV-1 early genes is similar to that of highly expressed host genes, but codon usage of HIV-1 late genes was better adapted to the selectively enriched tRNA pool, suggesting that alterations in the tRNA pool are induced late in viral infection. If HIV-1 genes are adapting to an altered tRNA pool, codon adaptation of HIV-1 may be better than previously thought. PMID:21216840

  20. Complete Genome Sequence of the WHO International Standard for HIV-2 RNA Determined by Deep Sequencing

    PubMed Central

    Ham, Claire; Morris, Clare

    2016-01-01

    The World Health Organization (WHO) International Standard for HIV-2 RNA nucleic acid assays was characterized by complete genome deep sequencing. The entire coding sequence and flanking long terminal repeats (LTRs), including minority species, were assigned subtype A. This information will aid design, development, and evaluation of HIV-2 RNA amplification assays. PMID:26847885

  1. 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. PMID:27448026

  2. 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.

  3. 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

  4. 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

  5. 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

  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. 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

  8. HIV-2 genomic RNA accumulates in stress granules in the absence of active translation

    PubMed Central

    Soto-Rifo, Ricardo; Valiente-Echeverria, Fernando; Rubilar, Paulina S.; Garcia-de-Gracia, Francisco; Ricci, Emiliano P.; Limousin, Taran; Décimo, Didier; Mouland, Andrew J.; Ohlmann, Théophile

    2014-01-01

    During the post-transcriptional events of the HIV-2 replication cycle, the full-length unspliced genomic RNA (gRNA) is first used as an mRNA to synthesize Gag and Gag-Pol proteins and then packaged into progeny virions. However, the mechanisms responsible for the coordinate usage of the gRNA during these two mutually exclusive events are poorly understood. Here, we present evidence showing that HIV-2 expression induces stress granule assembly in cultured cells. This contrasts with HIV-1, which interferes with stress granules assembly even upon induced cellular stress. Moreover, we observed that the RNA-binding protein and stress granules assembly factor TIAR associates with the gRNA to form a TIAR-HIV-2 ribonucleoprotein (TH2RNP) complex localizing diffuse in the cytoplasm or aggregated in stress granules. Although the assembly of TH2RNP in stress granules did not require the binding of the Gag protein to the gRNA, we observed that increased levels of Gag promoted both translational arrest and stress granule assembly. Moreover, HIV-2 Gag also localizes to stress granules in the absence of a ‘packageable’ gRNA. Our results indicate that the HIV-2 gRNA is compartmentalized in stress granules in the absence of active translation prior to being selected for packaging by the Gag polyprotein. PMID:25352557

  9. 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. PMID:26712001

  10. 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

  11. Differential Effects of Tra2ß Isoforms on HIV-1 RNA Processing and Expression

    PubMed Central

    Platt, Craig; Calimano, Maria; Nemet, Josip; Bubenik, Jodi; Cochrane, Alan

    2015-01-01

    Balanced processing of HIV-1 RNA is critical to virus replication and is regulated by host factors. In this report, we demonstrate that overexpression of either Tra2α or Tra2β results in a marked reduction in HIV-1 Gag/ Env expression, an effect associated with changes in HIV-1 RNA accumulation, altered viral splice site usage, and a block to export of HIV-1 genomic RNA. A natural isoform of Tra2β (Tra2ß3), lacking the N-terminal RS domain, also suppressed HIV-1 expression but had different effects on viral RNA processing. The functional differences between the Tra2β isoforms were also observed in the context of another RNA substrate indicating that these factors have distinct functions within the cell. Finally, we demonstrate that Tra2ß depletion results in a selective reduction in HIV-1 Env expression as well as an increase in multiply spliced viral RNA. Together, the findings indicate that Tra2α/β can play important roles in regulating HIV-1 RNA metabolism and expression. PMID:25970345

  12. 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

  13. 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

  14. Combination of microRNA expression profiling with genome-wide SNP genotyping to construct a coronary artery disease-related miRNA-miRNA synergistic network.

    PubMed

    Hua, Lin; Xia, Hong; Zhou, Ping; Li, Dongguo; Li, Lin

    2014-12-01

    In recent years, microRNAs (miRNAs) were found to play critical roles in many important biological processes. On the other hand, the rapid development of genome-wide association studies (GWAS) help identify potential genetic variants associated with the disease phenotypic variance. Therefore, we suggested a combined analysis of microRNA expression profiling with genome-wide Single Nucleotide Polymorphism (SNP) genotyping to identify potential disease-related biomarkers. Considering functional SNPs in miRNA genes or target sites might be important signals associated with human complex diseases, we constructed a miRNA-miRNA synergistic network related to coronary artery disease (CAD) by performing a genome-wide scan for SNPs in human miRNA 3' -untranslated regions (UTRs) target sites and computed potential SNP cooperation effects contributing to disease based on potential miRNA-SNP interactions reported recently. Furthermore, we identified some potential CAD-related miRNAs by analyzing the constructed miRNAmiRNA synergistic network. As a result, the predicted miRNA-miRNA network and miRNA clusters were validated by significantly high interaction effects of CAD-related miRNAs. Accurate classification performances were obtained for all of the identified miRNA clusters, and the sensitivity and specificity were all more than 90%. The network topological analysis confirmed some novel CAD-related miRNAs identified recently by experiments. Our method might help to understand miRNA function and CAD disease, as well as to explore the novel mechanisms involved. PMID:25641175

  15. Anal Human Papillomavirus Genotyping among HIV-Positive Men Who Have Sex with Men in Xi’an, China

    PubMed Central

    Li, Xiangwei; Yang, Yu; Xin, Henan; Li, Mufei; Feng, Boxuan; Gao, Lei

    2015-01-01

    Background Anal human papillomavirus (HPV) infection and its related diseases are relatively common in men who have sex with men (MSM), especially in those HIV positive. In China, molecular epidemiology of anal HPV infection among HIV-positive MSM has been sparsely studied. Methods A cross-sectional study was conducted among HIV-positive MSM in Xi’an, China between April and July 2014. Anal swabs were collected for HPV genotyping. Results A total of 195 HIV-positive MSM were included in this study. HPV genotyping showed that 99.0% (191/193) of participants were positive for at least one of the targeted 37 HPV genotypes. 183 (94.8%) of them were infected with multiple high-risk types and 154 (79.8%) of them with low-risk HPV types. HPV 18 was the most frequently identified high-risk type, followed by HPV 16 and HPV 51. As for low-risk types, HPV11, HPV 6 and HPV 81 were most commonly observe. High-risk HPV infection was found to be associated with the status of antiretroviral therapy (ART), the distribution of low-risk types was observed to be varied by CD4+ T cell level. Conclusion Almost all HIV-positive MSM were anal HPV infected in our study. It is highly recommended to consider regular active screening and preventive intervention of HPV infection among this high risk population. PMID:25923768

  16. 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

  17. Cytoplasmic HIV-1 RNA is mainly transported by diffusion in the presence or absence of Gag protein.

    PubMed

    Chen, Jianbo; Grunwald, David; Sardo, Luca; Galli, Andrea; Plisov, Sergey; Nikolaitchik, Olga A; Chen, De; Lockett, Stephen; Larson, Daniel R; Pathak, Vinay K; Hu, Wei-Shau

    2014-12-01

    Full-length HIV-1 RNA plays a central role in viral replication by serving as the mRNA for essential viral proteins and as the genome packaged into infectious virions. Proper RNA trafficking is required for the functions of RNA and its encoded proteins; however, the mechanism by which HIV-1 RNA is transported within the cytoplasm remains undefined. Full-length HIV-1 RNA transport is further complicated when group-specific antigen (Gag) protein is expressed, because a significant portion of HIV-1 RNA may be transported as Gag-RNA complexes, whose properties could differ greatly from Gag-free RNA. In this report, we visualized HIV-1 RNA and monitored its movement in the cytoplasm by using single-molecule tracking. We observed that most of the HIV-1 RNA molecules move in a nondirectional, random-walk manner, which does not require an intact cytoskeletal structure, and that the mean-squared distance traveled by the RNA increases linearly with time, indicative of diffusive movement. We also observed that a single HIV-1 RNA molecule can move at various speeds when traveling through the cytoplasm, indicating that its movement is strongly affected by the immediate environment. To examine the effect of Gag protein on HIV-1 RNA transport, we analyzed the cytoplasmic HIV-1 RNA movement in the presence of sufficient Gag for virion assembly and found that HIV-1 RNA is still transported by diffusion with mobility similar to the mobility of RNAs unable to express functional Gag. These studies define a major mechanism of HIV-1 gene expression and resolve the long-standing question of how the RNA genome is transported to the assembly site. PMID:25404326

  18. 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

  19. Pneumocystis jirovecii genotype associated with increased death rate of HIV-infected patients with pneumonia.

    PubMed

    Rabodonirina, Meja; Vaillant, Laetitia; Taffé, Patrick; Nahimana, Aimable; Gillibert, René-Pierre; Vanhems, Philippe; Hauser, Philippe M

    2013-01-01

    Pneumocystis jirovecii dihydropteroate synthase (DHPS) mutations have been associated with failure of sulfa prophylaxis; their effect on the outcome of patients with P. jirovecii pneumonia (PCP) remains controversial. P. jirovecii DHPS polymorphisms and genotypes were identified in 112 cases of PCP in 110 HIV-infected patients by using PCR single-strand conformation polymorphism. Of the 110 patients observed, 21 died; 18 of those deaths were attributed to PCP. Thirty-three percent of the PCP cases involved a P. jirovecii strain that had 1 or both DHPS mutations. The presence or absence of DHPS mutations had no effect on the PCP mortality rate within 1 month, whereas P.jirovecii type 7 and mechanical ventilation at PCP diagnosis were associated with an increased risk of death caused by PCP. Mechanical ventilation at PCP diagnosis was also associated with an increased risk of sulfa treatment failure at 5 days. PMID:23260763

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

    PubMed Central

    Wynn, Jessica E.

    2015-01-01

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

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

    PubMed

    Wynn, Jessica E; Santos, Webster L

    2015-06-01

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

  2. 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

  3. 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

  4. 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

  5. Nuclear Factor 90(NF90) targeted to TAR RNA inhibits transcriptional activation of HIV-1

    PubMed Central

    Agbottah, Emmanuel T; Traviss, Christine; McArdle, James; Karki, Sambhav; St Laurent, Georges C; Kumar, Ajit

    2007-01-01

    Background Examination of host cell-based inhibitors of HIV-1 transcription may be important for attenuating viral replication. We describe properties of a cellular double-stranded RNA binding protein with intrinsic affinity for HIV-1 TAR RNA that interferes with Tat/TAR interaction and inhibits viral gene expression. Results Utilizing TAR affinity fractionation, North-Western blotting, and mobility-shift assays, we show that the C-terminal variant of nuclear factor 90 (NF90ctv) with strong affinity for the TAR RNA, competes with Tat/TAR interaction in vitro. Analysis of the effect of NF90ctv-TAR RNA interaction in vivo showed significant inhibition of Tat-transactivation of HIV-1 LTR in cells expressing NF90ctv, as well as changes in histone H3 lysine-4 and lysine-9 methylation of HIV chromatin that are consistent with the epigenetic changes in transcriptionally repressed gene. Conclusion Structural integrity of the TAR element is crucial in HIV-1 gene expression. Our results show that perturbation Tat/TAR RNA interaction by the dsRNA binding protein is sufficient to inhibit transcriptional activation of HIV-1. PMID:17565699

  6. 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

  7. Cocaine Enhances HIV-1 Infectivity in Monocyte Derived Dendritic Cells by Suppressing microRNA-155

    PubMed Central

    Napuri, Jessica; Pilakka-Kanthikeel, Sudheesh; Raymond, Andrea; Agudelo, Marisela; Yndart-Arias, Adriana; Saxena, Shailendra K.; Nair, Madhavan

    2013-01-01

    Cocaine and other drugs of abuse increase HIV-induced immunopathogenesis; and neurobiological mechanisms of cocaine addiction implicate a key role for microRNAs (miRNAs), single-stranded non-coding RNAs that regulate gene expression and defend against viruses. In fact, HIV defends against miRNAs by actively suppressing the expression of polycistronic miRNA cluster miRNA-17/92, which encodes miRNAs including miR-20a. IFN-g production by natural killer cells is regulated by miR-155 and this miRNA is also critical to dendritic cell (DC) maturation. However, the impact of cocaine on miR-155 expression and subsequent HIV replication is unknown. We examined the impact of cocaine on two miRNAs, miR-20a and miR-155, which are integral to HIV replication, and immune activation. Using miRNA isolation and analysis, RNA interference, quantitative real time PCR, and reporter assays we explored the effects of cocaine on miR-155 and miR-20 in the context of HIV infection. Here we demonstrate using monocyte-derived dendritic cells (MDCCs) that cocaine significantly inhibited miR-155 and miR-20a expression in a dose dependent manner. Cocaine and HIV synergized to lower miR-155 and miR-20a in MDDCs by 90%. Cocaine treatment elevated LTR-mediated transcription and PU.1 levels in MDCCs. But in context of HIV infection, PU.1 was reduced in MDDCs regardless of cocaine presence. Cocaine increased DC-SIGN and and decreased CD83 expression in MDDC, respectively. Overall, we show that cocaine inhibited miR-155 and prevented maturation of MDDCs; potentially, resulting in increased susceptibility to HIV-1. Our findings could lead to the development of novel miRNA-based therapeutic strategies targeting HIV infected cocaine abusers. PMID:24391808

  8. A discrete element 3' of human immunodeficiency virus 1 (HIV-1) and HIV-2 mRNA initiation sites mediates transcriptional activation by an HIV trans activator

    SciTech Connect

    Jakobovits, A.; Smith, D.H.; Jakobovits, E.B.; Capon, D.J.

