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Sample records for 2-ltr hiv-1 dna

  1. Impact of the DNA extraction method on 2-LTR DNA circle recovery from HIV-1 infected cells

    PubMed Central

    Badralmaa, Yunden; Natarajan, Ven

    2013-01-01

    Detection of episomal 2-LTR DNA circles is used as a marker for the ongoing virus replication in patients infected with HIV-1, and efficient extraction of episomal DNA is critical for accurate estimation of the 2-LTR circles. The impact of different methods of DNA extraction on the recovery of 2-LTR circles was compared using mitochondrial DNA extracted as an internal control. The bacterial plasmid DNA isolation method extracted less than 10% of cellular DNA, 40% of mitochondrial DNA and 12-20 % of the input 2-LTR DNA. The total DNA isolation method recovered about 70% of mitochondrial DNA and 45% of the input 2-LTR DNA. The total nucleic acid isolation method recovered 90% of mitochondrial DNA and 60% of the input 2-LTR DNA. Similar results were obtained when the DNA was extracted from HIV-1 infected cells. Plasmid DNA isolation could not distinguish between 12 and 25 copies of 2-LTR DNA per million cells, whereas the total nucleic acid isolation showed a consistent and statistically significant difference between 12 and 25 copies. In conclusion, the total nucleic acid isolation method is more efficient than the plasmid DNA isolation method in recovering mitochondrial DNA and 2-LTR DNA circles from HIV-1 infected cells. PMID:23773807

  2. Low-Level Detection and Quantitation of Cellular HIV-1 DNA and 2-LTR Circles Using Droplet Digital PCR

    PubMed Central

    Henrich, Timothy J.; Gallien, Sebastien; Li, Jonathan Z.; Pereyra, Florencia; Kuritzkes, Daniel R.

    2012-01-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia was also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  3. Low-level detection and quantitation of cellular HIV-1 DNA and 2-LTR circles using droplet digital PCR.

    PubMed

    Henrich, Timothy J; Gallien, Sebastien; Li, Jonathan Z; Pereyra, Florencia; Kuritzkes, Daniel R

    2012-12-01

    Droplet digital PCR (ddPCR) is an emerging nucleic acid detection method that provides absolute quantitations of target sequences without relying on the use of standard curves. The ability of ddPCR to detect and quantitate total HIV-1 DNA and 2-LTR circles from a panel of patients on and off antiviral therapy was evaluated compared to established real-time (RT)-PCR methods. To calculate the dynamic range of ddPCR for HIV-1 DNA and 2-LTR circles, serial dilutions of DNA amplicons or episomes were determined by ddPCR as well as with RT-PCR. HIV-1 DNA from 3 viremic patients and 4 patients on suppressive antiretroviral therapy, and 2-LTR circles from 3 patients with low-level viremia were also quantitated. Copy numbers determined by ddPCR of serial dilutions of HIV-1 or human CCR5 DNA amplicon standards were comparable to nominal input copy number. The sensitivity of ddPCR to detect HIV-1 or CCR5 DNA was similar to that of RT-PCR. Low levels of 2-LTR circles were detected in samples from all 3 patients by both ddPCR and RT-PCR. ddPCR is a promising novel technology for the study of HIV-1 reservoirs and persistence, but further optimization of this novel technology would enhance the detection of very low-level viral genetic targets. PMID:22974526

  4. Accurate episomal HIV 2-LTR circles quantification using optimized DNA isolation and droplet digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2014-01-01

    Introduction In HIV-infected patients on combination antiretroviral therapy (cART), the detection of episomal HIV 2-LTR circles is a potential marker for ongoing viral replication. Quantification of 2-LTR circles is based on quantitative PCR or more recently on digital PCR assessment, but is hampered due to its low abundance. Sample pre-PCR processing is a critical step for 2-LTR circles quantification, which has not yet been sufficiently evaluated in patient derived samples. Materials and Methods We compared two sample processing procedures to more accurately quantify 2-LTR circles using droplet digital PCR (ddPCR). Episomal HIV 2-LTR circles were either isolated by genomic DNA isolation or by a modified plasmid DNA isolation, to separate the small episomal circular DNA from chromosomal DNA. This was performed in a dilution series of HIV-infected cells and HIV-1 infected patient derived samples (n=59). Samples for the plasmid DNA isolation method were spiked with an internal control plasmid. Results Genomic DNA isolation enables robust 2-LTR circles quantification. However, in the lower ranges of detection, PCR inhibition caused by high genomic DNA load substantially limits the amount of sample input and this impacts sensitivity and accuracy. Moreover, total genomic DNA isolation resulted in a lower recovery of 2-LTR templates per isolate, further reducing its sensitivity. The modified plasmid DNA isolation with a spiked reference for normalization was more accurate in these low ranges compared to genomic DNA isolation. A linear correlation of both methods was observed in the dilution series (R2=0.974) and in the patient derived samples with 2-LTR numbers above 10 copies per million peripheral blood mononuclear cells (PBMCs), (R2=0.671). Furthermore, Bland–Altman analysis revealed an average agreement between the methods within the 27 samples in which 2-LTR circles were detectable with both methods (bias: 0.3875±1.2657 log10). Conclusions 2-LTR circles

  5. Accurate quantification of episomal HIV-1 two-long terminal repeat circles by use of optimized DNA isolation and droplet digital PCR.

    PubMed

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; Vandekerckhove, Linos; De Spiegelaere, Ward

    2015-02-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. PMID:25502524

  6. Accurate Quantification of Episomal HIV-1 Two-Long Terminal Repeat Circles by Use of Optimized DNA Isolation and Droplet Digital PCR

    PubMed Central

    Malatinkova, Eva; Kiselinova, Maja; Bonczkowski, Pawel; Trypsteen, Wim; Messiaen, Peter; Vermeire, Jolien; Verhasselt, Bruno; Vervisch, Karen; De Spiegelaere, Ward

    2014-01-01

    Episomal HIV-1 two-long terminal repeat (2-LTR) circles are considered markers for ongoing viral replication. Two sample processing procedures were compared to accurately quantify 2-LTR in patients by using droplet digital PCR (ddPCR). Here, we show that plasmid isolation with a spiked non-HIV plasmid for normalization enables more accurate 2-LTR quantification than genomic DNA isolation. PMID:25502524

  7. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth.

    PubMed

    Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

    2016-03-01

    The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4-2.6) between time point 1 and 2; and median of 31 days (IQR: 28-36) between time point 2 and 3. Patients were median of 6 years (IQR: 3-12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2-8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the replication

  8. Integrated and Total HIV-1 DNA Predict Ex Vivo Viral Outgrowth

    PubMed Central

    Kiselinova, Maja; De Spiegelaere, Ward; Buzon, Maria Jose; Malatinkova, Eva; Lichterfeld, Mathias; Vandekerckhove, Linos

    2016-01-01

    The persistence of a reservoir of latently infected CD4 T cells remains one of the major obstacles to cure HIV. Numerous strategies are being explored to eliminate this reservoir. To translate these efforts into clinical trials, there is a strong need for validated biomarkers that can monitor the reservoir over time in vivo. A comprehensive study was designed to evaluate and compare potential HIV-1 reservoir biomarkers. A cohort of 25 patients, treated with suppressive antiretroviral therapy was sampled at three time points, with median of 2.5 years (IQR: 2.4–2.6) between time point 1 and 2; and median of 31 days (IQR: 28–36) between time point 2 and 3. Patients were median of 6 years (IQR: 3–12) on ART, and plasma viral load (<50 copies/ml) was suppressed for median of 4 years (IQR: 2–8). Total HIV-1 DNA, unspliced (us) and multiply spliced HIV-1 RNA, and 2LTR circles were quantified by digital PCR in peripheral blood, at 3 time points. At the second time point, a viral outgrowth assay (VOA) was performed, and integrated HIV-1 DNA and relative mRNA expression levels of HIV-1 restriction factors were quantified. No significant change was found for long- and short-term dynamics of all HIV-1 markers tested in peripheral blood. Integrated HIV-1 DNA was associated with total HIV-1 DNA (p<0.001, R² = 0.85), us HIV-1 RNA (p = 0.029, R² = 0.40), and VOA (p = 0.041, R2 = 0.44). Replication-competent virus was detected in 80% of patients by the VOA and it correlated with total HIV-1 DNA (p = 0.039, R² = 0.54). The mean quantification difference between Alu-PCR and VOA was 2.88 log10, and 2.23 log10 between total HIV-1 DNA and VOA. The levels of usHIV-1 RNA were inversely correlated with mRNA levels of several HIV-1 restriction factors (TRIM5α, SAMHD1, MX2, SLFN11, pSIP1). Our study reveals important correlations between the viral outgrowth and total and integrated HIV-1 DNA measures, suggesting that the total pool of HIV-1 DNA may predict the size of the

  9. 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).

  10. Human Macrophages Support Persistent Transcription From Unintegrated HIV-1 DNA

    PubMed Central

    Kelly, Jeremy; Beddall, Margaret H.; Yu, Dongyang; Iyer, Subashini R.; Marsh, Jon W.; Wu, Yuntao

    2008-01-01

    Retroviruses require integration of their RNA genomes for both stability and productive viral replication. In HIV infection of non-dividing, resting CD4 T cells, where integration is greatly impeded, the reverse transcribed HIV DNA has limited biological activity and a short half-life. In metabolically active and proliferating T cells unintegrated DNA rapidly diminishes with cell division. HIV also infects the non-dividing, but metabolically active macrophage population. In an in vitro examination of HIV infection of macrophages, we find that unintegrated viral DNA not only has an unusual stability, but also maintains biological activity. The unintegrated linear DNA, 1-LTR, and 2-LTR circles are stable for at least 30 days. Additionally there is persistent viral gene transcription, which is selective and skewed towards viral early genes such as nef and tat with highly diminished rev and vif. One viral early gene product Nef was measurably synthesized. We also find that independent of integration, the HIV infection process in macrophages leads to generation of numerous chemokines. PMID:18054979

  11. HIV-1 Integrase-DNA Recognition Mechanisms

    PubMed Central

    Kessl, Jacques J.; McKee, Christopher J.; Eidahl, Jocelyn O.; Shkriabai, Nikolozi; Katz, Ari; Kvaratskhelia, Mamuka

    2009-01-01

    Integration of a reverse transcribed DNA copy of the HIV viral genome into the host chromosome is essential for virus replication. This process is catalyzed by the virally encoded protein integrase. The catalytic activities, which involve DNA cutting and joining steps, have been recapitulated in vitro using recombinant integrase and synthetic DNA substrates. Biochemical and biophysical studies of these model reactions have been pivotal in advancing our understanding of mechanistic details for how IN interacts with viral and target DNAs, and are the focus of the present review. PMID:21994566

  12. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants

    PubMed Central

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M.; Sullivan, John L.; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  13. Early Combination Antiretroviral Therapy Limits Exposure to HIV-1 Replication and Cell-Associated HIV-1 DNA Levels in Infants.

    PubMed

    McManus, Margaret; Mick, Eric; Hudson, Richard; Mofenson, Lynne M; Sullivan, John L; Somasundaran, Mohan; Luzuriaga, Katherine

    2016-01-01

    The primary aim of this study was to measure HIV-1 persistence following combination antiretroviral therapy (cART) in infants and children. Peripheral blood mononuclear cell (PBMC) HIV-1 DNA was quantified prior to and after 1 year of cART in 30 children, stratified by time of initiation (early, age <3 months, ET; late, age >3 months-2 years, LT). Pre-therapy PBMC HIV-1 DNA levels correlated with pre-therapy plasma HIV-1 levels (r = 0.59, p<0.001), remaining statistically significant (p = 0.002) after adjustment for prior perinatal antiretroviral exposure and age at cART initiation. PBMC HIV-1 DNA declined significantly after 1 year of cART (Overall: -0.91±0.08 log10 copies per million PBMC, p<0.001; ET: -1.04±0.11 log10 DNA copies per million PBMC, p<0.001; LT: -0.74 ±0.13 log10 DNA copies per million PBMC, p<0.001) but rates of decline did not differ significantly between ET and LT. HIV-1 replication exposure over the first 12 months of cART, estimated as area-under-the-curve (AUC) of circulating plasma HIV-1 RNA levels, was significantly associated with PBMC HIV-1 DNA at one year (r = 0.51, p = 0.004). In 21 children with sustained virologic suppression after 1 year of cART, PBMC HIV-1 DNA levels continued to decline between years 1 and 4 (slope -0.21 log10 DNA copies per million PBMC per year); decline slopes did not differ significantly between ET and LT. PBMC HIV-1 DNA levels at 1 year and 4 years of cART correlated with age at cART initiation (1 year: p = 0.04; 4 years: p = 0.03) and age at virologic control (1 and 4 years, p = 0.02). Altogether, these data indicate that reducing exposure to HIV-1 replication and younger age at cART initiation are associated with lower HIV-1 DNA levels at and after one year of age, supporting the concept that HIV-1 diagnosis and cART initiation in infants should occur as early as possible. PMID:27104621

  14. 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

  15. HIV-1 DNA predicts disease progression and post-treatment virological control.

    PubMed

    Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan; Koelsch, Kersten K; Kelleher, Anthony D; Phillips, Rodney E; Frater, John

    2014-01-01

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. PMID:25217531

  16. HIV-1 DNA predicts disease progression and post-treatment virological control

    PubMed Central

    Williams, James P; Hurst, Jacob; Stöhr, Wolfgang; Robinson, Nicola; Brown, Helen; Fisher, Martin; Kinloch, Sabine; Cooper, David; Schechter, Mauro; Tambussi, Giuseppe; Fidler, Sarah; Carrington, Mary; Babiker, Abdel; Weber, Jonathan

    2014-01-01

    In HIV-1 infection, a population of latently infected cells facilitates viral persistence despite antiretroviral therapy (ART). With the aim of identifying individuals in whom ART might induce a period of viraemic control on stopping therapy, we hypothesised that quantification of the pool of latently infected cells in primary HIV-1 infection (PHI) would predict clinical progression and viral replication following ART. We measured HIV-1 DNA in a highly characterised randomised population of individuals with PHI. We explored associations between HIV-1 DNA and immunological and virological markers of clinical progression, including viral rebound in those interrupting therapy. In multivariable analyses, HIV-1 DNA was more predictive of disease progression than plasma viral load and, at treatment interruption, predicted time to plasma virus rebound. HIV-1 DNA may help identify individuals who could safely interrupt ART in future HIV-1 eradication trials. Clinical trial registration: ISRCTN76742797 and EudraCT2004-000446-20 DOI: http://dx.doi.org/10.7554/eLife.03821.001 PMID:25217531

  17. DNA Triplex-Based Complexes Display Anti-HIV-1-Cell Fusion Activity.

    PubMed

    Xu, Liang; Zhang, Tao; Xu, Xiaoyu; Chong, Huihui; Lai, Wenqing; Jiang, Xifeng; Wang, Chao; He, Yuxian; Liu, Keliang

    2015-08-01

    DNA triplexes with hydrophobic modifications were designed and evaluated for their activity as inhibitors of the cell fusion of human immunodeficiency virus type 1 (HIV-1). Triplex inhibitors displayed low micromolar activities in the cell-cell fusion assay and nanomolar activities in the anti-HIV-1 pseudovirus test. Helix structure and the presence of sufficient numbers of hydrophobic regions were essential for the antifusion activity. Results from native polyacrylamide gel electrophoresis and a fluorescent resonance energy transfer-based inhibitory assay indicated that these triplexes may interact with the primary pocket at the glycoprotein 41 (gp41) N-heptad repeat, thereby inhibiting formation of the HIV-1 gp41 6-helical bundle. Triplex-based complexes may represent a novel category of HIV-1 inhibitors in anti-HIV-1 drug discovery. PMID:26192705

  18. Rapid detection of HIV-1 proviral DNA for early infant diagnosis using recombinase polymerase amplification.

    PubMed

    Boyle, David S; Lehman, Dara A; Lillis, Lorraine; Peterson, Dylan; Singhal, Mitra; Armes, Niall; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie

    2013-01-01

    Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes. PMID:23549916

  19. Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification

    PubMed Central

    Boyle, David S.; Lehman, Dara A.; Lillis, Lorraine; Peterson, Dylan; Singhal, Mitra; Armes, Niall; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie

    2013-01-01

    ABSTRACT Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes. PMID:23549916

  20. HIV-1 and HIV-2 exhibit divergent interactions with HLTF and UNG2 DNA repair proteins.

    PubMed

    Hrecka, Kasia; Hao, Caili; Shun, Ming-Chieh; Kaur, Sarabpreet; Swanson, Selene K; Florens, Laurence; Washburn, Michael P; Skowronski, Jacek

    2016-07-01

    HIV replication in nondividing host cells occurs in the presence of high concentrations of noncanonical dUTP, apolipoprotein B mRNA-editing, enzyme-catalytic, polypeptide-like 3 (APOBEC3) cytidine deaminases, and SAMHD1 (a cell cycle-regulated dNTP triphosphohydrolase) dNTPase, which maintains low concentrations of canonical dNTPs in these cells. These conditions favor the introduction of marks of DNA damage into viral cDNA, and thereby prime it for processing by DNA repair enzymes. Accessory protein Vpr, found in all primate lentiviruses, and its HIV-2/simian immunodeficiency virus (SIV) SIVsm paralogue Vpx, hijack the CRL4(DCAF1) E3 ubiquitin ligase to alleviate some of these conditions, but the extent of their interactions with DNA repair proteins has not been thoroughly characterized. Here, we identify HLTF, a postreplication DNA repair helicase, as a common target of HIV-1/SIVcpz Vpr proteins. We show that HIV-1 Vpr reprograms CRL4(DCAF1) E3 to direct HLTF for proteasome-dependent degradation independent from previously reported Vpr interactions with base excision repair enzyme uracil DNA glycosylase (UNG2) and crossover junction endonuclease MUS81, which Vpr also directs for degradation via CRL4(DCAF1) E3. Thus, separate functions of HIV-1 Vpr usurp CRL4(DCAF1) E3 to remove key enzymes in three DNA repair pathways. In contrast, we find that HIV-2 Vpr is unable to efficiently program HLTF or UNG2 for degradation. Our findings reveal complex interactions between HIV-1 and the DNA repair machinery, suggesting that DNA repair plays important roles in the HIV-1 life cycle. The divergent interactions of HIV-1 and HIV-2 with DNA repair enzymes and SAMHD1 imply that these viruses use different strategies to guard their genomes and facilitate their replication in the host. PMID:27335459

  1. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities.

    PubMed

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  2. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    NASA Astrophysics Data System (ADS)

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1.

  3. Creation of chimeric human/rabbit APOBEC1 with HIV-1 restriction and DNA mutation activities

    PubMed Central

    Ikeda, Terumasa; Ong, Eugene Boon Beng; Watanabe, Nobumoto; Sakaguchi, Nobuo; Maeda, Kazuhiko; Koito, Atsushi

    2016-01-01

    APOBEC1 (A1) proteins from lagomorphs and rodents have deaminase-dependent restriction activity against HIV-1, whereas human A1 exerts a negligible effect. To investigate these differences in the restriction of HIV-1 by A1 proteins, a series of chimeric proteins combining rabbit and human A1s was constructed. Homology models of the A1s indicated that their activities derive from functional domains that likely act in tandem through a dimeric interface. The C-terminal region containing the leucine-rich motif and the dimerization domains of rabbit A1 is important for its anti-HIV-1 activity. The A1 chimeras with strong anti-HIV-1 activity were incorporated into virions more efficiently than those without anti-HIV-1 activity, and exhibited potent DNA-mutator activity. Therefore, the C-terminal region of rabbit A1 is involved in both its packaging into the HIV-1 virion and its deamination activity against both viral cDNA and genomic RNA. This study identifies the novel molecular mechanism underlying the target specificity of A1. PMID:26738439

  4. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

    PubMed

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients. PMID:25332688

  5. Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination

    PubMed Central

    Zhang, Shi-Meng; Zhang, He; Yang, Tian-Yi; Ying, Tian-Yi; Yang, Pei-Xiang; Liu, Xiao-Dan; Tang, Sheng-Jian; Zhou, Ping-Kun

    2014-01-01

    HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤4 µg/ml) and stimulates CSR at high concentrations (≥8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients. PMID:25332688

  6. Excision of HIV-1 DNA by gene editing: a proof-of-concept in vivo study.

    PubMed

    Kaminski, R; Bella, R; Yin, C; Otte, J; Ferrante, P; Gendelman, H E; Li, H; Booze, R; Gordon, J; Hu, W; Khalili, K