    1988-06-01

    An important point of regulation in the reproductive growth and latency of the human and simian immunodeficiency viruses (HIV and SIV, respectively) is provided by virally encoded trans-activators (tat), proteins capable of dramatically increasing viral gene expression. The mechanism of this autostimulatory pathway has remained unclear, however, with substantial effects having been reported at the level of either mRNA accumulation, translational efficiency, or both. The authors' previous findings indicated that trans-activation results primarily from induction of RNA levels but could not distinguish between the roles of transcriptional rate, RNA stabilization, and RNA transport in this event. In addition, the boundaries of tat-responding elements, which would be valuable in elucidating the mode of tat action, are not precisely known. In this study, HIV-1 and HIV-2 long terminal repeat-directed expression was characterized by using in an vitro nuclear transcription assay to clarify this mechanism, and a detailed mutational analysis was undertaken to localize precisely the sequences participating in this process. Two key findings were revealed: an increased transcription rate was the primary event in tat-mediated activation of HIV-1 and HIV-2, and trans-activation was impaired by mutations in two regions, the TATA box and sequences between +19 to +42, a region lacking enhancer activity. These results implicate a discrete 3' regulatory element in the transcriptional activation of the HIVs.

  9. 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

  10. 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

  11. 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

  12. 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

  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. 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

  15. 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

  16. Prognostic Value of HIV-1 RNA on CD4 Trajectories and Disease Progression Among Antiretroviral-Naive HIV-Infected Adults in Botswana: A Joint Modeling Analysis.

    PubMed

    Farahani, Mansour; Novitsky, Vladimir; Wang, Rui; Bussmann, Hermann; Moyo, Sikhulile; Musonda, Rosemary M; Moeti, Themba; Makhema, Joseph M; Essex, Max; Marlink, Richard

    2016-06-01

    Although HIV-1 RNA levels are measured at the time of initial diagnosis, the results are not used for the clinical follow-up of the patients. This study evaluates the prognostic value of the baseline HIV-1 RNA levels (above or below 10,000 copies/ml) on rate of disease progression, among antiretroviral therapy (ART)-naive patients in Botswana. A prospective cohort of 436 HIV-infected ART-naive adults with baseline CD4 > 400 cells/mm(3) were followed quarterly for 5 years in an urban clinic in Botswana. Baseline HIV-1 RNA levels and longitudinal CD4(+) T-cell count data were analyzed, using mixed-effects regression jointly modeled with the times to a composite endpoint defined by AIDS-defining clinical conditions or death. During 1,547 person-years (PYs) follow-up time, 106 individuals became eligible for ART initiation (incidence rate: 0.07 PYs) and 6 participants died of AIDS-related illness. There were 203 (47%) individuals with baseline HIV-1 RNA <10,000 copies/ml and 233 (53%) individuals with baseline RNA >10,000 copies/ml. The slope of the predicted CD4 trajectory for individuals with baseline HIV-1 RNA >10,000 copies/ml is 30% steeper than that for those with baseline RNA <10,000. The hazard of reaching the composite endpoint for the individuals with baseline HIV-1 RNA >10,000 copies/ml was 2.3 (95% confidence interval: 1.5-3.0) times higher than that for those with baseline HIV-1 RNA <10,000 copies/ml. CD4 decline in individuals with HIV-1 RNA >10,000 copies/ml is much faster than that in those with RNA <10,000. The elevated HIV-1 RNA can be used as a marker to identify individuals at risk of faster disease progression. PMID:26830351

  17. 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 Central

    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. PMID:26263394

  18. Early Antiretroviral Therapy in South African Children Reduces HIV-1-Infected Cells and Cell-Associated HIV-1 RNA in Blood Mononuclear Cells.

    PubMed

    van Zyl, Gert U; Bedison, Margaret A; van Rensburg, Anita Janse; Laughton, Barbara; Cotton, Mark F; Mellors, John W

    2015-07-01

    We measured cell-associated human immunodeficiency virus (HIV)-1 DNA (CAD) and RNA (CAR) and plasma HIV-1 RNA in blood samples from 20 children in the Children with HIV Early Antiretroviral (CHER) cohort after 7-8 years of suppressive combination antiretroviral therapy (cART). Children who initiated cART early (<2 months; n = 12) had lower HIV-1 CAD (median, 48 vs 216; P < .01) and CAR (median, 5 vs 436; P < .01) per million peripheral blood mononuclear cells than children who started later (≥ 2 months; n = 8). Plasma HIV-1 RNA levels were not significantly lower in early-treated children (0.5 vs 1.2 copies/mL; P = .16). Early treatment at <2 months of age reduces the number of HIV-infected cells and HIV CAR. PMID:25538273

  19. 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

  20. 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.

  1. Impact of Human Immunodeficiency Virus Type 1 (HIV-1) Genetic Diversity on Performance of Four Commercial Viral Load Assays: LCx HIV RNA Quantitative, AMPLICOR HIV-1 MONITOR v1.5, VERSANT HIV-1 RNA 3.0, and NucliSens HIV-1 QT

    PubMed Central

    Swanson, Priscilla; de Mendoza, Carmen; Joshi, Yagnya; Golden, Alan; Hodinka, Richard L.; Soriano, Vincent; Devare, Sushil G.; Hackett, John

    2005-01-01

    Human immunodeficiency virus type 1 (HIV-1) evolution and changing strain distribution present a challenge to nucleic acid-based assays. Reliable patient monitoring of viral loads requires the detection and accurate quantification of genetically diverse HIV-1. A panel of 97 HIV-1-seropositive plasma samples collected from Cameroon, Brazil, and South Africa was used to compare the performance of four commercially available HIV RNA quantitative tests: Abbott LCx HIV RNA Quantitative assay (LCx), Bayer Versant HIV-1 RNA 3.0 (bDNA), Roche AMPLICOR HIV-1 MONITOR v1.5 (Monitor v1.5), and bioMérieux NucliSens HIV-1 QT (NucliSens). The panel included group M, group O, and recombinant viruses based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified viral RNA in 97 (100%) of the samples. In comparison, bDNA, Monitor v1.5, and NucliSens quantified viral RNA in 96.9%, 94.8%, and 88.6% of the samples, respectively. The two group O specimens were quantified only by the LCx HIV assay. Analysis of nucleotide mismatches at the primer/probe binding sites for Monitor v1.5, NucliSens, and LCx assays revealed that performance characteristics reflected differences in the level of genetic conservation within the target regions. PMID:16081923

  2. An Evolved RNA Recognition Motif That Suppresses HIV-1 Tat/TAR-Dependent Transcription.

    PubMed

    Crawford, David W; Blakeley, Brett D; Chen, Po-Han; Sherpa, Chringma; Le Grice, Stuart F J; Laird-Offringa, Ite A; McNaughton, Brian R

    2016-08-19

    Potent and selective recognition and modulation of disease-relevant RNAs remain a daunting challenge. We previously examined the utility of the U1A N-terminal RNA recognition motif as a scaffold for tailoring new RNA hairpin recognition and showed that as few as one or two mutations can result in moderate affinity (low μM dissociation constant) for the human immunodeficiency virus (HIV) trans-activation response element (TAR) RNA, an RNA hairpin controlling transcription of the human immunodeficiency virus (HIV) genome. Here, we use yeast display and saturation mutagenesis of established RNA-binding regions in U1A to identify new synthetic proteins that potently and selectively bind TAR RNA. Our best candidate has truly altered, not simply broadened, RNA-binding selectivity; it binds TAR with subnanomolar affinity (apparent dissociation constant of ∼0.5 nM) but does not appreciably bind the original U1A RNA target (U1hpII). It specifically recognizes the TAR RNA hairpin in the context of the HIV-1 5'-untranslated region, inhibits the interaction between TAR RNA and an HIV trans-activator of transcription (Tat)-derived peptide, and suppresses Tat/TAR-dependent transcription. Proteins described in this work are among the tightest TAR RNA-binding reagents-small molecule, nucleic acid, or protein-reported to date and thus have potential utility as therapeutics and basic research tools. Moreover, our findings demonstrate how a naturally occurring RNA recognition motif can be dramatically resurfaced through mutation, leading to potent and selective recognition-and modulation-of disease-relevant RNA. PMID:27253715

  3. Architecture and Secondary Structure of an Entire HIV-1 RNA Genome

    PubMed Central

    Watts, Joseph M.; Dang, Kristen K.; Gorelick, Robert J.; Leonard, Christopher W.; Bess, Julian W.; Swanstrom, Ronald; Burch, Christina L.; Weeks, Kevin M.

    2009-01-01

    Single-stranded RNA viruses encompass broad classes of infectious agents and cause the common cold, cancer, AIDS, and other serious health threats. Viral replication is regulated at many levels, including using conserved genomic RNA structures. Most potential regulatory elements within viral RNA genomes are uncharacterized. Here we report the structure of an entire HIV-1 genome at single nucleotide resolution using SHAPE, a high-throughput RNA analysis technology. The genome encodes protein structure at two levels. In addition to the correspondence between RNA and protein primary sequences, a correlation exists between high levels of RNA structure and sequences that encode inter-domain loops in HIV proteins. This correlation suggests RNA structure modulates ribosome elongation to promote native protein folding. Some simple genome elements previously shown to be important, including the ribosomal gag-pol frameshift stem-loop, are components of larger RNA motifs. We also identify organizational principles for unstructured RNA regions. Highly used splice acceptors lie in unstructured motifs and hypervariable regions are sequestered from flanking genome regions by stable insulator helices. These results emphasize that the HIV-1 genome and, potentially, many coding RNAs are punctuated by numerous previously unrecognized regulatory motifs and that extensive RNA structure may constitute an additional level of the genetic code. PMID:19661910

  4. 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

  5. Exosomes derived from HIV-1-infected cells contain trans-activation response element RNA.

    PubMed

    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

    2013-07-01

    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 10(4)-10(6) copies/ml TAR RNA in exosomes derived from infected culture supernatants and 10(3) 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. Inhibition of HIV derived lentiviral production by TAR RNA binding domain of TAT protein

    PubMed Central

    Mi, Michael Y; Zhang, Jiying; He, Yukai

    2005-01-01

    Background A critical step in the production of new HIV virions involves the TAT protein binding to the TAR element. The TAT protein contains in close proximity its TAR RNA binding domain and protein transduction domain (PTD). The PTD domain of TAT has been identified as being instrumental in the protein's ability to cross mammalian cell and nuclear membranes. All together, this information led us to form the hypothesis that a protein containing the TAR RNA binding domain could compete with the native full length TAT protein and effectively block the TAR RNA binding site in transduced HIV infected cells. Results We synthesized a short peptide named Tat-P, which contained the TAR RNA binding and PTD domains to examine whether the peptide has the potential of inhibiting TAT dependent HIV replication. We investigated the inhibiting effects of Tat-P in vitro using a HIV derived lentiviral vector model. We found that the TAT PTD domain not only efficiently transduced test cells, but also effectively inhibited the production of lentiviral particles in a TAT dependent manner. These results were also supported by data derived from the TAT activated LTR-luciferase expression model and RNA binding assays. Conclusion Tat-P may become part of a category of anti-HIV drugs that competes with full length TAT proteins to inhibit HIV replication. In addition, this study indicates that the HIV derived lentiviral vector system is a safe and reliable screening method for anti-HIV drugs, especially for those targeting the interaction of TAT and TAR RNAs. PMID:16293193

  7. A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

    PubMed Central

    MCLAREN, MEREDITH; ASAI, KENGO; COCHRANE, ALAN

    2004-01-01

    Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3′ splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3′ end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3′ end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3′ end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression. PMID:15208447

  8. 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

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

    PubMed Central

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

    2009-01-01

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

  10. 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

  11. HIV-1 reverse transcriptase-associated RNase H cleaves RNA/RNA in arrested complexes: implications for the mechanism by which RNase H discriminates between RNA/RNA and RNA/DNA.

    PubMed Central

    Götte, M; Fackler, S; Hermann, T; Perola, E; Cellai, L; Gross, H J; Le Grice, S F; Heumann, H

    1995-01-01

    Reverse transcription of human immunodeficiency virus type 1 (HIV-1) is primed by tRNA(Lys3), which forms an 18 base pair RNA homoduplex with its 3' terminus and the primer binding site (PBS) of the viral genome. Using an in vitro system mimicking initiation of minus strand DNA synthesis, we analyzed the mechanism by which HIV-1 reverse transcriptase (RT)-associated ribonuclease H (RNase H) distinguishes between RNA/DNA and RNA/RNA (dsRNA). tRNA(Lys3) was hybridized to a PBS-containing RNA template and extended by addition of deoxynucleoside triphosphates (dNTPs). In the presence of all four dNTPs, initial cleavage of the RNA template occurred immediately downstream of the tRNA-DNA junction, reflecting RNase H specificity for RNA in a RNA/DNA hybrid. However, in the absence of DNA synthesis, or limiting this by chain termination, the PBS was cleaved at a constant distance of 18 nucleotides upstream of the nascent primer 3' terminus. The position of cleavage remained in register with the position of DNA synthesis arrest, indicating that hydrolysis of homoduplex RNA is spatialy co-ordinated with DNA synthesis. Kinetic studies comparing cleavage rates of an analogous DNA primer/PBS heteroduplex and the tRNA(Lys3)/PBS homoduplex showed that while the former is cleaved as rapidly as RT polymerizes, the latter proceeds 30-fold slower. Although the RNase H domain hydrolyzes dsRNA when RT is artificially arrested, specificity for RNA/DNA hybrids is maintained when DNA is actively synthesized, since residency of the RNase H domain at a single base position is not long enough to allow significant cleavage on dsRNA. Images PMID:7533725

  12. 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

  13. 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

  14. A role for RNA helicase A in post-transcriptional regulation of HIV type 1

    PubMed Central

    Li, Junzhou; Tang, Hengli; Mullen, Tina-Marie; Westberg, Christopher; Reddy, Thipparthi R.; Rose, David W.; Wong-Staal, Flossie