    2016-08-01

    A CRISPR/Cas9 gene editing strategy has been remarkable in excising segments of integrated HIV-1 DNA sequences from the genome of latently infected human cell lines and by introducing InDel mutations, suppressing HIV-1 replication in patient-derived CD4+ T-cells, ex vivo. Here, we employed a short version of the Cas9 endonuclease, saCas9, together with a multiplex of guide RNAs (gRNAs) for targeting the viral DNA sequences within the 5'-LTR and the Gag gene for removing critically important segments of the viral DNA in transgenic mice and rats encompassing the HIV-1 genome. Tail-vein injection of transgenic mice with a recombinant Adeno-associated virus 9 (rAAV9) vector expressing saCas9 and the gRNAs, rAAV:saCas9/gRNA, resulted in the cleavage of integrated HIV-1 DNA and excision of a 978 bp DNA fragment spanning between the LTR and Gag gene in the spleen, liver, heart, lung and kidney as well as in the circulating lymphocytes. Retro-orbital inoculation of rAAV9:saCas9/gRNA in transgenic rats eliminated a targeted segment of viral DNA and substantially decreased the level of viral gene expression in circulating blood lymphocytes. The results from the proof-of-concept studies, for the first time, demonstrate the in vivo eradication of HIV-1 DNA by CRISPR/Cas9 on delivery by an rAAV9 vector in a range of cells and tissues that harbor integrated copies of viral DNA. PMID:27194423

  7. Structural Insights into the HIV-1 Minus-strand Strong-stop DNA.

    PubMed

    Chen, Yingying; Maskri, Ouerdia; Chaminade, Françoise; René, Brigitte; Benkaroun, Jessica; Godet, Julien; Mély, Yves; Mauffret, Olivier; Fossé, Philippe

    2016-02-12

    An essential step of human immunodeficiency virus type 1 (HIV-1) reverse transcription is the first strand transfer that requires base pairing of the R region at the 3'-end of the genomic RNA with the complementary r region at the 3'-end of minus-strand strong-stop DNA (ssDNA). HIV-1 nucleocapsid protein (NC) facilitates this annealing process. Determination of the ssDNA structure is needed to understand the molecular basis of NC-mediated genomic RNA-ssDNA annealing. For this purpose, we investigated ssDNA using structural probes (nucleases and potassium permanganate). This study is the first to determine the secondary structure of the full-length HIV-1 ssDNA in the absence or presence of NC. The probing data and phylogenetic analysis support the folding of ssDNA into three stem-loop structures and the presence of four high-affinity binding sites for NC. Our results support a model for the NC-mediated annealing process in which the preferential binding of NC to four sites triggers unfolding of the three-dimensional structure of ssDNA, thus facilitating interaction of the r sequence of ssDNA with the R sequence of the genomic RNA. In addition, using gel retardation assays and ssDNA mutants, we show that the NC-mediated annealing process does not rely on a single pathway (zipper intermediate or kissing complex). PMID:26668324

  8. A polymerase chain reaction method for the amplification of full-length envelope genes of HIV-1 from DNA samples containing single molecules of HIV-1 provirus.

    PubMed

    McClure, P; Curran, R; Boneham, S; Ball, J K

    2000-07-01

    Polymerase chain reaction (PCR) amplification of full-length envelope genes from the human immunodeficiency virus type 1 (HIV-1) directly from uncultured clinical samples is difficult. This paper describes a comparative assessment of the performance of three thermostable polymerases in an HIV-1 full-length envelope gene PCR. The PCR method utilising Expand HiFi polymerase was successful when using DNA samples extracted from a variety of sources including blood, semen and various tissues. This method generated high and specific yields of product from samples containing as little as one copy of HIV-1 proviral DNA. The resulting PCR products were suitable for a variety of downstream analytical methods including DNA sequence analysis. PMID:10921844

  9. APOBEC3G restricts early HIV-1 replication in the cytoplasm of target cells

    SciTech Connect

    Anderson, Jenny L.; Hope, Thomas J.

    2008-05-25

    Cellular APOBEC3G (A3G) protein is packaged into human immunodeficiency virus type 1 (HIV-1) virions in producer cells yet restricts viral replication in target cells. To characterize this restriction in target cells, the effect of A3G on generating various HIV-1 cDNA products was measured by quantitative real-time PCR. A3G decreased cDNA products from Vif-deficient HIV-1, with minor effects on early reverse transcripts and larger declines in late reverse transcripts. However, the greatest decline was typically observed in nuclear 2-LTR circles. Moreover, the magnitude of these declines varied with A3G dose. Adding integration inhibitor did not stop the A3G-mediated loss in 2-LTR circles. Moreover, obstructing HIV-1 nuclear entry using vesicular stomatitis virus matrix protein did not stop the A3G-mediated decline in late reverse transcripts. Collectively, these data suggest that A3G has important restriction activity in the cytoplasm and progressively diminishes viral cytoplasmic and nuclear cDNA forms with increasing magnitude during restriction.

  10. Quantification of HIV-1 DNA using real-time recombinase polymerase amplification.

    PubMed

    Crannell, Zachary Austin; Rohrman, Brittany; Richards-Kortum, Rebecca

    2014-06-17

    Although recombinase polymerase amplification (RPA) has many advantages for the detection of pathogenic nucleic acids in point-of-care applications, RPA has not yet been implemented to quantify sample concentration using a standard curve. Here, we describe a real-time RPA assay with an internal positive control and an algorithm that analyzes real-time fluorescence data to quantify HIV-1 DNA. We show that DNA concentration and the onset of detectable amplification are correlated by an exponential standard curve. In a set of experiments in which the standard curve and algorithm were used to analyze and quantify additional DNA samples, the algorithm predicted an average concentration within 1 order of magnitude of the correct concentration for all HIV-1 DNA concentrations tested. These results suggest that quantitative RPA (qRPA) may serve as a powerful tool for quantifying nucleic acids and may be adapted for use in single-sample point-of-care diagnostic systems. PMID:24873435

  11. 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

  12. Autograft HIV-DNA Load Predicts HIV-1 Peripheral Reservoir After Stem Cell Transplantation for AIDS-Related Lymphoma Patients

    PubMed Central

    Bortolin, Maria Teresa; Pratesi, Chiara; Tedeschi, Rosamaria; Basaglia, Giancarlo; Abbruzzese, Luciano; Mazzucato, Mario; Spina, Michele; Vaccher, Emanuela; Tirelli, Umberto; Rupolo, Maurizio; Michieli, Mariagrazia; Di Mascio, Michele; De Paoli, Paolo

    2015-01-01

    Abstract Autologous stem cell transplantation (ASCT) is a widely used procedure for AIDS-related lymphomas, and it represents an opportunity to evaluate strategies curing HIV-1 infection. The association of autograft HIV-DNA load with peripheral blood HIV-1 reservoir before ASCT and its contribution in predicting HIV-1 reservoir size and stability during combination antiretroviral therapy (cART) after transplantation are unknown. Aiming to obtain information suggesting new functional cure strategies by ASCT, we retrospectively evaluated HIV-DNA load in autograft and in peripheral blood before and after transplantation in 13 cART-treated HIV-1 relapse/refractoring lymphoma patients. Among them seven discontinued cART after autograft infusion. HIV-DNA was evaluated by a sensitive quantitative real-time polymerase chain reaction (PCR). After debulking chemotherapy/mobilization, the autograft HIV-1 reservoir was higher than and not associated with the peripheral HIV-1 reservoir at baseline [median 215 HIV-DNA copies/106 autograft mononuclear cells, range 13–706 vs. 82 HIV-DNA copies/106 peripheral blood mononuclear cells (PBMCs), range 13–479, p=0.03]. After high dose chemotherapy and autograft infusion, HIV-DNA levels reached a plateau between month 6 and 12 of follow-up. No association was found between peripheral HIV-DNA levels at baseline and after infusion in both cART interrupting and not interrupting patients. Only in the last subgroup, a stable significant linear association between autograft and peripheral blood HIV-1 reservoir emerged from month 1 (R2=0.84, p=0.01) to month 12 follow-up (R2=0.99, p=0.0005). In summary, autograft HIV-1 reservoir size could be influenced by the mobilization phase and predicts posttransplant peripheral HIV-1 reservoir size in patients on continuous cART. These findings could promote new research on strategies reducing the HIV-1 reservoir by using the ASCT procedure. PMID:25581618

  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. Secondary Structure and Secondary Structure Dynamics of DNA Hairpins Complexed with HIV-1 NC Protein

    PubMed Central

    Cosa, Gonzalo; Harbron, Elizabeth J.; Zeng, Yining; Liu, Hsiao-Wei; O'Connor, Donald B.; Eta-Hosokawa, Chie; Musier-Forsyth, Karin; Barbara, Paul F.

    2004-01-01

    Reverse transcription of the HIV-1 RNA genome involves several complex nucleic acid rearrangement steps that are catalyzed by the HIV-1 nucleocapsid protein (NC), including for example, the annealing of the transactivation response (TAR) region of the viral RNA to the complementary region (TAR DNA) in minus-strand strong-stop DNA. We report herein single-molecule fluorescence resonance energy transfer measurements on single immobilized TAR DNA hairpins and hairpin mutants complexed with NC (i.e., TAR DNA/NC). Using this approach we have explored the conformational distribution and dynamics of the hairpins in the presence and absence of NC protein. The data demonstrate that NC shifts the equilibrium secondary structure of TAR DNA hairpins from a fully “closed” conformation to essentially one specific “partially open” conformation. In this specific conformation, the two terminal stems are “open” or unwound and the other stems are closed. This partially open conformation is arguably a key TAR DNA intermediate in the NC-induced annealing mechanism of TAR DNA. PMID:15454467

  15. Trichosanthin, a potent HIV-1 inhibitor, can cleave supercoiled DNA in vitro.

    PubMed Central

    Li, M X; Yeung, H W; Pan, L P; Chan, S I

    1991-01-01

    Trichosanthin, an abortifacient, immunosuppressive and anti-tumor protein purified from the traditional Chinese herb medicine Tian Hua Fen, is a potent inhibitor against HIV-1 replication. Under normal enzymatic digestion conditions, trichosanthin cleaves the supercoiled double-stranded DNA to produce nicked circular and linear DNA. Trichosanthin has no effect on linear double-stranded DNA. Neither does it convert relaxed circular duplex DNA into a supercoiled form in the presence of ATP. Thus trichosanthin is not a DNA gyrase. However, trichosanthin can cleave the relaxed circular DNA into a linear form, indicating that both the circular as well as the supercoiled forms are essential for trichosanthin recognition. In addition, trichosanthin contains one calcium metal ion per protein molecule, which presumably is related to its endonucleolytic activity. Images PMID:1659689

  16. Safety and Immunogenicity Study of Multiclade HIV-1 Adenoviral Vector Vaccine Alone or as Boost following a Multiclade HIV-1 DNA Vaccine in Africa

    PubMed Central

    Allen, Susan; Than, Soe; Adams, Elizabeth M.; Graham, Barney S.; Koup, Richard A.; Bailer, Robert T.; Smith, Carol; Dally, Len; Tarragona-Fiol, Tony; Bergin, Philip J.; Hayes, Peter; Ho, Martin; Loughran, Kelley; Komaroff, Wendy; Stevens, Gwynneth; Thomson, Helen; Boaz, Mark J.; Cox, Josephine H.; Schmidt, Claudia; Gilmour, Jill; Nabel, Gary J.; Fast, Patricia

    2010-01-01

    Background We conducted a double-blind, randomized, placebo-controlled Phase I study of a recombinant replication-defective adenovirus type 5 (rAd5) vector expressing HIV-1 Gag and Pol from subtype B and Env from subtypes A, B and C, given alone or as boost following a DNA plasmid vaccine expressing the same HIV-1 proteins plus Nef, in 114 healthy HIV-uninfected African adults. Methodology/Principal Findings Volunteers were randomized to 4 groups receiving the rAd5 vaccine intramuscularly at dosage levels of 1×1010 or 1×1011 particle units (PU) either alone or as boost following 3 injections of the DNA vaccine given at 4 mg/dose intramuscularly by needle-free injection using Biojector® 2000. Safety and immunogenicity were evaluated for 12 months. Both vaccines were well-tolerated. Overall, 62% and 86% of vaccine recipients in the rAd5 alone and DNA prime - rAd5 boost groups, respectively, responded to the HIV-1 proteins by an interferon-gamma (IFN-γ) ELISPOT. The frequency of immune responses was independent of rAd5 dosage levels. The highest frequency of responses after rAd5 alone was detected at 6 weeks; after DNA prime - rAd5 boost, at 6 months (end of study). At baseline, neutralizing antibodies against Ad5 were present in 81% of volunteers; the distribution was similar across the 4 groups. Pre-existing immunity to Ad5 did not appear to have a significant impact on reactogenicity or immune response rates to HIV antigens by IFN-γ ELISPOT. Binding antibodies against Env were detected in up to 100% recipients of DNA prime - rAd5 boost. One volunteer acquired HIV infection after the study ended, two years after receipt of rAd5 alone. Conclusions/Significance The HIV-1 rAd5 vaccine, either alone or as a boost following HIV-1 DNA vaccine, was well-tolerated and immunogenic in African adults. DNA priming increased the frequency and magnitude of cellular and humoral immune responses, but there was no effect of rAd5 dosage on immunogenicity endpoints. Trial

  17. Nucleic Acid Chaperone Activity of HIV-1 NC Proteins Investigated by Single Molecule DNA Stretching

    NASA Astrophysics Data System (ADS)

    Williams, Mark C.; Gorelick, Robert J.; Musier-Forsyth, Karin; Bloomfield, Victor A.

    2002-03-01

    HIV-1 Nucleocapsid Protein (NC) is a nucleic acid chaperone protein that is responsible for facilitating numerous nucleic acid rearrangements throughout the reverse transcription cycle of HIV-1. To understand the mechanism of NC’s chaperone function, we carried out single molecule DNA stretching studies in the presence of NC and mutant forms of NC. Using an optical tweezers instrument, we stretch single DNA molecules from the double-stranded helical state to the single-stranded (coil) state. Based on the observed cooperativity of DNA force-induced melting, we find that the fraction of melted base pairs at room temperature is increased dramatically in the presence of NC. Thus, upon NC binding, increased thermal fluctuations cause continuous melting and reannealing of base pairs so that DNA strands are able to rapidly sample configurations in order to find the lowest energy state. While NC destabilizes the double-stranded form of DNA, a mutant form of NC that lacks the zinc finger structures does not. DNA stretching experiments carried out in the presence of NC variants containing more subtle changes in the zinc finger structures were conducted to elucidate the contribution of each individual finger to NC’s chaperone activity, and these results will be reported.

  18. Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

    PubMed

    Alidjinou, E K; Bocket, L; Hober, D

    2015-02-01

    Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several "in-house" PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes. PMID:25201144

  19. Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul

    2003-03-01

    Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.

  20. DNA Physical Properties and Nucleosome Positions Are Major Determinants of HIV-1 Integrase Selectivity

    PubMed Central

    Naughtin, Monica; Haftek-Terreau, Zofia; Xavier, Johan; Meyer, Sam; Silvain, Maud; Jaszczyszyn, Yan; Levy, Nicolas; Miele, Vincent; Benleulmi, Mohamed Salah; Ruff, Marc; Parissi, Vincent; Vaillant, Cédric; Lavigne, Marc

    2015-01-01

    Retroviral integrases (INs) catalyse the integration of the reverse transcribed viral DNA into the host cell genome. This process is selective, and chromatin has been proposed to be a major factor regulating this step in the viral life cycle. However, the precise underlying mechanisms are still under investigation. We have developed a new in vitro integration assay using physiologically-relevant, reconstituted genomic acceptor chromatin and high-throughput determination of nucleosome positions and integration sites, in parallel. A quantitative analysis of the resulting data reveals a chromatin-dependent redistribution of the integration sites and establishes a link between integration sites and nucleosome positions. The co-activator LEDGF/p75 enhanced integration but did not modify the integration sites under these conditions. We also conducted an in cellulo genome-wide comparative study of nucleosome positions and human immunodeficiency virus type-1 (HIV-1) integration sites identified experimentally in vivo. These studies confirm a preferential integration in nucleosome-covered regions. Using a DNA mechanical energy model, we show that the physical properties of DNA probed by IN binding are important in determining IN selectivity. These novel in vitro and in vivo approaches confirm that IN has a preference for integration into a nucleosome, and suggest the existence of two levels of IN selectivity. The first depends on the physical properties of the target DNA and notably, the energy required to fit DNA into the IN catalytic pocket. The second depends on the DNA deformation associated with DNA wrapping around a nucleosome. Taken together, these results indicate that HIV-1 IN is a shape-readout DNA binding protein. PMID:26075397

  1. Total HIV-1 DNA, a Marker of Viral Reservoir Dynamics with Clinical Implications.

    PubMed

    Avettand-Fènoël, Véronique; Hocqueloux, Laurent; Ghosn, Jade; Cheret, Antoine; Frange, Pierre; Melard, Adeline; Viard, Jean-Paul; Rouzioux, Christine

    2016-10-01

    HIV-1 DNA persists in infected cells despite combined antiretroviral therapy (cART), forming viral reservoirs. Recent trials of strategies targeting latent HIV reservoirs have rekindled hopes of curing HIV infection, and reliable markers are thus needed to evaluate viral reservoirs. Total HIV DNA quantification is simple, standardized, sensitive, and reproducible. Total HIV DNA load influences the course of the infection and is therefore clinically relevant. In particular, it is predictive of progression to AIDS and death, independently of HIV RNA load and the CD4 cell count. Baseline total HIV DNA load is predictive of the response to cART. It declines during cART but remains quantifiable, at a level that reflects both the history of infection (HIV RNA zenith, CD4 cell count nadir) and treatment efficacy (residual viremia, cumulative viremia, immune restoration, immune cell activation). Total HIV DNA load in blood is also predictive of the presence and severity of some HIV-1-associated end-organ disorders. It can be useful to guide individual treatment, notably, therapeutic de-escalation. Although it does not distinguish between replication-competent and -defective latent viruses, the total HIV DNA load in blood, tissues, and cells provides insights into HIV pathogenesis, probably because all viral forms participate in host cell activation and HIV pathogenesis. Total HIV DNA is thus a biomarker of HIV reservoirs, which can be defined as all infected cells and tissues containing all forms of HIV persistence that participate in pathogenesis. This participation may occur through the production of new virions, creating new cycles of infection and disseminating infected cells; maintenance or amplification of reservoirs by homeostatic cell proliferation; and viral transcription and synthesis of viral proteins without new virion production. These proteins can induce immune activation, thus participating in the vicious circle of HIV pathogenesis. PMID:27559075

  2. Structural determinants of HIV-1 Vif susceptibility and DNA binding in APOBEC3F

    PubMed Central

    Siu, Karen K.; Sultana, Azmiri; Azimi, Farshad C.; Lee, Jeffrey E.

    2016-01-01

    The human APOBEC3 family of DNA cytosine deaminases serves as a front-line intrinsic immune response to inhibit the replication of diverse retroviruses. APOBEC3F and APOBEC3G are the most potent factors against HIV-1. As a countermeasure, HIV-1 viral infectivity factor (Vif) targets APOBEC3s for proteasomal degradation. Here, we report the crystal structure of the Vif-binding domain in APOBEC3F and a novel assay to assess Vif-APOBEC3 binding. Our results point to an amphipathic surface that is conserved in APOBEC3s as critical for Vif susceptibility in APOBEC3F. Electrostatic interactions likely mediate Vif binding. Moreover, structure-guided mutagenesis reveals a straight ssDNA-binding groove distinct from the Vif-binding site, and a novel ‘aromatic switch’ is proposed to explain DNA substrate specificities across the APOBEC3 family. This study opens new lines of inquiry that will further our understanding of APOBEC3-mediated retroviral restriction and provides an accurate template for structure-guided development of inhibitors targeting the APOBEC3-Vif axis. PMID:24185281

  3. HIV-1 Tat depresses DNA-PK{sub CS} expression and DNA repair, and sensitizes cells to ionizing radiation

    SciTech Connect

    Sun Yi; Huang Yuechen; Xu Qinzhi; Wang Huiping; Bai Bei; Sui Jianli; Zhou Pingkun . E-mail: zhoupk@nic.bmi.ac.cn

    2006-07-01

    Purpose There is accumulating evidence that cancer patients with human immmunodeficiency virus-1/acquired immunodeficency syndrome (HIV-1/AIDS) have more severe tissue reactions and often develop cutaneous toxic effects when subjected to radiotherapy. Here we explored the effects of the HIV-1 Tat protein on cellular responses to ionizing radiation. Methods and Materials Two Tat-expressing cell lines, TT2 and TE671-Tat, were derived from human rhabdomyosarcoma cells by transfecting with the HIV-1 tat gene. Radiosensitivity was determined using colony-forming ability. Gene expression was assessed by cDNA microarray and immunohybridization. The Comet assay and {gamma}-H2AX foci were use to detect DNA double-strand breaks (DSBs) and repair. Radiation-induced cell cycle changes were detected by flow cytometry. Results The radiosensitivity of TT2 and TE671-Tat cells was significantly increased as compared with parental TE671 cells or the control TE671-pCI cells. Tat also increased proliferation activity. The comet assay and {gamma}H2AX foci detection revealed a decreased capacity to repair radiation-induced DNA DSBs in Tat-expressing cells. Microarray assay demonstrated that the DNA repair gene DNA-PKcs, and cell cycle-related genes Cdc20, Cdc25C, KIF2C and CTS1 were downregulated in Tat-expressing cells. Depression of DNA-PKcs in Tat-expressing cells was further confirmed by RT-PCR and immuno-hybridization analysis. Tat-expressing cells exhibited a prolonged S phase arrest after 4 Gy {gamma}-irradiation, and a noticeable delay in the initiation and elimination of radiation-induced G{sub 2}/M arrest as compared with parental cells. In addition, the G{sub 2}/M arrest was incomplete in TT2 cells. Moreover, HIV-1 Tat resulted in a constitutive overexpression of cyclin B1 protein. Conclusion HIV-1 Tat protein sensitizes cells to ionizing radiation via depressing DNA repair and dysregulating cell cycle checkpoints. These observations provide new insight into the increased