    1999-01-01

    Retroviruses must bypass the tight coupling of splicing and nuclear export of mRNA in their replication cycle because unspliced genomic RNA and incompletely spliced mRNA must be exported to the cytoplasm for packaging or translation. This process is mediated by a cis-acting constitutive transport element (CTE) for simple retroviruses and by the trans-acting viral protein Rev in concert with its response element (RRE) for complex retroviruses (e.g., HIV). Recently, we identified RNA helicase A (RHA) as a potential cellular cofactor for CTE. Here, we report that RHA also plays a role in Rev/RRE-mediated gene expression and HIV replication. RHA binds weakly to HIV-1 RRE independently of Rev. Overexpression of RHA, but not of an RHA mutant lacking helicase activity, increased both Rev/RRE- and CTE-dependent gene expression and the levels of unspliced HIV mRNA. Microinjection of antibodies to RHA into nuclei dramatically inhibited both CTE- and Rev-dependent gene expression in human cells. Exogenous RHA cDNA, but not the mutant RHA, rescued this inhibition. We propose that RHA is required to release both CTE- and RRE-containing mRNA from spliceosomes before completion of splicing, thus freeing them for nuclear export. PMID:9892698

  15. TAR RNA decoys inhibit tat-activated HIV-1 transcription after preinitiation complex formation.

    PubMed Central

    Bohjanen, P R; Liu, Y; Garcia-Blanco, M A

    1997-01-01

    The ability of the HIV-1 Tat protein to trans -activate HIV-1 transcription in vitro is specifically inhibited by a circular TAR RNA decoy. This inhibition is not overcome by adding an excess of Tat to the reaction but is partially overcome by adding Tat in combination with nuclear extract, suggesting that TAR RNA might function by interacting with a complex containing Tat and cellular factor(s). A cell-free transcription system involving immobilized DNA templates was used to further define the factor(s) that interact with TAR RNA. Preinitiation complexes formed in the presence or absence of Tat were purified on immobilized templates containing the HIV-1 promoter. After washing, nucleotides and radiolabelled UTP were added and transcription was measured. The presence of Tat during preinitiation complex formation resulted in an increase in the level of full-length HIV-1 transcripts. This Tat-activated increase in HIV-1 transcription was not inhibited by circular TAR decoys added during preinitiation complex formation but was inhibited by circular TAR decoys subsequently added during the transcription reaction. These results suggest that TAR decoys inhibit Tat-activated HIV-1 transcription after preinitiation complex formation, perhaps by interacting with components of transcription complexes. PMID:9358155

  16. 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

  17. Anogenital HIV RNA in Thai men who have sex with men in Bangkok during acute HIV infection and after randomization to standard vs. intensified antiretroviral regimens

    PubMed Central

    Phanuphak, Nittaya; Teeratakulpisarn, Nipat; van Griensven, Frits; Chomchey, Nitiya; Pinyakorn, Suteeraporn; Fletcher, James LK; Trichavaroj, Rapee; Pattanachaiwit, Supanit; Michael, Nelson; Phanuphak, Praphan; Kim, Jerome H; Ananworanich, Jintanat

    2015-01-01

    Introduction HIV transmission risk is highest during acute HIV infection (AHI). We evaluated HIV RNA in the anogenital compartment in men who have sex with men (MSM) during AHI and compared time to undetectable HIV RNA after three-drug versus five-drug antiretroviral therapy (ART) to understand risk for onward HIV transmission. Methods MSM with AHI (n=54) had blood, seminal plasma and anal lavage collected for HIV RNA at baseline, days 3 and 7, and weeks 2, 4, 12 and 24. Data were compared between AHI stages: 1 (fourth-generation antigen-antibody combo immunoassay [IA]–, third-generation IA–, n=15), 2 (fourth-generation IA+, third-generation IA–, n=9) and 3 (fourth-generation IA+, third-generation IA+, western blot–/indeterminate, n=30) by randomization to five-drug (tenofovir+emtricitabine+efavirenz+raltegravir+maraviroc, n=18) versus three-drug (tenofovir+emtricitabine+efavirenz, n=18) regimens. Results Mean age was 29 years and mean duration since HIV exposure was 15.4 days. Mean baseline HIV RNA was 5.5 in blood, 3.9 in seminal plasma and 2.6 log10 copies/ml in anal lavage (p<0.001). Blood and seminal plasma HIV RNA were higher in AHI Stage 3 compared to Stage 1 (p<0.01). Median time from ART initiation to HIV RNA <50 copies/ml was 60 days in blood, 15 days in seminal plasma and three days in anal lavage. Compared with the three-drug ART, the five-drug ART had a shorter time to HIV RNA <1500 copies/ml in blood (15 vs. 29 days, p=0.005) and <50 copies/ml in seminal plasma (13 vs. 24 days, p=0.048). Conclusions Among MSM with AHI, HIV RNA was highest in blood, followed by seminal plasma and anal lavage. ART rapidly reduced HIV RNA in all compartments, with regimen intensified by raltegravir and maraviroc showing faster HIV RNA reductions in blood and seminal plasma. PMID:25956171

  18. 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. PMID:26631810

  19. 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

  20. 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

  1. 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

  2. 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

  3. Dimerization of HIV-1 genomic RNA of subtypes A and B: RNA loop structure and magnesium binding.

    PubMed Central

    Jossinet, F; Paillart, J C; Westhof, E; Hermann, T; Skripkin, E; Lodmell, J S; Ehresmann, C; Ehresmann, B; Marquet, R

    1999-01-01

    Retroviruses encapsidate their genome as a dimer of homologous RNA molecules noncovalently linked close to their 5' ends. The dimerization initiation site (DIS) of human immunodeficiency virus type 1 (HIV-1) RNA is a hairpin structure that contains in the loop a 6-nt self-complementary sequence flanked by two 5' and one 3' purines. The self-complementary sequence, as well as the flanking purines, are crucial for dimerization of HIV-1 RNA, which is mediated by formation of a "kissing-loop" complex between the DIS of each monomer. Here, we used chemical modification interference, lead-induced cleavage, and three-dimensional modeling to compare dimerization of subtype A and B HIV-1 RNAs. The DIS loop sequences of these RNAs are AGGUGCACA and AAGCGCGCA, respectively. In both RNAs, ethylation of most but not all phosphate groups in the loop and methylation of the N7 position of the G residues in the self-complementary sequence inhibited dimerization. These results demonstrate that small perturbations of the loop structure are detrimental to dimerization. Conversely, methylation of the N1 position of the first and last As in the loop were neutral or enhanced dimerization, a result consistent with these residues forming a noncanonical sheared base pair. Phosphorothioate interference, lead-induced cleavage, and Brownian-dynamics simulation revealed an unexpected difference in the dimerization mechanism of these RNAs. Unlike subtype B, subtype A requires binding of a divalent cation in the loop to promote RNA dimerization. This difference should be taken into consideration in the design of antidimerization molecules aimed at inhibiting HIV-1 replication. PMID:10496223

  4. 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

  5. A Re-Examination of Global Suppression of RNA Interference by HIV-1

    PubMed Central

    Sanghvi, Viraj R.; Steel, Laura F.

    2011-01-01

    The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing. PMID:21386885

  6. 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

  7. 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

  8. Quantification of HIV RNA and Human Herpesvirus DNA in Seminal Plasma

    PubMed Central

    Vargas-Meneses, Milenka V.; Massanella, Marta; Ignacio, Caroline C.; Gianella, Sara

    2016-01-01

    Multiple viruses can co-infect the genital tract, modifying the immunologic and virologic milieu and possibly playing a role in viral transmission and pathogenesis. The aim of our studies has been to understand the complex relationships between HIV-1 RNA, and multiple human herpesviruses known to frequently replicate in the genital tract of HIV-infected men (i.e. cytomegalovirus [CMV], Epstein Bar virus [EBV], herpes simplex virus [HSV] types 1 and 2, and human herpesviruses [HHV] 6, 7 and 8) (Gianella et al., 2013a; Gianella et al., 2013b; Gianella et al., 2013c; Gianella et al., 2014). This protocol was designed to collect and process male genital secretion (GS), and to isolate and further quantify HIV RNA and DNA of seven HHV from seminal plasma using quantitative real time PCR technology. PMID:27376109

  9. Cooperative and specific binding of Vif to the 5' region of HIV-1 genomic RNA.

    PubMed

    Henriet, Simon; Richer, Delphine; Bernacchi, Serena; Decroly, Etienne; Vigne, Robert; Ehresmann, Bernard; Ehresmann, Chantal; Paillart, Jean-Christophe; Marquet, Roland

    2005-11-18

    The viral infectivity factor (Vif) protein of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication in vivo. Packaging of Vif into viral particles is mediated by an interaction with viral genomic RNA and association with viral nucleoprotein complexes. Despite recent findings on the RNA-binding properties of Vif suggesting that Vif could be involved in retroviral assembly, no RNA sequence or structure specificity has been determined so far. To gain further insight into the mechanisms by which Vif might regulate viral replication, we studied the interactions of Vif with HIV-1 genomic RNA in vitro. Using extensive biochemical analysis, we have measured the affinity of recombinant Vif proteins for synthetic RNAs corresponding to various regions of the HIV-1 genome. We found that recombinant Vif proteins bind specifically to HIV-1 viral RNA fragments corresponding to the 5'-untranslated region (5'-UTR), gag and the 5' part of pol (K(d) between 45 nM and 65 nM). RNA encompassing nucleotides 1-497 or 499-996 of the HIV-1 genomic RNA bind 9+/-2 and 21+/-3 Vif molecules, respectively, and at least some of these proteins bind in a cooperative manner (Hill constant alpha(H) = 2.3). In contrast, RNAs corresponding to other parts of the HIV-1 genome or heterologous RNAs showed poor binding capacity and weak cooperativity (K(d) > 200 nM). Moreover, RNase T1 footprinting revealed a hierarchical binding of Vif, pointing to TAR and the poly(A) stem-loop structures as primary strong affinity targets, and downstream structures as secondary sites with moderate affinity. Taken together, our findings suggest that Vif may assist other proteins to maintain a correct folding of the genomic RNA in order to facilitate its packaging and further steps such as reverse transcription. Interestingly, our results suggest also that Vif could bind the viral RNA in order to protect it from the action of the antiviral factor APOBEC-3G/3F. PMID:16236319

  10. Identification of Biologically Active, HIV TAR RNA-Binding Small Molecules Using Small Molecule Microarrays

    PubMed Central

    2015-01-01

    Identifying small molecules that selectively bind to structured RNA motifs remains an important challenge in developing potent and specific therapeutics. Most strategies to find RNA-binding molecules have identified highly charged compounds or aminoglycosides that commonly have modest selectivity. Here we demonstrate a strategy to screen a large unbiased library of druglike small molecules in a microarray format against an RNA target. This approach has enabled the identification of a novel chemotype that selectively targets the HIV transactivation response (TAR) RNA hairpin in a manner not dependent on cationic charge. Thienopyridine 4 binds to and stabilizes the TAR hairpin with a Kd of 2.4 μM. Structure–activity relationships demonstrate that this compound achieves activity through hydrophobic and aromatic substituents on a heterocyclic core, rather than cationic groups typically required. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE) analysis was performed on a 365-nucleotide sequence derived from the 5′ untranslated region (UTR) of the HIV-1 genome to determine global structural changes in the presence of the molecule. Importantly, the interaction of compound 4 can be mapped to the TAR hairpin without broadly disrupting any other structured elements of the 5′ UTR. Cell-based anti-HIV assays indicated that 4 inhibits HIV-induced cytopathicity in T lymphocytes with an EC50 of 28 μM, while cytotoxicity was not observed at concentrations approaching 1 mM. PMID:24820959

  11. 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

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

    PubMed Central

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

    2014-01-01

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

  13. 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

  14. 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

  15. 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

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-06-01

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

  18. 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. PMID:27267944

  19. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein

    PubMed Central

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisné, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55Gag, reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNALys3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  20. 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

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

    PubMed

    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

  2. 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

  3. Specific recognition of the HIV-1 genomic RNA by the Gag precursor.

    PubMed

    Abd El-Wahab, Ekram W; Smyth, Redmond P; Mailler, Elodie; Bernacchi, Serena; Vivet-Boudou, Valérie; Hijnen, Marcel; Jossinet, Fabrice; Mak, Johnson; Paillart, Jean-Christophe; Marquet, Roland

    2014-01-01

    During assembly of HIV-1 particles in infected cells, the viral Pr55(Gag) protein (or Gag precursor) must select the viral genomic RNA (gRNA) from a variety of cellular and viral spliced RNAs. However, there is no consensus on how Pr55(Gag) achieves this selection. Here, by using RNA binding and footprinting assays, we demonstrate that the primary Pr55(Gag) binding site on the gRNA consists of the internal loop and the lower part of stem-loop 1 (SL1), the upper part of which initiates gRNA dimerization. A double regulation ensures specific binding of Pr55(Gag) to the gRNA despite the fact that SL1 is also present in spliced viral RNAs. The region upstream of SL1, which is present in all HIV-1 RNAs, prevents binding to SL1, but this negative effect is counteracted by sequences downstream of SL4, which are unique to the gRNA. PMID:24986025

  4. The Major Human Immunodeficiency Virus Type 2 (HIV-2) Packaging Signal Is Present on All HIV-2 RNA Species: Cotranslational RNA Encapsidation and Limitation of Gag Protein Confer Specificity

    PubMed Central

    Griffin, Stephen D. C.; Allen, Jane F.; Lever, Andrew M. L.

    2001-01-01

    Deletion of a region of the human immunodeficiency virus type 2 (HIV-2) 5′ leader RNA reduces genomic RNA encapsidation to about 5% that of wild-type virus with no defect in viral protein production but severely limits virus spread in Jurkat T cells, indicating that this region contains a major cis-acting encapsidation signal, or psi (Ψ). Being upstream of the major splice donor, it is present on all viral transcripts. We have shown that HIV-2 selects its genomic RNA for encapsidation cotranslationally, rendering wild-type HIV-2 unable to encapsidate vector RNAs in trans . Virus with Ψ deleted, however, encapsidates an HIV-2 vector, demonstrating competition for Gag protein. HIV-2 overcomes the lack of packaging signal location specificity by two novel mechanisms, cotranslational packaging and competition for limiting Gag polyprotein. PMID:11711596

  5. 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. PMID:26896646

  6. 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

  7. 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

  8. 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

  9. 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.