  4. Solution structure of the DNA binding domain of HIV-1 integrase.

    PubMed

    Lodi, P J; Ernst, J A; Kuszewski, J; Hickman, A B; Engelman, A; Craigie, R; Clore, G M; Gronenborn, A M

    1995-08-01

    The solution structure of the DNA binding domain of HIV-1 integrase (residues 220-270) has been determined by multidimensional NMR spectroscopy. The protein is a dimer in solution, and each subunit is composed of a five-stranded beta-barrel with a topology very similar to that of the SH3 domain. The dimer is formed by a stacked beta-interface comprising strands 2, 3, and 4, with the two triple-stranded antiparallel beta-sheets, one from each subunit, oriented antiparallel to each other. One surface of the dimer, bounded by the loop between strands beta 1 and beta 2, forms a saddle-shaped groove with dimensions of approximately 24 x 23 x 12 A in cross section. Lys264, which has been shown from mutational data to be involved in DNA binding, protrudes from this surface, implicating the saddle-shaped groove as the potential DNA binding site. PMID:7632683

  5. Cellular HIV-1 DNA quantitation in patients during simplification therapy with protease inhibitor-sparing regimens.

    PubMed

    Sarmati, Loredana; Parisi, Saverio Giuseppe; Nicastri, Emanuele; d'Ettorre, Gabriella; Andreoni, Carolina; Dori, Luca; Gatti, Francesca; Montano, Marco; Buonomini, Anna Rita; Boldrin, Caterina; Palù, Giorgio; Vullo, Vincenzo; Andreoni, Massimo

    2007-07-01

    Simplified regimens containing protease-inhibitors (PI)-sparing combinations were used in patients with virological suppression after prolonged highly active antiretroviral therapy. This study evaluated the total HIV-1 DNA quantitation as a predictor of long-term success for PI-sparing simplified therapy. Sixty-two patients were enrolled in a prospective non-randomized cohort. All patients have been receiving a triple-therapy regimen, two nucleoside reverse transcriptase inhibitors (NRTIs) plus one PI, for at least 9 months and were characterized by undetectable plasma HIV-1 RNA levels (<50 cp/ml) for at least 6 months. Patients were changed to a simplified PI-sparing regimen to overcome PI-associated adverse effects. HIV-DNA levels in peripheral blood mononuclear cells (PBMCs) were evaluated at baseline and at the end of follow-up. Patients with proviral DNA levels below the median value (226 copies/10(6) PBMCs) had a significant higher CD4 cell count at nadir (P = 0.003) and at enrolment (P = 0.001) with respect to patients with HIV-DNA levels above the median value. At month 18, 53 out of 62 (85%) patients on simplified regimen showed virological success, 4 (6.4%) patients experienced virological failure and 5 (8%) patients showed viral blip. At logistic regression analysis, HIV-DNA levels below 226 copies/10(6) PBMCs at baseline were associated independently to a reduced risk of virological failure or viral blip during simplified therapy (OR 0.002, 95% CI 0.001-0.46, P = 0.025). The substitution of PI with NRTI or non-NRTIs may represent an effective treatment option. Indeed, treatment failure or viral blip were experienced by 6% and 8% of the patients on simplified therapy, respectively. In addition, sustained suppression of the plasma viral load was significantly correlated with low levels of proviral DNA before treatment simplification. PMID:17516532

  6. Autograft HIV-DNA load predicts HIV-1 peripheral reservoir after stem cell transplantation for AIDS-related lymphoma patients.

    PubMed

    Zanussi, Stefania; Bortolin, Maria Teresa; Pratesi, Chiara; Tedeschi, Rosamaria; Basaglia, Giancarlo; Abbruzzese, Luciano; Mazzucato, Mario; Spina, Michele; Vaccher, Emanuela; Tirelli, Umberto; Rupolo, Maurizio; Michieli, Mariagrazia; Di Mascio, Michele; De Paoli, Paolo

    2015-01-01

    Autologous stem cell transplantation (ASCT) is a widely used procedure for AIDS-related lymphomas, and it represents an opportunity to evaluate strategies curing HIV-1 infection. The association of autograft HIV-DNA load with peripheral blood HIV-1 reservoir before ASCT and its contribution in predicting HIV-1 reservoir size and stability during combination antiretroviral therapy (cART) after transplantation are unknown. Aiming to obtain information suggesting new functional cure strategies by ASCT, we retrospectively evaluated HIV-DNA load in autograft and in peripheral blood before and after transplantation in 13 cART-treated HIV-1 relapse/refractoring lymphoma patients. Among them seven discontinued cART after autograft infusion. HIV-DNA was evaluated by a sensitive quantitative real-time polymerase chain reaction (PCR). After debulking chemotherapy/mobilization, the autograft HIV-1 reservoir was higher than and not associated with the peripheral HIV-1 reservoir at baseline [median 215 HIV-DNA copies/10(6) autograft mononuclear cells, range 13-706 vs. 82 HIV-DNA copies/10(6) peripheral blood mononuclear cells (PBMCs), range 13-479, p = 0.03]. After high dose chemotherapy and autograft infusion, HIV-DNA levels reached a plateau between month 6 and 12 of follow-up. No association was found between peripheral HIV-DNA levels at baseline and after infusion in both cART interrupting and not interrupting patients. Only in the last subgroup, a stable significant linear association between autograft and peripheral blood HIV-1 reservoir emerged from month 1 (R(2) = 0.84, p = 0.01) to month 12 follow-up (R(2) = 0.99, p = 0.0005). In summary, autograft HIV-1 reservoir size could be influenced by the mobilization phase and predicts posttransplant peripheral HIV-1 reservoir size in patients on continuous cART. These findings could promote new research on strategies reducing the HIV-1 reservoir by using the ASCT procedure. PMID:25581618

  7. NLRX1 Sequesters STING to Negatively Regulate the Interferon Response, Thereby Facilitating the Replication of HIV-1 and DNA Viruses.

    PubMed

    Guo, Haitao; König, Renate; Deng, Meng; Riess, Maximilian; Mo, Jinyao; Zhang, Lu; Petrucelli, Alex; Yoh, Sunnie M; Barefoot, Brice; Samo, Melissa; Sempowski, Gregory D; Zhang, Aiping; Colberg-Poley, Anamaris M; Feng, Hui; Lemon, Stanley M; Liu, Yong; Zhang, Yanping; Wen, Haitao; Zhang, Zhigang; Damania, Blossom; Tsao, Li-Chung; Wang, Qi; Su, Lishan; Duncan, Joseph A; Chanda, Sumit K; Ting, Jenny P-Y

    2016-04-13

    Understanding the negative regulators of antiviral immune responses will be critical for advancing immune-modulated antiviral strategies. NLRX1, an NLR protein that negatively regulates innate immunity, was previously identified in an unbiased siRNA screen as required for HIV infection. We find that NLRX1 depletion results in impaired nuclear import of HIV-1 DNA in human monocytic cells. Additionally, NLRX1 was observed to reduce type-I interferon (IFN-I) and cytokines in response to HIV-1 reverse-transcribed DNA. NLRX1 sequesters the DNA-sensing adaptor STING from interaction with TANK-binding kinase 1 (TBK1), which is a requisite for IFN-1 induction in response to DNA. NLRX1-deficient cells generate an amplified STING-dependent host response to cytosolic DNA, c-di-GMP, cGAMP, HIV-1, and DNA viruses. Accordingly, Nlrx1(-/-) mice infected with DNA viruses exhibit enhanced innate immunity and reduced viral load. Thus, NLRX1 is a negative regulator of the host innate immune response to HIV-1 and DNA viruses. PMID:27078069

  8. Interaction of HIV-1 Gag protein components with single DNA molecules

    NASA Astrophysics Data System (ADS)

    Cruceanu, Margareta; Gorelick, Robert J.; Williams, Mark C.

    2003-03-01

    The Gag protein of the HIV-1 retrovirus is cleaved into three major proteins as part of viral maturation: nucleocapsid (NC), capsid, and matrix. NC is the first of these proteins to be cleaved, and it is cleaved in three stages into NCp15, followed by NCp9, and finally NCp7. In this study, we use optical tweezers to investigate the capability of these NC proteins to alter the helix-coil transition of single DNA molecules. We have previously shown that the capability to alter the DNA helix-coil transition is an excellent probe of the nucleic acid chaperone activity of NC proteins, in which the secondary structure of nucleic acids is rearranged to facilitate reverse transcription. By examining the capability of NCp15, NCp9, and NCp7 to alter DNA stretching, the current studies will test the role of proteolytic cleavage of Gag in regulating the nucleic acid chaperone activity of NC. Whereas binding studies suggest that NCp9 and NCp15 bind more strongly to DNA than NCp7, our DNA stretching results indicate that these proteins all have similar effects on DNA stretching.

  9. A post-entry role for CD63 in early HIV-1 replication

    SciTech Connect

    Li Guangyu; Dziuba, Natallia; Friedrich, Brian; Murray, James L.; Ferguson, Monique R.

    2011-04-10

    Macrophages and CD4{sup +} lymphocytes are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in early HIV-1 replication events in both macrophages and a CD4{sup +} cell line. Further analysis revealed that viral attachment and cell-cell fusion were unaffected by CD63 silencing. However, CD63-depleted macrophages showed a significant decrease in the initiation and completion of HIV-1 reverse transcription, affecting subsequent events of the HIV-1 life cycle. Integration of HIV-1 cDNA as well as the formation of 2-LTR circles was notably reduced. Reporter assays showed that CD63 down regulation reduced production of the early HIV protein Tat. In agreement, CD63 silencing also inhibited production of the late protein p24. These findings suggest that CD63 plays an early post-entry role prior to or at the reverse transcription step.

  10. Expanded breadth of the T-cell response to mosaic HIV-1 envelope DNA vaccination

    SciTech Connect

    Korber, Bette; Fischer, William; Wallstrom, Timothy

    2009-01-01

    An effective AIDS vaccine must control highly diverse circulating strains of HIV-1. Among HIV -I gene products, the envelope (Env) protein contains variable as well as conserved regions. In this report, an informatic approach to the design of T-cell vaccines directed to HIV -I Env M group global sequences was tested. Synthetic Env antigens were designed to express mosaics that maximize the inclusion of common potential Tcell epitope (PTE) 9-mers and minimize the inclusion of rare epitopes likely to elicit strain-specific responses. DNA vaccines were evaluated using intracellular cytokine staining (ICS) in inbred mice with a standardized panel of highly conserved 15-mer PTE peptides. I, 2 and 3 mosaic sets were developed that increased theoretical epitope coverage. The breadth and magnitude ofT-cell immunity stimulated by these vaccines were compared to natural strain Env's; additional comparisons were performed on mutant Env's, including gpl60 or gpl45 with or without V regions and gp41 deletions. Among them, the 2 or 3 mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the 3 mosaic set elicited responses to an average of 8 peptide pools compared to 2 pools for a set of3 natural Env's. Synthetic mosaic HIV -I antigens can therefore induce T-cell responses with expanded breadth and may facilitate the development of effective T -cell-based HIV -1 vaccines.

  11. The MSHA strain of Pseudomonas aeruginosa activated TLR pathway and enhanced HIV-1 DNA vaccine immunoreactivity.

    PubMed

    Hou, Jue; Liu, Yong; Liu, Ying; Shao, Yiming

    2012-01-01

    The mannose-sensitive hemagglutination pilus strain of Pseudomonas aeruginosa (PA-MSHA) has been shown to trigger naïve immune responses through the activation of monocytes, macrophages, natural killer cells (NK cells) and antigen presenting cells (APCs). Based on the hypothesis that PA-MSHA activates natural immunity through the Toll-like receptor (TLR) pathway, we scanned several critical TLR pathway molecules in mouse splenocytes using high-throughput real-time QRT-PCR and co-stimulatory molecule in bone marrow-derived dendritic cells (BMDCs) following in vitro stimulation by PA-MSHA. PA-MSHA enabled activation of the TLR pathway mediated by NF-κB and JNK signaling in splenocytes, and the co-stimulatory molecule CD86 was up-regulated in BMDCs. We then assessed the adjuvant effect of PA-MSHA for HIV-1 DNA vaccines. In comparison to DNA inoculation alone, co-inoculation with low dosage of PA-MSHA enhanced specific immunoreactivity against HIV-1 Env in both cellular and humoral responses, and promoted antibody avidity maturation. However, high doses of adjuvant resulted in an immunosuppressive effect; a two- or three-inoculation regimen yielded low antibody responses and the two-inoculation regimen exhibited only a slight cellular immunity response. To our knowledge, this is the first report demonstrating the utility of PA-MSHA as an adjuvant to a DNA vaccine. Further research is needed to investigate the exact mechanisms through which PA-MSHA achieves its adjuvant effects on innate immune responses, especially on dendritic cells. PMID:23077664

  12. Combined Antiviral Therapy Using Designed Molecular Scaffolds Targeting Two Distinct Viral Functions, HIV-1 Genome Integration and Capsid Assembly

    PubMed Central

    Khamaikawin, Wannisa; Saoin, Somphot; Nangola, Sawitree; Chupradit, Koollawat; Sakkhachornphop, Supachai; Hadpech, Sudarat; Onlamoon, Nattawat; Ansari, Aftab A; Byrareddy, Siddappa N; Boulanger, Pierre; Hong, Saw-See; Torbett, Bruce E; Tayapiwatana, Chatchai

    2015-01-01

    Designed molecular scaffolds have been proposed as alternative therapeutic agents against HIV-1. The ankyrin repeat protein (AnkGAG1D4) and the zinc finger protein (2LTRZFP) have recently been characterized as intracellular antivirals, but these molecules, used individually, do not completely block HIV-1 replication and propagation. The capsid-binder AnkGAG1D4, which inhibits HIV-1 assembly, does not prevent the genome integration of newly incoming viruses. 2LTRZFP, designed to target the 2-LTR-circle junction of HIV-1 cDNA and block HIV-1 integration, would have no antiviral effect on HIV-1-infected cells. However, simultaneous expression of these two molecules should combine the advantage of preventive and curative treatments. To test this hypothesis, the genes encoding the N-myristoylated Myr(+)AnkGAG1D4 protein and the 2LTRZFP were introduced into human T-cells, using a third-generation lentiviral vector. SupT1 cells stably expressing 2LTRZFP alone or with Myr(+)AnkGAG1D4 showed a complete resistance to HIV-1 in viral challenge. Administration of the Myr(+)AnkGAG1D4 vector to HIV-1-preinfected SupT1 cells resulted in a significant antiviral effect. Resistance to viral infection was also observed in primary human CD4+ T-cells stably expressing Myr(+)AnkGAG1D4, and challenged with HIV-1, SIVmac, or SHIV. Our data suggest that our two anti-HIV-1 molecular scaffold prototypes are promising antiviral agents for anti-HIV-1 gene therapy. PMID:26305555

  13. A Sensitive Branched DNA HIV-1 Signal Amplification Viral Load Assay with Single Day Turnaround

    PubMed Central

    Baumeister, Mark A.; Zhang, Nan; Beas, Hilda; Brooks, Jesse R.; Canchola, Jesse A.; Cosenza, Carlo; Kleshik, Felix; Rampersad, Vinod; Surtihadi, Johan; Battersby, Thomas R.

    2012-01-01

    Branched DNA (bDNA) is a signal amplification technology used in clinical and research laboratories to quantitatively detect nucleic acids. An overnight incubation is a significant drawback of highly sensitive bDNA assays. The VERSANT® HIV-1 RNA 3.0 Assay (bDNA) (“Versant Assay”) currently used in clinical laboratories was modified to allow shorter target incubation, enabling the viral load assay to be run in a single day. To dramatically reduce the target incubation from 16–18 h to 2.5 h, composition of only the “Lysis Diluent” solution was modified. Nucleic acid probes in the assay were unchanged. Performance of the modified assay (assay in development; not commercially available) was evaluated and compared to the Versant Assay. Dilution series replicates (>950 results) were used to demonstrate that analytical sensitivity, linearity, accuracy, and precision for the shorter modified assay are comparable to the Versant Assay. HIV RNA-positive clinical specimens (n = 135) showed no significant difference in quantification between the modified assay and the Versant Assay. Equivalent relative quantification of samples of eight genotypes was demonstrated for the two assays. Elevated levels of several potentially interfering endogenous substances had no effect on quantification or specificity of the modified assay. The modified assay with drastically improved turnaround time demonstrates the viability of signal-amplifying technology, such as bDNA, as an alternative to the PCR-based assays dominating viral load monitoring in clinical laboratories. Highly sensitive bDNA assays with a single day turnaround may be ideal for laboratories with especially stringent cost, contamination, or reliability requirements. PMID:22479381

  14. Single DNA molecule stretching measures the activity of chemicals that target the HIV-1 nucleocapsid protein

    PubMed Central

    Cruceanu, Margareta; Stephen, Andrew G.; Beuning, Penny J.; Gorelick, Robert J.; Fisher, Robert J.; Williams, Mark C.

    2006-01-01

    We develop a biophysical method for investigating chemical compounds that target the nucleic acid chaperone activity of HIV-1 nucleocapsid protein (NCp7). We used an optical tweezers instrument to stretch single λ-DNA molecules through the helix-to-coil transition in the presence of NCp7 and various chemical compounds. The change in the helix-coil transition width induced by wild-type NCp7 and its zinc finger variants correlates with in vitro nucleic acid chaperone activity measurements and in vivo assays. The compound-NC interaction measured here reduces NCp7’s capability to alter the transition width. Purified compounds from the NCI Diversity set, 119889, 119911, and 119913 reduce the chaperone activity of 5 nM NC in aqueous solution at 10 nM, 25 nM, and 100 nM concentration, respectively. Similarly, gallein reduced the activity of 4 nM NC at 100 nM concentration. Further analysis allows us to dissect the impact of each compound on both sequence-specific and non-sequence-specific DNA binding of NC, two of the main components of NC’s nucleic acid chaperone activity. These results suggest that DNA stretching experiments can be used to screen chemical compounds targeting NC proteins, and to further explore the mechanisms by which these compounds interact with NC and alter its nucleic acid chaperone activity. PMID:17034752

  15. Mechanism of polyoxometalate-mediated inactivation of DNA polymerases: an analysis with HIV-1 reverse transcriptase indicates specificity for the DNA-binding cleft.

    PubMed Central

    Sarafianos, S G; Kortz, U; Pope, M T; Modak, M J

    1996-01-01

    The anti-DNA polymerase activity of a structural family of polyoxometalates has been determined. Two representative compounds of this family, possessing a saddle-like structure [(O3POPO3)4W12O36]16- (polyoxometalate I) and [(O3PCH2PO3)4W12O36]16- (polyoxometalate II) were found to inhibit all the DNA polymerases tested, with IC50 values ranging from 2 to 10 microM. A comparative study with HIV-1 reverse transcriptase (RT) and Klenow polymerase as representative DNA polymerases indicated that protection from inactivation was achieved by inclusion of DNA but not by deoxynucleotide triphosphates (dNTPs). Kinetic analysis revealed that the mode of HIV-1 RT inhibition is competitive with respect to DNA, and non-competitive with respect to dNTP binding. Cross-linking experiments confirmed that the inhibitors interfere with the DNA-binding function of HIV-1 reverse transcriptase. Interestingly, a number of drug-resistant mutants of HIV-1 RT exhibit a sensitivity to polyoxometalate comparable to the wild-type HIV-1 RT, suggesting that these polyoxometalates interact at a novel site. Because different polymerases contain DNA-binding clefts of various dimensions, it should be possible to modify polyoxometalates or to add a link to an enzyme-specific drug so that more effective inhibitors could be developed. Using a computer model of HIV-1 RT we performed docking studies in a binary complex (enzyme-polyoxometalate I) to propose tentatively a possible interacting site in HIV-1 RT consistent with the available biochemical results as well as with the geometric and charge constraints of the two molecules. PMID:8912703

  16. Novel bimodular DNA aptamers with guanosine quadruplexes inhibit phylogenetically diverse HIV-1 reverse transcriptases

    PubMed Central

    Michalowski, Daniel; Chitima-Matsiga, Rebecca; Held, Daniel M.; Burke, Donald H.