  10. Development of a reverse genetics system based on RNA polymerase II for Newcastle disease virus genotype VII.

    PubMed

    Wang, Jianzhong; Wang, Chunfeng; Feng, Na; Wang, Hualei; Zheng, Xuexing; Yang, Songtao; Gao, Yuwei; Xia, Xianzhu; Yin, Renfu; Liu, Xiufan; Hu, Shunlin; Ding, Chan; Yu, Shengqing; Cong, Yanlong; Ding, Zhuang

    2015-02-01

    Newcastle disease virus (NDV) has only a single serotype but diversified genotypes. Genotype VII strains are the prevalent currently circulating genotype worldwide, and in particular, these strains cause outbreaks in waterfowl. In this study, a reverse genetics system for highly virulent NDV isolated from goose flocks was developed independent of conventional T7 RNA polymerase. Infectious virus was successfully generated by an RNA polymerase II promoter to drive transcription of the full-length virus antigenome. A green fluorescent protein (GFP)-expressing virus was generated by inserting an additional transcription cassette coding for the enhanced GFP between the P and M genes of the genome. The expression of GFP was confirmed by western blotting and fluorescence microscopy. The replication kinetics and pathogenicity of the recombinant viruses are indistinguishable from the parental wild-type virus. This reverse genetics system will provide a powerful tool for the analysis of goose-origin NDV dissemination and pathogenesis, as well as preparation for genotype-matched NDV attenuated vaccines. PMID:25384536

  11. 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

  12. 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

  13. RNA and DNA binding properties of HIV-1 Vif protein: a fluorescence study.

    PubMed

    Bernacchi, Serena; Henriet, Simon; Dumas, Philippe; Paillart, Jean-Christophe; Marquet, Roland

    2007-09-01

    The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Some "non-permissive" cell lines cannot sustain replication of Vif(-) HIV-1 virions. In these cells, Vif counteracts the natural antiretroviral activity of the DNA-editing enzymes APOBEC3G/3F. Moreover, Vif is packaged into viral particles through a strong interaction with genomic RNA in viral nucleoprotein complexes. To gain insights into determinants of this binding process, we performed the first characterization of Vif/nucleic acid interactions using Vif intrinsic fluorescence. We determined the affinity of Vif for RNA fragments corresponding to various regions of the HIV-1 genome. Our results demonstrated preferential and moderately cooperative binding for RNAs corresponding to the 5'-untranslated region of HIV-1 (5'-untranslated region) and gag (cooperativity parameter omega approximately 65-80, and K(d) = 45-55 nM). In addition, fluorescence spectroscopy allowed us to point out the TAR apical loop and a short region in gag as primary strong affinity binding sites (K(d) = 9.5-14 nM). Interestingly, beside its RNA binding properties, the Vif protein can also bind the corresponding DNA oligonucleotides and their complementary counterparts with an affinity similar to the one observed for the RNA sequences, while other DNA sequences displayed reduced affinity. Taken together, our results suggest that Vif binding to RNA and DNA offers several non-exclusive ways to counteract APOBEC3G/3F factors, in addition to the well documented Vif-induced degradation by the proteasome and to the Vif-mediated repression of translation of these antiviral factors. PMID:17609216

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

    PubMed

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

    2016-07-01

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

  15. 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

  16. 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

  17. 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

  18. A structure-based approach for targeting the HIV-1 genomic RNA dimerization initiation site.

    PubMed

    Ennifar, Eric; Paillart, Jean-Christophe; Bernacchi, Serena; Walter, Philippe; Pale, Patrick; Decout, Jean-Luc; Marquet, Roland; Dumas, Philippe

    2007-10-01

    Dimerization of the genomic RNA is an important step of the HIV-1 replication cycle. The Dimerization Initiation Site (DIS) promotes dimerization of the viral genome by forming a loop-loop complex between two DIS hairpins. Crystal structures of the DIS loop-loop complex revealed an unexpected and strong similitude with the bacterial 16S ribosomal aminoacyl-tRNA site (A site), which is the target of aminoglycoside antibiotics. As a consequence of these structural and sequence similarities, the HIV-1 DIS also binds some aminoglycosides, not only in vitro, but also ex vivo, in lymphoid cells and in viral particles. Crystal structures of the DIS loop-loop in complex with several aminoglycoside antibiotics provide a detailed-view of the DIS/drug interaction and reveal some hints about possible modifications to increase the drug affinity and/or specificity. PMID:17434658

  19. HIV-1 RRE RNA acts as an RNA silencing suppressor by competing with TRBP-bound siRNAs

    PubMed Central

    Daniels, Sylvanne M; Sinck, Lucile; Ward, Natalie J; Melendez-Peña, Carlos E; Scarborough, Robert J; Azar, Ibrahim; Rance, Elodie; Daher, Aïcha; Pang, Ka-Ming; Rossi, John J; Gatignol, Anne

    2015-01-01

    Several proteins and RNAs expressed by mammalian viruses have been reported to interfere with RNA interference (RNAi) activity. We investigated the ability of the HIV-1-encoded RNA elements Trans-Activation Response (TAR) and Rev-Response Element (RRE) to alter RNAi. MicroRNA let7-based assays showed that RRE is a potent suppressor of RNAi activity, while TAR displayed moderate RNAi suppression. We demonstrate that RRE binds to TAR-RNA Binding Protein (TRBP), an essential component of the RNA Induced Silencing Complex (RISC). The binding of TAR and RRE to TRBP displaces small interfering (si)RNAs from binding to TRBP. Several stem-deleted RRE mutants lost their ability to suppress RNAi activity, which correlated with a reduced ability to compete with siRNA-TRBP binding. A lentiviral vector expressing TAR and RRE restricted RNAi, but RNAi was restored when Rev or GagPol were coexpressed. Adenoviruses are restricted by RNAi and encode their own suppressors of RNAi, the Virus-Associated (VA) RNA elements. RRE enhanced the replication of wild-type and VA-deficient adenovirus. Our work describes RRE as a novel suppressor of RNAi that acts by competing with siRNAs rather than by disrupting the RISC. This function is masked in lentiviral vectors co-expressed with viral proteins and thus will not affect their use in gene therapy. The potent RNAi suppressive effects of RRE identified in this study could be used to enhance the expression of RNAi restricted viruses used in oncolysis such as adenoviruses. PMID:25668122

  20. Demonstration of de novo HIV type 1 production by detection of multiply spliced and unspliced HIV type 1 RNA in paraffin-embedded tonsils.

    PubMed

    Brachtel, Elena F; Mascola, John R; Wear, Douglas J; Ehrenberg, Philip K; Dayhoff, Deborah E; Sanders-Buell, Eric; Michael, Nelson L; Frankel, Sarah Schlesinger

    2002-07-20

    HIV-1 infection of tonsils takes place when virus spreads systemically, and may occur when tonsillar tissue serves as the initial portal of HIV-1 entry. The HIV replication cycle includes the production of regulatory and accessory gene mRNAs, produced by splicing of genomic mRNA, that are hallmarks of de novo virus production. We sought to demonstrate, for the first time, the presence of multiply spliced viral RNA transcripts in archival tissue as a marker for active virus replication. Further, amplified cDNA sequences from unspliced pol gene mRNA were used to define the genetic subtype of HIV-1 within these tissues. RNA was extracted from surgical pathological, formalin-fixed, paraffin-embedded specimens, and RT-PCR was performed with primers for unspliced and multiply spliced HIV-1 transcripts. Amplification products were analyzed by agarose gel electrophoresis and their specificity was confirmed by sequencing and Southern blot hybridization. Unspliced HIV-1 pol transcripts yielded cDNA amplicons of 184 base pairs (bp) that were cloned and sequenced. Phylogenetic analysis revealed these sequences to be of HIV-1 subtype B. Multiply spliced transcripts specific for the tat/rev (173 bp), tat (268 bp), and tat/rev/nef (146 bp) regulatory gene mRNAs could be demonstrated in all cases. These results support the demonstration of active replication of HIV-1 in archival tonsillar tissues previously shown by p24 antigen staining. They also show the feasibility of performing molecular epidemiologic studies on HIV-1 cDNA sequences from archived pathologic specimens. PMID:12167270

  1. 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. PMID:25258395

  2. 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

  3. The role of Vif oligomerization and RNA chaperone activity in HIV-1 replication.

    PubMed

    Batisse, Julien; Guerrero, Santiago; Bernacchi, Serena; Sleiman, Dona; Gabus, Caroline; Darlix, Jean-Luc; Marquet, Roland; Tisné, Carine; Paillart, Jean-Christophe

    2012-11-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells that involve most natural HIV-1 target cells. Vif counteracts the packaging of two cellular cytidine deaminases named APOBEC3G (A3G) and A3F by diverse mechanisms including the recruitment of an E3 ubiquitin ligase complex and the proteasomal degradation of A3G/A3F, the inhibition of A3G mRNA translation or by a direct competition mechanism. In addition, Vif appears to be an active partner of the late steps of viral replication by participating in virus assembly and Gag processing, thus regulating the final stage of virion formation notably genomic RNA dimerization and by inhibiting the initiation of reverse transcription. Vif is a small pleiotropic protein with multiple domains, and recent studies highlighted the importance of Vif conformation and flexibility in counteracting A3G and in binding RNA. In this review, we will focus on the oligomerization and RNA chaperone properties of Vif and show that the intrinsic disordered nature of some Vif domains could play an important role in virus assembly and replication. Experimental evidence demonstrating the RNA chaperone activity of Vif will be presented. PMID:22728817

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

    PubMed

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

    1999-10-01

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

  5. 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

  6. 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. PMID:26714709

  7. 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

  8. Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA.

    PubMed

    Kiselinova, Maja; Pasternak, Alexander O; De Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

    2014-01-01

    Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used

  9. Comparison of Droplet Digital PCR and Seminested Real-Time PCR for Quantification of Cell-Associated HIV-1 RNA

    PubMed Central

    De Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

    2014-01-01

    Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been recently described as an alternative PCR-based technique for absolute quantification with higher accuracy compared to qPCR. Here, a comparison was made between the droplet digital PCR (ddPCR) and the seminested qPCR for quantification of unspliced (us) and multiply spliced (ms) CA HIV-1 RNA. Synthetic RNA standards and CA HIV-1 RNA from infected patients on and off ART (N = 34) were quantified with both methods. Correlations were observed between the methods both for serially diluted synthetic standards (usRNA: R2 = 0.97, msRNA: R2 = 0.92) and patient-derived samples (usRNA: R2 = 0.51, msRNA: R2 = 0.87). Seminested qPCR showed better quantitative linearity, accuracy and sensitivity in the quantification of synthetic standards than ddPCR, especially in the lower quantification ranges. Both methods demonstrated equally high detection rate of usRNA in patient samples on and off ART (91%), whereas ddPCR detected msRNA in larger proportion of samples from ART-treated patients (p = 0.13). We observed an average agreement between the methods for usRNA quantification in patient samples, albeit with a large standard deviation (bias = 0.05±0.75 log10). However, a bias of 0.94±0.36 log10 was observed for msRNA. No-template controls were consistently negative in the seminested qPCR, but yielded a positive ddPCR signal for some wells. Therefore, the false positive signals may have affected the detection power of ddPCR in this study. Digital PCR is promising for HIV nucleic acid quantification, but the false positive signals need further attention. Quantitative assays for CA HIV RNA have the potential to improve monitoring of patients on ART and to be used

  10. 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

  11. The unique HCV genotype distribution and the discovery of a novel subtype 6u among IDUs co-infected with HIV-1 in Yunnan, China.

    PubMed

    Xia, Xueshan; Lu, Ling; Tee, Kok Keng; Zhao, Wenhua; Wu, Jianguo; Yu, Jing; Li, Xiaojie; Lin, Yixiong; Mukhtar, Muhammad Mahmood; Hagedorn, Curt H; Takebe, Yutaka

    2008-07-01

    The Yunnan province is the epicenter of HIV-1 epidemics in China and a center for drug trafficking to the other parts of the world. In six prefectures of this province, a total of 132 IDUs were recruited to determine the sero-prevalence of HCV and HIV-1 and the positive rates were 93.94% and 68.18%, respectively (P<0.001). Co-infection with HCV and HIV-1 was found among 89 IDUs, of whom several HCV fragments were amplified and sequenced. Sequences of the HCV 5'NCR-C and NS5B region were determined from 82 IDUs. Phylogenetic analyses showed consistent genotyping among 80 IDUs. Among them HCV genotypes 1a, 1b, 3a, 3b, 6a, 6n, and a tentatively assigned novel 6u subtype were found in 1 (1.25%), 16 (20%), 19 (23.75%), 24 (30%), 4 (5%), 9 (11.25%) and 7 (8.75%) individuals, respectively. In two IDUs, genotyping results were discordant, suggesting mixed HCV infections or recombination. The proportion of patients with HCV 1b tended to decrease from the north to south and from the east to west in this province. Genotype 3 and 6 strains were more frequent in the southern prefectures. The novel subtype 6u strains were only detected in Dehong which borders Myanmar. Our findings showed a unique pattern of HCV genotype distribution, which is similar to that in the southeastern Asian countries but distinct from that among the general population in China. Routes of drug trafficking and the resulting high prevalence of HIV-1 infection may have contributed to this pattern of HCV genotype distribution. PMID:18461611

  12. 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. PMID:25825936

  13. PCR-based identification of eight Lactobacillus species and 18 hr-HPV genotypes in fixed cervical samples of South African women at risk of HIV and BV.