    2008-01-01

    DNA aptamers RT5, RT6 and RT47 form a group of related sequences that inhibit HIV-1 reverse transcriptase (RT). The essential inhibitory structure is identified here as bimodular, with a 5′ stem–loop module physically connected to a 3′-guanosine quadruplex module. The stem–loop tolerates considerable sequence plasticity. Connections between the guanosine triplets in the quadruplex could be simplified to a single nucleotide or a nonnucleic acid linker, such as hexaethylene glycol. All 12 quadruplex guanosines are required in an aptamer retaining most of the original loop sequence from RT6; only 11 are required for aptamer R1T (single T residue in intra-quadruplex loops). Circular dichroism (CD) spectroscopy gave ellipticity minima and maxima at 240 nm and 264 nm, indicating a parallel arrangement of the quadruplex strands. The simplified aptamers displayed increased overall stability. An aptamer carrying the original intra-quadruplex loops from RT6 inhibited RT in K+ buffers but not in Na+ buffers and displayed significant CD spectral broadening in Na+ buffers, while R1T inhibited RT in both buffers and displayed less broadening in Na+ buffers. The bimodular ssDNA aptamers inhibited RT from diverse primate lentiviruses with low nM IC50 values. These data provide insight into the requirements for broad-spectrum RT inhibition by nucleic acid aptamers. PMID:18996899

  17. Innate Immune Activity Correlates with CD4 T Cell-Associated HIV-1 DNA Decline during Latency-Reversing Treatment with Panobinostat

    PubMed Central

    Olesen, Rikke; Vigano, Selena; Rasmussen, Thomas A.; Søgaard, Ole S.; Ouyang, Zhengyu; Buzon, Maria; Bashirova, Arman; Carrington, Mary; Palmer, Sarah; Brinkmann, Christel R.; Yu, Xu G.; Østergaard, Lars; Tolstrup, Martin

    2015-01-01

    ABSTRACT The pharmaceutical reactivation of dormant HIV-1 proviruses by histone deacetylase inhibitors (HDACi) represents a possible strategy to reduce the reservoir of HIV-1-infected cells in individuals treated with suppressive combination antiretroviral therapy (cART). However, the effects of such latency-reversing agents on the viral reservoir size are likely to be influenced by host immune responses. Here, we analyzed the immune factors associated with changes in proviral HIV-1 DNA levels during treatment with the potent HDACi panobinostat in a human clinical trial involving 15 cART-treated HIV-1-infected patients. We observed that the magnitude, breadth, and cytokine secretion profile of HIV-1-specific CD8 T cell responses were unrelated to changes in HIV-1 DNA levels in CD4 T cells during panobinostat treatment. In contrast, the proportions of CD3− CD56+ total NK cells and CD16+ CD56dim NK cells were inversely correlated with HIV-1 DNA levels throughout the study, and changes in HIV-1 DNA levels during panobinostat treatment were negatively associated with the corresponding changes in CD69+ NK cells. Decreasing levels of HIV-1 DNA during latency-reversing treatment were also related to the proportions of plasmacytoid dendritic cells, to distinct expression patterns of interferon-stimulated genes, and to the expression of the IL28B CC genotype. Together, these data suggest that innate immune activity can critically modulate the effects of latency-reversing agents on the viral reservoir and may represent a target for future immunotherapeutic interventions in HIV-1 eradication studies. IMPORTANCE Currently available antiretroviral drugs are highly effective in suppressing HIV-1 replication, but the virus persists, despite treatment, in a latent form that does not actively express HIV-1 gene products. One approach to eliminate these cells, colloquially termed the “shock-and-kill” strategy, focuses on the use of latency-reversing agents that induce active

  18. HIV-1 p24(gag) derived conserved element DNA vaccine increases the breadth of immune response in mice.

    PubMed

    Kulkarni, Viraj; Rosati, Margherita; Valentin, Antonio; Ganneru, Brunda; Singh, Ashish K; Yan, Jian; Rolland, Morgane; Alicea, Candido; Beach, Rachel Kelly; Zhang, Gen-Mu; Le Gall, Sylvie; Broderick, Kate E; Sardesai, Niranjan Y; Heckerman, David; Mothe, Beatriz; Brander, Christian; Weiner, David B; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2013-01-01

    Viral diversity is considered a major impediment to the development of an effective HIV-1 vaccine. Despite this diversity, certain protein segments are nearly invariant across the known HIV-1 Group M sequences. We developed immunogens based on the highly conserved elements from the p24(gag) region according to two principles: the immunogen must (i) include strictly conserved elements of the virus that cannot mutate readily, and (ii) exclude both HIV regions capable of mutating without limiting virus viability, and also immunodominant epitopes located in variable regions. We engineered two HIV-1 p24(gag) DNA immunogens that express 7 highly Conserved Elements (CE) of 12-24 amino acids in length and differ by only 1 amino acid in each CE ('toggle site'), together covering >99% of the HIV-1 Group M sequences. Altering intracellular trafficking of the immunogens changed protein localization, stability, and also the nature of elicited immune responses. Immunization of C57BL/6 mice with p55(gag) DNA induced poor, CD4(+) mediated cellular responses, to only 2 of the 7 CE; in contrast, vaccination with p24CE DNA induced cross-clade reactive, robust T cell responses to 4 of the 7 CE. The responses were multifunctional and composed of both CD4(+) and CD8(+) T cells with mature cytotoxic phenotype. These findings provide a method to increase immune response to universally conserved Gag epitopes, using the p24CE immunogen. p24CE DNA vaccination induced humoral immune responses similar in magnitude to those induced by p55(gag), which recognize the virus encoded p24(gag) protein. The inclusion of DNA immunogens composed of conserved elements is a promising vaccine strategy to induce broader immunity by CD4(+) and CD8(+) T cells to additional regions of Gag compared to vaccination with p55(gag) DNA, achieving maximal cross-clade reactive cellular and humoral responses. PMID:23555935

  19. Mitochondrial DNA mutations in blood samples from HIV-1-infected children undergoing long-term antiretroviral therapy.

    PubMed

    Ouyang, Yabo; Qiao, Luxin; Liu, Kai; Zang, Yunjin; Sun, Yu; Dong, Yaowu; Liu, Daojie; Guo, Xianghua; Wei, Feili; Lin, Minghua; Zhang, Fujie; Chen, Dexi

    2016-07-01

    We have analyzed mutations in whole mitochondrial (mt) genomes of blood samples from HIV-1-infected children treated with long-term antiretroviral therapy (ART), who had an excellent virological response. HIV-1-infected children who have undergone ART for 4 y with an excellent virological response (group A; 15 children) and ten healthy children (controls) without HIV-1 infection were enrolled retrospectively. Peripheral blood mononuclear cells (PBMCs) were obtained and mt DNA mutations were studied. The total number of mtDNA mutations in group A was 3 H more than in the controls (59 vs. 19, P<0.001) and the same trend was seen in all mtDNA regions. Among these mtDNA mutations, 140 and 28 mutations were detected in group A and the controls, respectively. The D-loop, CYTB and 12s rRNA were the three most common mutation regions in both groups, with significant differences between the groups observed at nucleotide positions C309CC, T489C CA514deletion, T16249C and G16474GG (D-loop); T14783C, G15043A, G15301A, and A15662G (CYTB); and G709A (12s rRNA). G15043A and A15662G had been associated with mitochondrial diseases. Our findings suggest that mtDNA mutations occur frequently in long-term ART-treated, HIV-1-infected children who have an excellent virological response, although they did not have obvious current symptoms. The CYTB region may play an important role in mtDNA mutation during ART, which might contribute to the development of subsequent mitochondrial diseases. PMID:27402477

  20. Intranasal HIV-1-gp160-DNA/gp41 peptide prime-boost immunization regimen in mice results in long-term HIV-1 neutralizing humoral mucosal and systemic immunity.

    PubMed

    Devito, Claudia; Zuber, Bartek; Schröder, Ulf; Benthin, Reinhold; Okuda, Kenji; Broliden, Kristina; Wahren, Britta; Hinkula, Jorma

    2004-12-01

    An intranasal DNA vaccine prime followed by a gp41 peptide booster immunization was compared with gp41 peptide and control immunizations. Serum HIV-1-specific IgG and IgA as well as IgA in feces and vaginal and lung secretions were detected after immunizations. Long-term humoral immunity was studied for up to 12 mo after the booster immunization by testing the presence of HIV-1 gp41- and CCR5-specific Abs and IgG/IgA-secreting B lymphocytes in spleen and regional lymph nodes in immunized mice. A long-term IgA-specific response in the intestines, vagina, and lungs was obtained in addition to a systemic immune response. Mice immunized only with gp41 peptides and L3 adjuvant developed a long-term gp41-specific serum IgG response systemically, although over a shorter period (1-9 mo), and long-term mucosal gp41-specific IgA immunity. HIV-1-neutralizing serum Abs were induced that were still present 12 mo after booster immunization. HIV-1 SF2-neutralizing fecal and lung IgA was detectable only in the DNA-primed mouse groups. Intranasal DNA prime followed by one peptide/L3 adjuvant booster immunization, but not a peptide prime followed by a DNA booster, was able to induce B cell memory and HIV-1-neutralizing Abs for at least half of a mouse's life span. PMID:15557206

  1. 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

  2. Uracil DNA glycosylase initiates degradation of HIV-1 cDNA containing misincorporated dUTP and prevents viral integration

    PubMed Central

    Weil, Amy F.; Ghosh, Devlina; Zhou, Yan; Seiple, Lauren; McMahon, Moira A.; Spivak, Adam M.; Siliciano, Robert F.; Stivers, James T.

    2013-01-01

    HIV-1 reverse transcriptase discriminates poorly between dUTP and dTTP, and accordingly, viral DNA products become heavily uracilated when viruses infect host cells that contain high ratios of dUTP:dTTP. Uracilation of invading retroviral DNA is thought to be an innate immunity barrier to retroviral infection, but the mechanistic features of this immune pathway and the cellular fate of uracilated retroviral DNA products is not known. Here we developed a model system in which the cellular dUTP:dTTP ratio can be pharmacologically increased to favor dUTP incorporation, allowing dissection of this innate immunity pathway. When the virus-infected cells contained elevated dUTP levels, reverse transcription was found to proceed unperturbed, but integration and viral protein expression were largely blocked. Furthermore, successfully integrated proviruses lacked detectable uracil, suggesting that only nonuracilated viral DNA products were integration competent. Integration of the uracilated proviruses was restored using an isogenic cell line that had no detectable human uracil DNA glycosylase (hUNG2) activity, establishing that hUNG2 is a host restriction factor in cells that contain high dUTP. Biochemical studies in primary cells established that this immune pathway is not operative in CD4+ T cells, because these cells have high dUTPase activity (low dUTP), and only modest levels of hUNG activity. Although monocyte-derived macrophages have high dUTP levels, these cells have low hUNG activity, which may diminish the effectiveness of this restriction pathway. These findings establish the essential elements of this pathway and reconcile diverse observations in the literature. PMID:23341616

  3. Single genome amplification of proviral HIV-1 DNA from dried blood spot specimens collected during early infant screening programs in Lusaka, Zambia