    PubMed

    Dols, Joke A M; Reid, Gregor; Kort, Remco; Schuren, Frank H J; Tempelman, Hugo; Bontekoe, Tj Romke; Korporaal, Hans; Van der Veer, E M; Smit, Pieter W; Boon, Mathilde E

    2012-06-01

    Vaginal lactobacilli assessed by PCR-based microarray and PCR-based genotyping of HPV in South African women at risk for HIV and BV. Vaginal lactobacilli can be defined by microarray techniques in fixed cervical samples of South African women. Cervical brush samples suspended in the coagulant fixative BoonFix of one hundred women attending a health centre for HIV testing in South Africa were available for this study. In the Ndlovu Medical Centre in Elandsdoorn, South Africa, identification of 18 hr-HPV genotypes was done using the INNO-LiPA method. An inventory of lactobacilli organisms was performed using microarray technology. On the basis of the Lactobacillus and Lactobacillus biofilm scoring, the cases were identified as Leiden bacterial vaginosis (BV) negative (BV-; n = 41), Leiden BV intermediate (BV±; n = 25), and Leiden BV positive (BV+; n = 34). Fifty-one women were HIV positive and 49 HIV negative. Out of the 51 HIV positive women, 35 were HPV infected. These 51 HIV positive women were frequently infected with HPV16 and HPV18. In addition, HPV35, HPV52, HPV33, and HPV66 were often detected in these samples. Lactobacillus salivarius and Lactobacillus iners were the most prevalent lactobacilli as established by the microarray technique. In women with HPV infection, the prevalence of Lactobacillus crispatus was significantly reduced. In both HIV and HPV infection, a similar (but not identical) shift in the composition of the lactobacillus flora was observed. We conclude that there is a shift in the composition of vaginal lactobacilli in HIV-infected women. Because of the prominence of HPV35, HPV52, HPV33, and HPV66, vaccination for exclusively HPV16 and HPV18 might be insufficient in South African HIV+ women. PMID:22021225

  14. 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

  15. 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

  16. Antiretroviral Therapy (ART) Use, Human Immunodeficiency Virus (HIV)-1 RNA Suppression, and Medical Causes of Hospitalization Among HIV-Infected Intravenous Drug Users in the Late ART Era

    PubMed Central

    Vallecillo, Gabriel; Mojal, Sergio; Torrens, Marta; Muga, Roberto

    2014-01-01

    Background.  Antiretroviral therapy (ART) has reduced the rates and changed the causes of hospital admission. However, human immunodeficiency virus-positive intravenous drug users (HIV-IDU) continue to have increased hospitalizations and discharge diagnosis are less defined in the late ART era. Our aim was to examine ART use, HIV-1 RNA suppression, and hospital discharge diagnoses among HIV-IDU admitted to an urban hospital. Methods.  A retrospective analysis was made of HIV-IDU admitted for medical causes for the first time (2006–2010). Surgical, obstetric, or mental (except HIV-associated neurocognitive disorder) diagnoses were excluded. Clinical characteristics, number of admissions, and primary discharge diagnoses were determined for each patient. Results.  Three hundred and seventy-five admissions were recorded among 197 hospitalized HIV-IDU. Lifetime prevalence of ART use was 83.2% (164 of 197) and the rate of HIV-1 RNA <50 copies/mL was 38.1% (75 of 197). Primary discharge diagnosis groups were as follows: bacterial infections (59.2%), chronic end-organ damage (16.8%), complications derived from injected drug use (16.8%), malignancies (9.1%), and opportunistic infections (6.6%). Chronic end-organ damage was diagnosed more frequently in patients with HIV-1 RNA <50 copies/mL (36% vs 4.9%; P < .000), and complications derived from injected drug use (23.8% vs 5.3%; P < .0008) and acquired immune deficiency syndrome (AIDS) opportunistic infections (19.8% vs 1.3% P < .019) were usually diagnosed in patients with HIV-1 RNA detectable viral load. Conclusions.  Human immunodeficiency virus-positive intravenous drug users are admitted to hospitals mainly for non-AIDS-related illnesses; however, sustained HIV-1 RNA viral load suppression is poor and determines hospital discharge diagnoses. Providers need to be aware of the management of HIV-related comorbidities and reinforce strategies to improve ART retention in this population. PMID:25734084

  17. 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

  18. 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

  19. 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.

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

    PubMed

    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

  1. 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

  2. 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. PMID:26553869

  3. MicroRNA-mediated regulation of p21 and TASK1 cellular restriction factors enhances HIV-1 infection

    PubMed Central

    Farberov, Luba; Herzig, Eytan; Modai, Shira; Isakov, Ofer; Hizi, Amnon; Shomron, Noam

    2015-01-01

    MicroRNAs (miRNAs) are short non-coding RNAs that play a central role in the regulation of gene expression by binding to target mRNAs. Several studies have revealed alterations in cellular miRNA profiles following HIV-1 infection, mostly for miRNAs involved in inhibiting viral infection. These miRNA expression modifications might also serve to block the innate HIV-1 inhibition mechanism. As a result, it is expected that during HIV-1 infection miRNAs target genes that hinder or prevent the progression of the HIV-1 replication cycle. One of the major sets of genes known to inhibit the progression of HIV-1 infection are cellular restriction factors. In this study, we identified a direct miRNA target gene that modulates viral spread in T-lymphocytes and HeLa-CCR5 cell lines. Following infection, let-7c, miR-34a or miR-124a were upregulated, and they targeted and downregulated p21 and TASK1 (also known as CDKN1A and KCNK3, respectively) cellular proteins. This eventually led to increased virion release and higher copy number of viral genome transcripts in infected cells. Conversely, by downregulating these miRNAs, we could suppress viral replication and spread. Our data suggest that HIV-1 exploits the host miRNA cellular systems in order to block the innate inhibition mechanism, allowing a more efficient infection process. PMID:25717002

  4. 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. PMID:26733199

  5. 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. PMID:23966501

  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. Characteristics and genotype profiles of antiretroviral-naïve patients entering a Southern US HIV outpatient clinic 2009-2012.

    PubMed

    Seal, Paula S; Frontini, Maria; Jhita, Preya K; Deichmann, Paige C; Clark, Rebecca A

    2016-06-01

    The US city of New Orleans was ranked second in the nation for estimated HIV case rates in 2011. Opt-out testing was established at the Interim Louisiana Hospital in New Orleans in 2013. The majority of new diagnoses were referred to the HIV outpatient program. We conducted a retrospective chart review of newly referred antiretroviral-naïve patients establishing HIV care between January 2009 and June 2013 to characterise demographic and genotype profiles to assist in clinical management and needed services. Of the eligible 226 patients, 68% were men, and 88% were African American. Nearly half of the study patients were younger than 35 years of age. Forty-six percent had an initial CD4 count <200 cells/mm(3), and 39% had a HIV viral load >100,000 copies/mL. The antiretroviral class with the most common major mutation was the non-nucleoside reverse transcriptase inhibitors (NNRTIs) where K103N was the most common major NNRTI mutation at presentation. We observed that male patients showed more advanced disease with later presentation to care, confirming the need for earlier HIV diagnosis. When considering initial antiretroviral therapy, baseline genotype information is encouraged, particularly if considering a NNRTI-based regimen. PMID:26016726

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

    PubMed Central

    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

  10. Etravirine as a Switching Option for Patients with HIV RNA Suppression: A Review of Recent Trials

    PubMed Central

    Nelson, Mark; Hill, Andrew; van Delft, Yvon; Moecklinghoff, Christiane

    2014-01-01

    Unlike other nonnucleoside reverse transcriptase inhibitors, etravirine is only approved for use in treatment-experienced patients. In the DUET 1 and 2 trials, 1203 highly treatment-experienced patients were randomized to etravirine or placebo, in combination with darunavir/ritonavir and optimized background treatment. In these trials, etravirine showed significantly higher rates of HIV RNA suppression when compared with placebo (61% versus 40% at Week 48). There was no significant rise of lipids or neuropsychiatric adverse events, but there was an increase in the risk of rash with etravirine treatment. In the SENSE trial, which evaluated etravirine and efavirenz in 157 treatment-naïve patients in combination with 2 nucleoside analogues, there was a lower risk of lipid elevations and neuropsychiatric adverse events with etravirine when compared to efavirenz. Etravirine has been evaluated in three randomized switching studies. In the SSAT029 switch trial, 38 patients who had neuropsychiatric adverse events possibly related to efavirenz showed an improvement in these after switching to etravirine. The Swiss Switch-EE recruited 58 individuals without neuropsychiatric adverse events who were receiving efavirenz, and no benefit was shown when switching to etravirine. In the Spanish ETRA-SWITCH trial (n = 46), there were improvements in lipids when individuals switched from a protease inhibitor to etravirine. These switching trials were conducted in patients with full HIV RNA suppression: <50 copies/mL and with no history of virological failure or resistance to therapy. The results from these three randomized switching studies suggest a possible new role for etravirine, in combination with two nucleoside analogues, as a switching option for those with HIV RNA suppression but who are reporting adverse events possibly related to antiretroviral therapy. However a large well-powered trial would need to be conducted to strengthen the evidence from the pilot studies conducted

  11. Humoral, Mucosal, and Cell-Mediated Immunity Against Vaccine and Nonvaccine Genotypes After Administration of Quadrivalent Human Papillomavirus Vaccine to HIV-Infected Children

    PubMed Central

    Weinberg, Adriana; Song, Lin-Ye; Saah, Alfred; Brown, Martha; Moscicki, Anna B.; Meyer, William A.; Bryan, Janine; Levin, Myron J.

    2012-01-01

    Objectives. To characterize the immunogenicity of a quadrivalent human papillomavirus vaccine (QHPV) in human immunodeficiency virus (HIV)–infected children, we studied their immune responses to 3 or 4 doses. Methods. HIV-infected children aged 7–12 years with a CD4 cell percentage of ≥15% of lymphocytes, received 3 doses of QHPV with or without a fourth dose after 72 weeks. Type-specific and cross-reactive antibodies and cell-mediated immunity were measured. Results. Type-specific antibodies to HPV6, 11, and 16 were detected in 100% and ≥94% of children at 4 and 72 weeks, respectively, after the third QHPV dose. Corresponding numbers for HPV18 were 97% and 76%, respectively. A fourth QHPV dose increased seropositivity to ≥96% for all vaccine genotypes. Four weeks after the third QHPV dose, 67% of vaccinees seroconverted to HPV31, an HPV16-related genotype not in the vaccine; 69% and 39% of vaccinees developed mucosal HPV16 and 18 immunoglobulin G antibodies, respectively; and 60% and 52% of vaccinees developed cytotoxic T lymphocytes (CTLs) for HPV16 and 31, respectively. Conclusions. Three QHPV doses generated robust and persistent antibodies to HPV6, 11, and 16 but comparatively weaker responses to HPV18. A fourth dose increased antibodies against all vaccine genotypes in an anamnestic fashion. CTLs and mucosal antibodies against vaccine genotypes, as well as cross-reactive antibodies and CTL against nonvaccine genotypes, were detected. PMID:22859825

  12. Targeted binding of nucleocapsid protein transforms the folding landscape of HIV-1 TAR RNA

    PubMed Central

    McCauley, Micah J.; Rouzina, Ioulia; Manthei, Kelly A.; Gorelick, Robert J.; Musier-Forsyth, Karin; Williams, Mark C.

    2015-01-01

    Retroviral nucleocapsid (NC) proteins are nucleic acid chaperones that play a key role in the viral life cycle. During reverse transcription, HIV-1 NC facilitates the rearrangement of nucleic acid secondary structure, allowing the transactivation response (TAR) RNA hairpin to be transiently destabilized and annealed to a cDNA hairpin. It is not clear how NC specifically destabilizes TAR RNA but does not strongly destabilize the resulting annealed RNA–DNA hybrid structure, which must be formed for reverse transcription to continue. By combining single-molecule optical tweezers measurements with a quantitative mfold-based model, we characterize the equilibrium TAR stability and unfolding barrier for TAR RNA. Experiments show that adding NC lowers the transition state barrier height while also dramatically shifting the barrier location. Incorporating TAR destabilization by NC into the mfold-based model reveals that a subset of preferential protein binding sites is responsible for the observed changes in the unfolding landscape, including the unusual shift in the transition state. We measure the destabilization induced at these NC binding sites and find that NC preferentially targets TAR RNA by binding to specific sequence contexts that are not present on the final annealed RNA–DNA hybrid structure. Thus, specific binding alters the entire RNA unfolding landscape, resulting in the dramatic destabilization of this specific structure that is required for reverse transcription. PMID:26483503

  13. Assessment of RNA carrier function in peptide amphiphiles derived from the HIV fusion peptide.

    PubMed

    Pratumyot, Yaowalak; Torres, Oscar B; Bong, Dennis

    2016-05-01

    A small library of amphiphilic peptides has been evaluated for duplex RNA carrier function into A549 cells. We studied peptides in which a C-terminal 7-residue cationic domain is attached to a neutral/hydrophobic 23-residue domain that is based on the viral fusion peptide of HIV. We also examined peptides in which the cationic charge was evenly distributed throughout the peptide. Strikingly, subtle sequence variations in the hydrophobic domain that do not alter net hydrophobicity result in wide variation in RNA uptake. Additionally, cyclic cystine variants are much less active as RNA carriers than their open-chain cysteine analogs. With regard to electrostatic effects, we find that lysine is less effective than arginine in facilitating uptake, and that even distribution of cationic residues throughout the peptide sequence results in especially effective RNA carrier function. Overall, minor changes in peptide hydrophobicity, flexibility and charge distribution can significantly alter carrier function. We hypothesize this is due to altered properties of the peptide-RNA assembly rather than peptide secondary structure. PMID:26988874

  14. Dense Genotyping of Immune-Related Loci Identifies Variants Associated with Clearance of HPV among HIV-Positive Women in the HIV Epidemiology Research Study (HERS)

    PubMed Central

    Sudenga, Staci L.; Wiener, Howard W.; King, Caroline C.; Rompalo, Anne M.; Cu-Uvin, Susan; Klein, Robert S.; Shah, Keerti V.; Sobel, Jack D.; Jamieson, Denise J.; Shrestha, Sadeep

    2014-01-01

    Persistent high-risk human papillomavirus (HR-HPV) is a necessary and causal factor of cervical cancer. Most women naturally clear HPV infections; however, the biological mechanisms related to HPV pathogenesis have not been clearly elucidated. Host genetic factors that specifically regulate immune response could play an important role. All HIV-positive women in the HIV Epidemiology Research Study (HERS) with a HR-HPV infection and at least one follow-up biannual visit were included in the study. Cervicovaginal lavage samples were tested for HPV using type-specific HPV hybridization assays. Type-specific HPV clearance was defined as two consecutive HPV-negative tests after a positive test. DNA from participants was genotyped for 196,524 variants within 186 known immune related loci using the custom ImmunoChip microarray. To assess the influence of each single-nucleotide polymorphism (SNP) with HR-HPV clearance, the Cox proportional hazards model with the Wei-Lin-Weissfeld approach was used, adjusting for CD4+ count, low risk HPV (LR-HPV) co-infection, and relevant confounders. Three analytical models were performed: race-specific (African Americans (n = 258), European Americans (n = 87), Hispanics (n = 55), race-adjusted combined analysis, and meta-analysis of pooled independent race-specific analyses. Women were followed for a median time of 1,617 days. Overall, three SNPs (rs1112085, rs11102637, and rs12030900) in the MAGI-3 gene and one SNP (rs8031627) in the SMAD3 gene were associated with HR-HPV clearance (p<10−6). A variant (rs1633038) in HLA-G were also significantly associated in African American. Results from this study support associations of immune-related genes, having potential biological mechanism, with differential cervical HR-HPV infection outcomes. PMID:24918582

  15. Preclinical In Vivo Evaluation of the Safety of a Multi-shRNA-based Gene Therapy Against HIV-1

    PubMed Central

    Centlivre, Mireille; Legrand, Nicolas; Klamer, Sofieke; Liu, Ying Poi; Eije, Karin Jasmijn von; Bohne, Martino; Rijnstra, Esther Siteur-van; Weijer, Kees; Blom, Bianca; Voermans, Carlijn; Spits, Hergen; Berkhout, Ben

    2013-01-01

    Highly active antiretroviral therapy (HAART) has significantly improved the quality of life and the life expectancy of HIV-infected individuals. Still, drug-induced side effects and emergence of drug-resistant viral variants remain important issues that justify the exploration of alternative therapeutic options. One strategy consists of a gene therapy based on RNA interference to induce the sequence-specific degradation of the HIV-1 RNA genome. We have selected four potent short hairpin RNA (shRNA) candidates targeting the viral capside, integrase, protease and tat/rev open-reading frames and screened the safety of them during human hematopoietic cell development, both in vitro and in vivo. Although the four shRNA candidates appeared to be safe in vitro, one shRNA candidate impaired the in vivo development of the human immune system in Balb/c Rag2−/−IL-2Rγc−/− (BRG) mice. The three remaining shRNA candidates were combined into one single lentiviral vector (LV), and safety of the shRNA combination during human hematopoietic cell development was confirmed. Overall, we demonstrate here the preclinical in vivo safety of a LV expressing three shRNAs against HIV-1, which is proposed for a future Phase I clinical trial. PMID:24002730

  16. 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

  17. Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

    PubMed Central

    Wheeler, Lee Adam; Trifonova, Radiana; Vrbanac, Vladimir; Basar, Emre; McKernan, Shannon; Xu, Zhan; Seung, Edward; Deruaz, Maud; Dudek, Tim; Einarsson, Jon Ivar; Yang, Linda; Allen, Todd M.; Luster, Andrew D.; Tager, Andrew M.; Dykxhoorn, Derek M.; Lieberman, Judy

    2011-01-01

    The continued spread of the HIV epidemic underscores the need to interrupt transmission. One attractive strategy is a topical vaginal microbicide. Sexual transmission of herpes simplex virus type 2 (HSV-2) in mice can be inhibited by intravaginal siRNA application. To overcome the challenges of knocking down gene expression in immune cells susceptible to HIV infection, we used chimeric RNAs composed of an aptamer fused to an siRNA for targeted gene knockdown in cells bearing an aptamer-binding receptor. Here, we showed that CD4 aptamer-siRNA chimeras (CD4-AsiCs) specifically suppress gene expression in CD4+ T cells and macrophages in vitro, in polarized cervicovaginal tissue explants, and in the female genital tract of humanized mice. CD4-AsiCs do not activate lymphocytes or stimulate innate immunity. CD4-AsiCs that knock down HIV genes and/or CCR5 inhibited HIV infection in vitro and in tissue explants. When applied intravaginally to humanized mice, CD4-AsiCs protected against HIV vaginal transmission. Thus, CD4-AsiCs could be used as the active ingredient of a microbicide to prevent HIV sexual transmission. PMID:21576818

  18. APOBEC3G inhibits HIV-1 RNA elongation by inactivating the viral trans-activation response element.

    PubMed

    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-07-29

    Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

  19. APOBEC3G Inhibits HIV-1 RNA Elongation by Inactivating the Viral Trans-Activation Response Element

    PubMed Central

    Nowarski, Roni; Prabhu, Ponnandy; Kenig, Edan; Smith, Yoav; Britan-Rosich, Elena; Kotler, Moshe

    2014-01-01

    Deamination of cytidine residues in viral DNA (vDNA) is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient HIV-1 replication. dC to dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here we demonstrate that A3G provides an additional layer of defence against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. Finally, we show that free ssDNA termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3′+5′ ends is sufficient for A3G deamination, identifying A3G as an efficient mutator, and that deamination of (−)SSDNA results in an early block of HIV-1 transcription. PMID:24859335

  20. siRNA Against KIR3DL1 as a Potential Gene Therapeutic Agent in Controlling HIV-1 Infection

    PubMed Central

    Fu, Geng-Feng; Pan, Ji-Cheng; Lin, Nan; Hu, Hai-Yang; Tang, Wei-Ming; Xu, Jin-Shui; Wang, Xiao-Liang; Xu, Xiao-Qin; Qiu, Tao; Liu, Xiao-Yan; Chen, Guo-Hong; Mahapatra, Tanmay; Huan, Xi-Ping

    2014-01-01

    Abstract Objectives: The aim of this study was to develop a small interfering RNA (siRNA) against the expression of KIR3DL1 receptor on natural killer (NK) cells, in order to promote the ability of NK cells to destroy human immunodeficiency virus (HIV)-infected cells and thus prevent failure of siRNA therapy targeting human immunodeficiency virus type 1 (HIV-1) virus among HIV-1 infected patients in vitro. Methods: A siRNA targeting KIR3DL1 was synthesized and then modified with cholesterol, methylene, and sulfate. The inhibitory action of the siRNAs on primary cultured NK cells was detected. The amount of IFN-γ and TNF-α secretions in NK cells was measured. The intended functions of NK cells in vitro were analyzed by CFSE and PI methods. Results: There were no significant differences in inhibiting the expression of KIR3DL1 on NK cells between the modified and unmodified siRNAs, while inhibition by each of them differed significantly from controls. The amount of IFN-γ and TNF-α secretions in the NK cells was abundant due to unsuccessful expression of KIR3DL1 on NK cells, which further promoted function of the NK cells. Conclusion: The siRNA against KIR3DL1 could enhance the ability of the NK cells to kill the HIV-1 infected cells in vitro and successfully prevented the failure of siRNA therapy targeting the HIV-1 virus. Therefore, it can act as a potential gene therapeutic agent among HIV-1 infected people. PMID:24834927

  1. Dual mechanisms of translation initiation of the full-length HIV-1 mRNA contribute to gag synthesis.

    PubMed

    Monette, Anne; Valiente-Echeverría, Fernando; Rivero, Matias; Cohen, Éric A; Lopez-Lastra, Marcelo; Mouland, Andrew J

    2013-01-01

    The precursor group-specific antigen (pr55(Gag)) is central to HIV-1 assembly. Its expression alone is sufficient to assemble into virus-like particles. It also selects the genomic RNA for encapsidation and is involved in several important virus-host interactions for viral assembly and restriction, making its synthesis essential for aspects of viral replication. Here, we show that the initiation of translation of the HIV-1 genomic RNA is mediated through both a cap-dependent and an internal ribosome entry site (IRES)-mediated mechanisms. In support of this notion, pr55(Gag) synthesis was maintained at 70% when cap-dependent translation initiation was blocked by the expression of eIF4G- and PABP targeting viral proteases in two in vitro systems and in HIV-1-expressing cells directly infected with poliovirus. While our data reveal that IRES-dependent translation of the viral genomic RNA ensures pr55(Gag) expression, the synthesis of other HIV-1 proteins, including that of pr160(Gag/Pol), Vpr and Tat is suppressed early during progressive poliovirus infection. The data presented herein implies that the unspliced HIV-1 genomic RNA utilizes both cap-dependent and IRES-dependent translation initiation to supply pr55(Gag) for virus assembly and production. PMID:23861855

  2. DHX9/RHA Binding to the PBS-Segment of the Genomic RNA during HIV-1 Assembly Bolsters Virion Infectivity.

    PubMed

    Boeras, Ioana; Song, Zhenwei; Moran, Andrew; Franklin, Jarryd; Brown, William Clay; Johnson, Marc; Boris-Lawrie, Kathleen; Heng, Xiao

    2016-06-01

    Cellular RNA-binding proteins incorporated into virions during human immunodeficiency virus type 1 (HIV-1) assembly promote the replication efficiency of progeny virions. Despite its critical role in bolstering virion infectivity, the molecular basis for the incorporation of DHX9/RNA helicase A (RHA) to virions remains unclear. Here, cell-based experiments demonstrate that the truncation of segments of the HIV-1 5'-untranslated region (5'-UTR) distinct from the core encapsidation sequence eliminated virion incorporation of RHA, indicating that RHA recruitment is mediated by specific interactions with the HIV-1 5'-UTR. In agreement with biological data, isothermal titration calorimetry determined that the dimer conformation of the 5'-UTR binds one RHA molecule per RNA strand, and the interaction is independent of nucleocapsid protein binding. NMR spectra employing a deuterium-labeling approach enabled resolution of the dimeric 5'-UTR in complex with the RHA N-terminal domain. The structure of the large molecular mass complex was dependent on RHA binding to a double-stranded region of the primer binding site (PBS)-segment of the 5'-UTR. A single A-to-C substitution was sufficient to disrupt biophysical conformation and attenuate virion infectivity in cell-based assays. Taken together, our studies demonstrate the structural basis for HIV-1 genomic RNA to recruit beneficial cellular cofactor to virions. The support of progeny virion infectivity by RHA is attributable to structure-dependent binding at the PBS-segment of the HIV-1 5'-UTR during virus assembly. PMID:27107641

  3. 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

  4. 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

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

    PubMed Central

    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 Kd = 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. PMID:19910370

  6. 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. PMID:19910370

  7. Human Papillomavirus Genotype Attribution and Estimation of Preventable Fraction of Anal Intraepithelial Neoplasia Cases Among HIV-Infected Men Who Have Sex With Men

    PubMed Central

    Sahasrabuddhe, Vikrant V.; Castle, Philip E.; Follansbee, Stephen; Borgonovo, Sylvia; Tokugawa, Diane; Schwartz, Lauren M.; Lorey, Thomas S.; LaMere, Brandon J.; Gage, Julia C.; Fetterman, Barbara; Boyle, Sean; Sadorra, Mark; Tang, Scott Dahai; Darragh, Teresa M.; Wentzensen, Nicolas

    2013-01-01

    Background. The prevention of human papillomavirus (HPV)–induced anal cancer in high-risk populations such as human immunodeficiency virus (HIV)-infected men who have sex with men (MSM) remains an urgent priority, given rising incidence rates despite widespread antiretroviral therapy use. Methods. HPV genotypes and anal disease prevalence, by cytology and histopathologic findings, were evaluated among 363 HIV-infected MSM. We modeled fractions of high-grade anal intraepithelial neoplasia (HGAIN) attributable to individual carcinogenic HPV genotypes and estimated the range of the proportion of HGAIN cases potentially preventable by prophylactic HPV vaccines. Results. HPV16 was the most common genotype overall (26.4% of cases) and among HGAIN cases (55%). Prevalence of multiple (≥2) carcinogenic HPV genotypes increased from 30.9% in cases of AIN grade <1 to 76.3% in cases of AIN grade 3 (Ptrend < .001). The fractions of HGAIN cases attributable to carcinogenic HPV16/18 targeted by currently licensed bivalent and quadrivalent HPV vaccines ranged from 12% to 61.5%, and the fractions attributable to carcinogenic HPV16/18/31/33/45/52/58 targeted by an investigational nonavalent HPV vaccine ranged from 39% to 89.4%. Conclusions. Our analytical framework allows estimation of HGAIN cases attributable to individual HPV genotypes in the context of multiple concurrent HPV infections, which are very common among HIV-infected MSM. Our results suggest that licensed and investigational HPV prophylactic vaccines have the potential to prevent a substantial proportion of HGAIN cases in this population. PMID:23162133

  8. Performance Characteristics of the QUANTIPLEX HIV-1 RNA 3.0 Assay for Detection and Quantitation of Human Immunodeficiency Virus Type 1 RNA in Plasma

    PubMed Central

    Erice, Alejo; Brambilla, Donald; Bremer, James; Jackson, J. Brooks; Kokka, Robert; Yen-Lieberman, Belinda; Coombs, Robert W.

    2000-01-01

    The QUANTIPLEX HIV-1 RNA assay, version 3.0 (a branched DNA, version 3.0, assay [bDNA 3.0 assay]), was evaluated by analyzing spiked and clinical plasma samples and was compared with the AMPLICOR HIV-1 MONITOR Ultrasensitive (ultrasensitive reverse transcription-PCR [US-RT-PCR]) method. A panel of spiked plasma samples that contained 0 to 750,000 copies of human immunodeficiency virus type 1 (HIV-1) RNA per ml was tested four times in each of four laboratories (1,344 assays). Negative results (<50 copies/ml) were obtained in 30 of 32 (94%) assays with seronegative samples, 66 of 128 (52%) assays with HIV-1 RNA at 50 copies/ml, and 5 of 128 (4%) assays with HIV-1 RNA at 100 copies/ml. The assay was linear from 100 to 500,000 copies/ml. The within-run standard deviation (SD) of the log10 estimated HIV-1 RNA concentration was 0.08 at 1,000 to 500,000 copies/ml, increased below 1,000 copies/ml, and was 0.17 at 100 copies/ml. Between-run reproducibility at 100 to 500 copies/ml was <0.10 log10 in most comparisons. Interlaboratory differences across runs were ≤0.10 log10 at all concentrations examined. A subset of the panel (25 to 500 copies/ml) was also analyzed by the US-RT-PCR assay. The within-run SD varied inversely with the log10 HIV-1 RNA concentration but was higher than the SD for the bDNA 3.0 assay at all concentrations. Log-log regression analysis indicated that the two methods produced very similar estimates at 100 to 500 copies/ml. In parallel testing of clinical specimens with low HIV-1 RNA levels, 80 plasma samples with <50 copies/ml by the US-RT-PCR assay had <50 copies/ml when they were retested by the bDNA 3.0 assay. In contrast, 11 of 78 (14%) plasma samples with <50 copies/ml by the bDNA 3.0 assay had ≥50 copies/ml when they were retested by the US-RT-PCR assay (median, 86 copies/ml; range, 50 to 217 copies/ml). Estimation of bDNA 3.0 values of <50 copies/ml by extending the standard curve of the assay showed that these samples with discrepant

  9. 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

  10. HIV-1 TAR RNA-binding proteins control TAT activation of translation in Xenopus oocytes.

    PubMed

    Braddock, M; Powell, R; Blanchard, A D; Kingsman, A J; Kingsman, S M

    1993-01-01

    Human immunodeficiency virus (HIV-1) gene expression is activated by the viral TAT protein that interacts with an RNA sequence, TAR, located at the 5' end of all viral mRNAs. TAT functions primarily as a transcriptional activator in mammalian cells. However, in Xenopus oocytes TAT functions primarily as a translational activator. TAR is an RNA structure comprising a partially base-paired stem, a tripyrimidine bulge in the upper stem, and an unpaired six-nucleotide loop. In vitro, TAT binds directly to the bulge with no requirement for the loop. In vivo, however, mutations in the loop abolish TAT activation of transcription and translation, implying a requirement for TAR-binding cellular factors. We now provide genetic evidence for the presence of two TAR-specific cellular factors in Xenopus oocytes. These factors display independent and mutually exclusive interactions with either the loop or the bulge region of TAR. Furthermore, by using in vivo RNA competition assays we show that the cellular factors regulate the accessibility of the TAT binding site. The fact that Xenopus oocytes contain factors that specifically interact with a human viral RNA sequence might indicate that the TAT/TAR interaction is subverting a conserved pathway in the cell. PMID:8422967

  11. 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).

  12. New sensitive one-step real-time duplex PCR method for group A and B HIV-2 RNA load.

    PubMed

    Avettand-Fenoel, Véronique; Damond, Florence; Gueudin, Marie; Matheron, Sophie; Mélard, Adeline; Collin, Gilles; Descamps, Diane; Chaix, Marie-Laure; Rouzioux, Christine; Plantier, Jean-Christophe

    2014-08-01

    The Agence Nationale de Recherche sur le Sida et les hépatites virales (ANRS) previously developed a widely used method for HIV-1 RNA quantification (Biocentric). Here, we report the development of a new specific and sensitive method for HIV-2 RNA quantification, based on an adaptation of the existing HIV-1 protocol. The new test is based on TaqMan one-step reverse transcription-quantitative PCR (qRT-PCR) targeting two conserved consensus regions of HIV-2 (long terminal repeat [LTR] and gag). Analytic performances were determined in three laboratories. Clinical performances were evaluated on 100 plasma samples from HIV-2-infected patients (groups A, B, and H) by comparison with the assay currently used for the ANRS HIV-2 cohort. The specificity was 100%. Sensitivity was 50 copies/ml (cp/ml) and was optimized to 10 cp/ml. The within-run coefficients of variation in the three laboratories varied from 0.54% to 1.61% at 4 log10 copies/ml and from 7.24% to 14.32% at 2 log10 cp/ml. The between-run coefficients of variation varied from 2.28% to 6.43%. Of the 39 clinical samples below 2 log10 in the current assay, the new test improved the detection or quantification of 17 samples, including eight group B samples. For quantifiable samples, similar loads were obtained with the two assays for group A samples. The median difference between the two assays for group B samples was +0.18 but with greater heterogeneity than for group A. The HIV-2 group H sample had similar results with the two assays. This new assay is highly sensitive and accurately quantifies the most prevalent HIV-2 groups. This test will be useful for monitoring low viral loads in HIV-2-infected patients. PMID:24920771

  13. Engineering and Validation of a Vector for Concomitant Expression of Rare Transfer RNA (tRNA) and HIV-1 nef Genes in Escherichia coli.

    PubMed

    Mualif, Siti Aisyah; Teow, Sin-Yeang; Omar, Tasyriq Che; Chew, Yik Wei; Yusoff, Narazah Mohd; Ali, Syed A

    2015-01-01

    Relative ease in handling and manipulation of Escherichia coli strains make them primary candidate to express proteins heterologously. Overexpression of heterologous genes that contain codons infrequently used by E. coli is related with difficulties such as mRNA instability, early termination of transcription and/or translation, deletions and/or misincorporation, and cell growth inhibition. These codon bias -associated problems are addressed by co-expressing ColE1-compatible, rare tRNA expressing helper plasmids. However, this approach has inadequacies, which we have addressed by engineering an expression vector that concomitantly expresses the heterologous protein of interest, and rare tRNA genes in E. coli. The expression vector contains three (argU, ileY, leuW) rare tRNA genes and a useful multiple cloning site for easy in-frame cloning. To maintain the overall size of the parental plasmid vector, the rare tRNA genes replaced the non-essential DNA segments in the vector. The cloned gene is expressed under the control of T7 promoter and resulting recombinant protein has a C-terminal 6His tag for IMAC-mediated purification. We have evaluated the usefulness of this expression vector by expressing three HIV-1 genes namely HIV-1 p27 (nef), HIV-1 p24 (ca), and HIV-1 vif in NiCo21(DE3) E.coli and demonstrated the advantages of using expression vector that concomitantly expresses rare tRNA and heterologous genes. PMID:26147991

  14. The contribution of viral genotype to plasma viral set-point in HIV infection.

    PubMed

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

    2014-05-01

    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

  15. 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

  16. 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

  17. Comparison of Genotypic and Phylogenetic Relationships of Environmental Enterococcus Isolates by BOX-PCR Typing and 16S rRNA Gene Sequencing ▿

    PubMed Central

    Nayak, Bina S.; Badgley, Brian; Harwood, Valerie J.

    2011-01-01

    Environmental Enterococcus spp. were compared by BOX-PCR genotyping and 16S rRNA gene sequencing to clarify the predictive relationship of BOX-PCR fingerprints to species designation. BOX-PCR and 16S rRNA gene relationships agreed for 77% of strains. BOX-PCR provided superior intraspecies discrimination but incorrectly identified some strains to the species level and divided some species into multiple groups. PMID:21622792

  18. 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

  19. Characterization of RNA binding and chaperoning activities of HIV-1 Vif protein. Importance of the C-terminal unstructured tail.

    PubMed

    Sleiman, Dona; Bernacchi, Serena; Xavier Guerrero, Santiago; Brachet, Franck; Larue, Valéry; Paillart, Jean-Christophe; Tisne, Carine

    2014-01-01

    The viral infectivity factor (Vif) is essential for the productive infection and dissemination of HIV-1 in non-permissive cells, containing the cellular anti-HIV defense cytosine deaminases APOBEC3 (A3G and A3F). Vif neutralizes the antiviral activities of the APOBEC3G/F by diverse mechanisms including their degradation through the ubiquitin/proteasome pathway and their translational inhibition. In addition, Vif appears to be an active partner of the late steps of viral replication by interacting with Pr55(Gag), reverse transcriptase and genomic RNA. Here, we expressed and purified full-length and truncated Vif proteins, and analyzed their RNA binding and chaperone properties. First, we showed by CD and NMR spectroscopies that the N-terminal domain of Vif is highly structured in solution, whereas the C-terminal domain remains mainly unfolded. Both domains exhibited substantial RNA binding capacities with dissociation constants in the nanomolar range, whereas the basic unfolded C-terminal domain of Vif was responsible in part for its RNA chaperone activity. Second, we showed by NMR chemical shift mapping that Vif and NCp7 share the same binding sites on tRNA(Lys) 3, the primer of HIV-1 reverse transcriptase. Finally, our results indicate that Vif has potent RNA chaperone activity and provide direct evidence for an important role of the unstructured C-terminal domain of Vif in this capacity. PMID:25144404

  20. Epstein-Barr and cytomegalovirus DNA salivary shedding correlate with long-term plasma HIV RNA detection in HIV-infected men who have sex with men.

    PubMed

    Scaggiante, Renzo; Andreis, Samantha; Basso, Monica; Franchin, Elisa; Franzetti, Marzia; Del Vecchio, Claudia; Torti, Carlo; Mengoli, Carlo; Cruciani, Mario; Sarmati, Loredana; Palù, Giorgio; Parisi, Saverio Giuseppe

    2016-07-01

    The aim of the study was to evaluate cytomegalovirus (CMV) and Epstein-Barr virus (EBV) DNA salivary shedding in HIV-positive men who have sex with men (MSM) and to determine whether viro-immunological parameters and long-term (24 months) plasma HIV RNA (pHIV) detection may predict herpesviruses replication. A total of 193 HIV-positive MSM were consecutively recruited (mean CD4+ cell count 607 cells/mm(3) and mean nadir value 333 cells/mm(3) ); pHIV was analyzed for 24 months prior to saliva sampling: patients were categorized as successfully suppressed (SS) and not suppressed (NS). The EBV viral load was categorized as high viral load (HVL), intermediate (IVL), or low (LVL), CMV DNA as positive or negative. NS patients experienced both herpesviruses detectability more frequently respect to SS patients (P = 0.034); conversely, no salivary shedding was more frequent in SS patients (P = 0.014). HVL EBV was more frequent in NS patients than in SS subjects (P = 0.038 for isolated EBV detection and P = 0.001 when CMV shedding was associated). NS subjects with HVL EBV had a median pHIV of 43,820 copies/ml, significantly higher respect to IVL and LVL patients (P = 0.027 and P = 0.0005, respectively). CMV shedding was mostly associated to EBV shedding. NS patients showed a significantly higher frequency of saliva HVL EBV detection compared to SS patients; moreover, NS patients with HVL EBV had a higher pHIV respect to those with IVL and LVL shedding. Our results suggest that a successful pHIV suppression could reduce the burden of salivary EBV replication and likely the risk of herpesviruses-related cancers. PMID:26636290

  1. 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. PMID:25772204

  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. Identification of a novel HIV-1 TAR RNA bulge binding protein.

    PubMed

    Baker, B; Muckenthaler, M; Vives, E; Blanchard, A; Braddock, M; Nacken, W; Kingsman, A J; Kingsman, S M

    1994-08-25

    The Tat protein binds to TAR RNA to stimulate the expression of the human immunodeficiency virus type 1 (HIV-1) genome. Tat is an 86 amino acid protein that contains a short region of basic residues (aa49-aa57) that are required for RNA binding and TAR is a 59 nucleotide stem-loop with a tripyrimidine bulge in the upper stem. TAR is located at the 5' end of all viral RNAs. In vitro, Tat specifically interacts with TAR by recognising the sequence of the bulge and upper stem, with no requirement for the loop. However, in vivo the loop sequence is critical for activation, implying a requirement for accessory cellular TAR RNA binding factors. A number of TAR binding cellular factors have been identified in cell extracts and various models for the function of these factors have been suggested, including roles as coactivators and inhibitors. We have now identified a novel 38 kD cellular factor that has little general, single-stranded or double-stranded RNA binding activity, but that specifically recognises the bulge and upper stem region of TAR. The protein, referred to as BBP (bulge binding protein), is conserved in mammalian and amphibian cells and in Schizosaccharomyces pombe but is not found in Saccharomyces cerevisiae. BBP is an effective competitive inhibitor of Tat binding to TAR in vitro. Our data suggest that the bulge-stem recognition motif in TAR is used to mediate cellular factor/RNA interactions and indicates that Tat action might be inhibited by such competing reactions in vivo. PMID:8078772

  4. 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 ...

  5. 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

  6. Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device.

    PubMed

    Root, Brian E; Agarwal, Abhishek K; Kelso, David M; Barron, Annelise E

    2011-02-01

    In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the 2-propanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g., serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotides selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g., SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers. PMID:21214255

  7. Purification of HIV RNA from serum using a polymer capture matrix in a microfluidic device

    PubMed Central

    Root, Brian E.; Agarwal, Abhishek K.; Kelso, David M.; Barron, Annelise E.

    2012-01-01

    In this report, we demonstrate the purification of DNA and RNA from a 10% serum sample using an oligonucleotide capture matrix. This approach provides a one-stage, completely aqueous system capable of purifying both RNA and DNA for downstream PCR amplification. The advantages of utilizing the polymer capture matrix method in place of the solid-phase extraction method is that the capture matrix eliminates both guanidine and the isopropanol wash that can inhibit downstream PCR and competition with proteins for the binding sites that can limit the capacity of the device. This method electrophoreses a biological sample (e.g. serum) containing the nucleic acid target through a polymer matrix with covalently bound oligonucleotides. These capture oligonucleotide selectively hybridize and retain the target nucleic acid, while the other biomolecules and reagents (e.g. SDS) pass through the matrix to waste. Following this purification step, the solution can be heated above the melting temperature of the capture sequence to release the target molecule, which is then electrophoresed to a recovery chamber for subsequent PCR amplification. We demonstrate that the device can be applied to purify both DNA and RNA from serum. The gag region of HIV at a starting concentration of 37.5 copies per microliter was successfully purified from a 10% serum sample demonstrating the applicability of this method to detect viruses present in low copy numbers. PMID:21214255

  8. IL-21 induces antiviral microRNA-29 in CD4 T cells to limit HIV-1 infection.

    PubMed

    Adoro, Stanley; Cubillos-Ruiz, Juan R; Chen, Xi; Deruaz, Maud; Vrbanac, Vladimir D; Song, Minkyung; Park, Suna; Murooka, Thomas T; Dudek, Timothy E; Luster, Andrew D; Tager, Andrew M; Streeck, Hendrik; Bowman, Brittany; Walker, Bruce D; Kwon, Douglas S; Lazarevic, Vanja; Glimcher, Laurie H

    2015-01-01

    Initial events after exposure determine HIV-1 disease progression, underscoring a critical need to understand host mechanisms that interfere with initial viral replication. Although associated with chronic HIV-1 control, it is not known whether interleukin-21 (IL-21) contributes to early HIV-1 immunity. Here we take advantage of tractable primary human lymphoid organ aggregate cultures to show that IL-21 directly suppresses HIV-1 replication, and identify microRNA-29 (miR-29) as an antiviral factor induced by IL-21 in CD4 T cells. IL-21 promotes transcription of all miR-29 species through STAT3, whose binding to putative regulatory regions within the MIR29 gene is enriched by IL-21 signalling. Notably, exogenous IL-21 limits early HIV-1 infection in humanized mice, and lower viremia in vivo is associated with higher miR-29 expression. Together, these findings reveal a novel antiviral IL-21-miR-29 axis that promotes CD4 T-cell-intrinsic resistance to HIV-1 infection, and suggest a role for IL-21 in initial HIV-1 control in vivo. PMID:26108174

  9. 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

  10. 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.

  11. Internal translation initiation from HIV-1 transcripts is conferred by a common RNA structure

    PubMed Central

    Plank, Terra-Dawn M; Whitehurst, James T; Cencic, Regina; Pelletier, Jerry; Kieft, Jeffrey S

    2014-01-01

    Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG. PMID:26779399

  12. Internal translation initiation from HIV-1 transcripts is conferred by a common RNA structure.

    PubMed

    Plank, Terra-Dawn M; Whitehurst, James T; Cencic, Regina; Pelletier, Jerry; Kieft, Jeffrey S

    2014-01-01

    Alternative splicing of the human immunodeficiency virus 1 (HIV-1) RNA transcripts produces mRNAs encoding nine different viral proteins. The leader of each contains a common non-coding exon at the 5' end. Previous studies showed that the leaders from the common exon-containing transcripts gag, nef, vif, vpr and vpu can direct protein synthesis through internal ribosome entry sites (IRESs) with varying efficiencies. Here we explored whether the common exon acts as an IRES element in the context of all the 5' leaders or if each harbors a distinct IRES. We also explored the relationship between the IRESs and initiation codon selection. We find that the common exon adopts a similar conformation in every leader we explored and that the sequence and structure is required for IRES activity. We also find that each leader uses a scanning mechanism for start codon identification. Together, our data point to a model in which the common exon on HIV-1 transcripts acts as the ribosome landing pad, recruiting preinitiation complexes upstream of the initiation codon, followed by scanning to each transcript's initiator AUG. PMID:26779399

  13. Cyclic Peptide Inhibitors of HIV-1 Capsid-Human Lysyl-tRNA Synthetase Interaction

    PubMed Central

    2012-01-01

    The human immunodeficiency virus type 1 (HIV-1) capsid protein (CA) plays a critical role in the viral life cycle. The C-terminal domain (CTD) of CA binds to human lysyl-tRNA synthetase (hLysRS), and this interaction facilitates packaging of host cell tRNALys,3, which serves as the primer for reverse transcription. Here, we report the library synthesis, high-throughput screening, and identification of cyclic peptides (CPs) that bind HIV-1 CA. Scrambling or single-residue changes of the selected peptide sequences eliminated binding, suggesting a sequence-specific mode of interaction. Two peptides (CP2 and CP4) subjected to detailed analysis also inhibited hLysRS/CA interaction in vitro. Nuclear magnetic resonance spectroscopy and mutagenesis studies revealed that both CPs bind to a site proximal to helix 4 of the CA-CTD, which is the known site of hLysRS interaction. These results extend the current repertoire of CA-binding molecules to a new class of peptides targeting a novel site with potential for development into novel antiviral agents. PMID:22276994

  14. Viroids: From Genotype to Phenotype Just Relying on RNA Sequence and Structural Motifs

    PubMed Central

    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. PMID:22719735

  15. Heterogeneous loss of HIV transcription and proviral DNA from 8E5/LAV lymphoblastic leukemia cells revealed by RNA FISH:FLOW analyses.

    PubMed

    Wilburn, Kaley M; Mwandumba, Henry C; Jambo, Kondwani C; Boliar, Saikat; Solouki, Sabrina; Russell, David G; Gludish, David W

    2016-01-01

    8E5/LAV cells harbor a single HIV provirus, and are used frequently to generate standards for HIV genome quantification. Using flow cytometry-based in situ mRNA hybridization validated by qPCR, we find that different batches of 8E5 cells contain varying numbers of cells lacking viral mRNA and/or viral genomes. These findings raise concerns for studies employing 8E5 cells for quantitation, and highlight the value of mRNA FISH and flow cytometry in the detection and enumeration of HIV-positive cells. PMID:27515378

  16. 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

  17. 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

  18. Validation of an ultrasensitive digital droplet PCR assay for HIV-2 plasma RNA quantification

    PubMed Central

    Ruelle, Jean; Yfantis, Vasilieios; Duquenne, Armelle; Goubau, Patrick

    2014-01-01

    Introduction Low or undetectable plasma viral load (VL) using current qPCR assays is common for HIV-2 patients. Digital PCR is an emerging technology enabling more precision and reproducibility than qPCR at low DNA/RNA copy numbers. Available data related to digital droplet PCR (ddPCR, Bio-Rad) underscore issues linked to the threshold definition of positivity, coupled to the specificity of low copy results [1]. Materials and Methods A RT-PCR protocol was set up using the One-Step RT-ddPCR Kit for Probes on the QX200 platform (Bio-Rad, Hercules, CA) in an accredited environment (ISO15189:2012 norm). Parameters tested were in line with the digital MIQE guidelines [2]. Inter-run coefficient of variation (CV) was established using synthetic RNA controls diluted in HIV-negative plasma. The ddPCR assay was compared to a qRT-PCR previously used in routine (LOQ 50 cop/mL [3]) using 46 clinical samples and the NIBSC international HIV-2 RNA standard. Results The optimal PCR efficiency and the best separation between positive and negative droplets were obtained with a mixture containing 0.5 mM manganese acetate, 700 nM primers and 250 nM of the 5'FAM-probe. Using a manual threshold to define positivity, 7.74% of negative controls (n=168) were scored as positive due to one positive droplet. The presence of two positive droplets or more was not observed for negative controls. Serial dilutions of a positive control showed excellent linearity (R2=0.999) and enabled us to define a limit of quantification of two positives droplets, which corresponds to 0.14 copies/μL in the reaction mixture and to seven copies per mL of plasma. The inter-run coefficient of variation was 3.37% at a mean value of 4,468 cop/mL, 19.59% at 416 cop/mL and 32.28% at 8 cop/mL. The NIBSC standard of 1,000 IU was quantified 1,400 copies by ddPCR and close to 5,000 copies by qPCR (delta log superior to 0.5). Among 46 clinical samples, 22 were undetectable with both qPCR and ddPCR, 12 were detected with both

  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.

  20. Endogenous MCM7 MicroRNA Cluster as a Novel Platform to Multiplex Small Interfering and Nucleolar RNAs for Combinational HIV-1 Gene Therapy

    PubMed Central

    Chung, Janet; Zhang, Jane; Li, Haitang; Ouellet, Dominique L.; DiGiusto, David L.

    2012-01-01

    Abstract Combinational therapy with small RNA inhibitory agents against multiple viral targets allows efficient inhibition of viral production by controlling gene expression at critical time points. Here we explore combinations of different classes of therapeutic anti-HIV-1 RNAs expressed from within the context of an intronic MCM7 (minichromosome maintenance complex component-7) platform that naturally harbors 3 microRNAs (miRNAs). We replaced the endogenous miRNAs with anti-HIV small RNAs, including small interfering RNAs (siRNAs) targeting HIV-1 tat and rev messages that function to induce post-transcriptional gene silencing by the RNA interference pathway, a nucleolar-localizing RNA ribozyme that targets the conserved U5 region of HIV-1 transcripts for degradation, and finally nucleolar trans-activation response (TAR) and Rev-binding element (RBE) RNA decoys designed to sequester HIV-1 Tat and Rev proteins inside the nucleolus. We demonstrate the versatility of the MCM7 platform in expressing and efficiently processing the siRNAs as miRNA mimics along with nucleolar small RNAs. Furthermore, three of the combinatorial constructs tested potently suppressed viral replication during a 1-month HIV challenge, with greater than 5-log inhibition compared with untransduced, HIV-1-infected CEM T lymphocytes. One of the most effective constructs contains an anti-HIV siRNA combined with a nucleolar-localizing U5 ribozyme and TAR decoy. This represents the first efficacious example of combining Drosha-processed siRNAs with small nucleolar ribonucleoprotein (snoRNP)-processed nucleolar RNA chimeras from a single intron platform for effective inhibition of viral replication. Moreover, we demonstrated enrichment/selection for cells expressing levels of the antiviral RNAs that provide optimal inhibition under the selective pressure of HIV. The combinations of si/snoRNAs represent a new paradigm for combinatorial RNA-based gene therapy applications. PMID:22834872

  1. 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

  2. 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

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

    DOE PAGESBeta

    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

  4. Inhibition of HIV-1 gene expression by novel macrophage-tropic DNA enzymes targeted to cleave HIV-1 TAT/Rev RNA.

    PubMed Central

    Unwalla, H; Banerjea, A C

    2001-01-01

    Many regions of the HIV-1 genome have been targeted in earlier studies by RNA-cleaving DNA enzymes possessing the 10-23 catalytic motif, and efficient inhibition of HIV-1 gene expression was reported. All these studies employed charged synthetic lipids to introduce the catalytic DNA into the mammalian cells, which severely limits its practical application and usefulness in vivo. Taking advantage of the ability of G residues to interact directly with the scavenger receptors on the macrophages, we synthesized a DNA enzyme 5970 that contained 10 G residues at the 3' end. With the aim of improving the intracellular stability of the DNA enzyme 5970, we added two short stretches of stem-loop structures that were 12 bases long on either side of the DNA enzyme 5970. DNA enzyme 5970 without the poly-G tracts cleaved the synthetic RNA of HIV-1 TAT/Rev, two important regulatory proteins of HIV, very efficiently in a sequence-specific manner. Addition of 10 G residues at the 3' end of the DNA enzyme affected the cleavage efficiency only marginally whereas the same DNA enzyme with stem-loop structures on either end was significantly less efficient. The DNA enzyme with the poly-G tract at its 3' end was taken up specifically by a human macrophage-specific cell line directly in the absence of Lipofectin and was also able to inhibit HIV-1 gene expression in a transient-expression system as well as when challenged with the virus. The potential applications of these novel macrophage-tropic DNA enzymes are discussed. PMID:11415445

  5. 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

  6. 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

  7. Use of Human Papillomavirus DNA, E6/E7 mRNA, and p16 Immunocytochemistry to Detect and Predict anal High-Grade Squamous Intraepithelial Lesions in HIV-Positive and HIV-Negative Men Who Have Sex with Men

    PubMed Central

    Phanuphak, Nittaya; Teeratakulpisarn, Nipat; Keelawat, Somboon; Pankam, Tippawan; Barisri, Jiranuwat; Triratanachat, Surang; Deesua, Amornrat; Rodbamrung, Piyanee; Wongsabut, Jiratchaya; Tantbirojn, Patou; Numto, Saranya; Ruangvejvorachai, Preecha; Phanuphak, Praphan; Palefsky, Joel M.; Ananworanich, Jintanat; Kerr, Stephen J.

    2013-01-01

    Background Men who have sex with men (MSM) are at high risk of having anal cancer. Anal high-grade squamous intraepithelial lesion (HSIL) is the precursor of anal cancer. We explored the use of different biomarkers associated with human papillomavirus (HPV) infection and HPV-mediated cell transformation to detect and predict HSIL among HIV-positive and HIV-negative MSM. Methodology/Principal Findings A total of 123 HIV-positive and 123 HIV-negative MSM were enrolled and followed for 12 months. High-resolution anoscopy (HRA) with biopsies were performed at every visit along with anal sample collection for cytology, high-risk HPV DNA genotyping, HPV E6/E7 mRNA, and p16 immunocytochemistry. Performance characteristics and area under the receiver operator characteristics curve were calculated for these biomarkers at baseline, and Cox regression compared the usefulness of these biomarkers in predicting incident HSIL. High-risk HPV DNA, E6/E7 mRNA, and p16 immunocytochemistry each identified 43–46% of MSM whose baseline test positivity would trigger HRA referral. E6/E7 mRNA had the highest sensitivity (64.7%) and correctly classified the highest number of prevalent HSIL cases. With the exception of p16 immunochemistry, most tests showed significant increases in sensitivity but decreases specificity versus anal cytology, while the overall number of correctly classified cases was not significantly different. Baseline or persistent type 16 and/or 18 HPV DNA was the only test significantly predicting incident histologic HSIL within 12 months in models adjusted for HIV status and low-grade squamous intraepithelial lesions at baseline. Conclusions/Significance Countries with a high HIV prevalence among MSM and limited HRA resources may consider using biomarkers to identify individuals at high risk of HSIL. E6/E7 mRNA had the highest sensitivity for prevalent HSIL detection regardless of HIV status, whereas type 16 and/or 18 HPV DNA performed best in predicting development of

  8. 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

  9. 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. PMID:26196543

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

    PubMed Central

    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. PMID:26196543