    PubMed Central

    Seu, Lillian; Mwape, Innocent; Guffey, M. Bradford

    2014-01-01

    The ability to evaluate individual HIV-1 virions from the quasispecies of vertically infected infants was evaluated in a field setting at the Centre for Infectious Disease Research in Zambia. Infant heel-prick blood specimens were spotted onto dried blood spot (DBS) filter paper cards at government health clinics. Nucleic acid was extracted and used as a template for HIV-1 proviral DNA detection by a commercial Amplicor HIV-1 PCR test (Roche, version 1.5). On samples that tested positive by commercial diagnostic assay, amplification of DNA was performed using an in-house assay of the 5′ and 3′ region of the HIV-1 genome. Additionally, fragments covering 1200 nucleotides within pol (full length protease and partial reverse transcriptase) and 1400 nucleotides within env (variable 1-variable 5 region) were further analyzed by single genome amplification (SGA). In summary, we have demonstrated an in-house assay for amplifying the 5′ and 3′ proviral HIV-1 DNA as well as pol and env proviral DNA fragments from DBS cards collected and analyzed entirely in Zambia. In conclusion, this study shows the feasibility of utilizing DBS cards to amplify the whole proviral HIV-1 genome as well as perform SGA on key HIV-1 genes. PMID:24667303

  4. Integrase-independent HIV-1 infection is augmented under conditions of DNA damage and produces a viral reservoir

    SciTech Connect

    Ebina, Hirotaka Kanemura, Yuka; Suzuki, Yasutsugu; Urata, Kozue; Misawa, Naoko; Koyanagi, Yoshio

    2012-05-25

    HIV-1 possesses a viral protein, integrase (IN), which is necessary for its efficient integration in target cells. However, it has been reported that an IN-defective HIV strain is still capable of integration. Here, we assessed the ability of wild type (WT) HIV-1 to establish infection in the presence of IN inhibitors. We observed a low, yet clear infection of inhibitor-incubated cells infected with WT HIV which was identical to cells infected with IN-deficient HIV, D64A. Furthermore, the IN-independent integration could be enhanced by the pretreatment of cells with DNA-damaging agents suggesting that integration is mediated by a DNA repair system. Moreover, significantly faster viral replication kinetics with augmented viral DNA integration was observed after infection in irradiated cells treated with IN inhibitor compared to nonirradiated cells. Altogether, our results suggest that HIV DNA has integration potential in the presence of an IN inhibitor and may serve as a virus reservoir.

  5. Docking of anti-HIV-1 oxoquinoline-acylhydrazone derivatives as potential HSV-1 DNA polymerase inhibitors

    NASA Astrophysics Data System (ADS)

    Yoneda, Julliane Diniz; Albuquerque, Magaly Girão; Leal, Kátia Zaccur; Santos, Fernanda da Costa; Batalha, Pedro Netto; Brozeguini, Leonardo; Seidl, Peter R.; de Alencastro, Ricardo Bicca; Cunha, Anna Cláudia; de Souza, Maria Cecília B. V.; Ferreira, Vitor F.; Giongo, Viveca A.; Cirne-Santos, Cláudio; Paixão, Izabel C. P.

    2014-09-01

    Although there are many antiviral drugs available for the treatment of herpes simplex virus (HSV) infections, still the synthesis of new anti-HSV candidates is an important strategy to be pursued, due to the emergency of resistant HSV strains mainly in human immunodeficiency virus (HIV) co-infected patients. Some 1,4-dihydro-4-oxoquinolines, such as PNU-183792 (1), show a broad spectrum antiviral activity against human herpes viruses, inhibiting the viral DNA polymerase (POL) without affecting the human POLs. Thus, on an ongoing antiviral research project, our group has synthesized ribonucleosides containing the 1,4-dihydro-4-oxoquinoline (quinolone) heterocyclic moiety, such as the 6-Cl derivative (2), which is a dual antiviral agent (HSV-1 and HIV-1). Molecular dynamics simulations of the complexes of 1 and 2 with the HSV-1 POL suggest that structural modifications of 2 should increase its experimental anti-HSV-1 activity, since its ribosyl and carboxyl groups are highly hydrophilic to interact with a hydrophobic pocket of this enzyme. Therefore, in this work, comparative molecular docking simulations of 1 and three new synthesized oxoquinoline-acylhydrazone HIV-1 inhibitors (3-5), which do not contain those hydrophilic groups, were carried out, in order to access these modifications in the proposition of new potential anti-HSV-1 agents, but maintaining the anti-HIV-1 activity. Among the docked compounds, the oxoquinoline-acylhydrazone 3 is the best candidate for an anti-HSV-1 agent, and, in addition, it showed anti-HIV-1 activity (EC50 = 3.4 ± 0.3 μM). Compounds 2 and 3 were used as templates in the design of four new oxoquinoline-acylhydrazones (6-9) as potential anti-HSV-1 agents to increase the antiviral activity of 2. Among the docked compounds, oxoquinoline-acylhydrazone 7 was selected as the best candidate for further development of dual anti-HIV/HSV activity.

  6. Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein

    PubMed Central

    Kanevsky, Igor; Chaminade, Françoise; Chen, Yingying; Godet, Julien; René, Brigitte; Darlix, Jean-Luc; Mély, Yves; Mauffret, Olivier; Fossé, Philippe

    2011-01-01

    Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA–DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop–loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop–loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open ‘Y’ conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the ‘Y’ conformation exhibiting at least 12 unpaired nucleotides in its lower part. PMID:21724607

  7. Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein.

    PubMed

    Kanevsky, Igor; Chaminade, Françoise; Chen, Yingying; Godet, Julien; René, Brigitte; Darlix, Jean-Luc; Mély, Yves; Mauffret, Olivier; Fossé, Philippe

    2011-10-01

    Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA-DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop-loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop-loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open 'Y' conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the 'Y' conformation exhibiting at least 12 unpaired nucleotides in its lower part. PMID:21724607

  8. Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA

    PubMed Central

    Masuda, Takao; Sato, Yoko; Huang, Yu-Lun; Koi, Satoshi; Takahata, Tatsuro; Hasegawa, Atsuhiko; Kawai, Gota; Kannagi, Mari

    2015-01-01

    Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription. PMID:26631448

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

    PubMed

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

    2009-05-19

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

  10. DNA prime-protein boost using subtype consensus Env was effective in eliciting neutralizing antibody responses against subtype BC HIV-1 viruses circulating in China

    PubMed Central

    Zhang, Mingshun; Zhang, Lu; Zhang, Chunhua; Hong, Kunxue; Shao, Yiming; Huang, Zuhu; Wang, Shixia; Lu, Shan

    2012-01-01

    Previously, we have shown that DNA prime-protein boost is effective in eliciting neutralizing antibodies (NAb) against randomly selected HIV-1 isolates. Given the genetic diversity of HIV-1 viruses and the unique predominant subtypes in different geographic regions, it is critical to test the DNA prime-protein boost approach against circulating viral isolates in key HIV endemic areas. In the current study, the same DNA prime-protein boost vaccine was used as in previous studies to investigate the induction of NAb responses against HIV-1 clade BC, a major subtype circulating in China. A codon optimized gp120-BC DNA vaccine, based on the consensus envelope (Env) antigen sequence of clade BC, was constructed and a stable CHO cell line expressing the same consensus BC gp120 protein was produced. The immunogenicity of this consensus gp120-BC was examined in New Zealand White rabbits by either DNA prime-protein boost or protein alone vaccination approaches. High levels of Env-specific antibody responses were elicited by both approaches. However, DNA prime-protein boost but not the protein alone immune sera contained significant levels of NAb against pseudotyped viruses expressing HIV-1 BC Env antigens. Furthermore, high frequencies of CD4 binding site-targeted antibodies were found in the DNA prime- protein boost rabbit sera indicating that the positive NAb may be the result of antibodies against conformationally sensitive epitopes on HIV-1 Env. The findings support that DNA prime-protein boost was effective in eliciting NAb against a key HIV-1 virus subtype in China. This result may lead to the development of regional HIV vaccines through this approach. PMID:23111170

  11. RING Domain Mutations Uncouple TRIM5α Restriction of HIV-1 from Inhibition of Reverse Transcription and Acceleration of Uncoating

    PubMed Central

    Roa, Amanda; Hayashi, Fumiaki; Yang, Yang; Lienlaf, Maritza; Zhou, Jing; Shi, Jiong; Watanabe, Satoru; Kigawa, Takanori; Yokoyama, Shigeyuki; Aiken, Christopher

    2012-01-01

    Rhesus TRIM5α (TRIM5αrh) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5αrh to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5α to block reverse transcription, we studied a set of TRIM5αrh RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5αrh RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5α. Similar effects of this particular group of mutations were observed with human TRIM5α inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5αrh RING domain variants also prevented the degradation of TRIM5αrh that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5α to accelerate uncoating. Collectively, these results suggest that TRIM5αrh blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells. PMID:22114335

  12. RING domain mutations uncouple TRIM5α restriction of HIV-1 from inhibition of reverse transcription and acceleration of uncoating.

    PubMed

    Roa, Amanda; Hayashi, Fumiaki; Yang, Yang; Lienlaf, Maritza; Zhou, Jing; Shi, Jiong; Watanabe, Satoru; Kigawa, Takanori; Yokoyama, Shigeyuki; Aiken, Christopher; Diaz-Griffero, Felipe

    2012-02-01

    Rhesus TRIM5α (TRIM5α(rh)) is a cytosolic protein that potently restricts HIV-1 at an early postentry stage, prior to reverse transcription. The ability of TRIM5α(rh) to block HIV-1 infection has been correlated with a decrease of pelletable HIV-1 capsid during infection. To genetically dissect the ability of TRIM5α to block reverse transcription, we studied a set of TRIM5α(rh) RING domain mutants that potently restrict HIV-1 but allow the occurrence of reverse transcription. These TRIM5α(rh) RING variants blocked HIV-1 infection after reverse transcription but prior to integration, as suggested by the routing of nuclear viral DNA to circularization in the form of 2-long terminal repeat (2-LTR) circles. The folding of RING domain variants was similar to that of the wild type, as evaluated by nuclear magnetic resonance. RING domain changes that allowed the occurrence of reverse transcription were impaired in their ability to decrease the amount of pelletable capsid compared with wild-type TRIM5α. Similar effects of this particular group of mutations were observed with human TRIM5α inhibition of N-tropic murine leukemia virus (N-MLV). Interestingly, TRIM5α(rh) RING domain variants also prevented the degradation of TRIM5α(rh) that occurs following cell entry of HIV-1. These data correlated the block of reverse transcription with the ability of TRIM5α to accelerate uncoating. Collectively, these results suggest that TRIM5α(rh) blocks HIV-1 reverse transcription by inducing premature viral uncoating in target cells. PMID:22114335

  13. Four-tiered {pi} interaction at the dimeric interface of HIV-1 integrase critical for DNA integration and viral infectivity

    SciTech Connect

    Al-Mawsawi, Laith Q.; Hombrouck, Anneleen; Dayam, Raveendra; Debyser, Zeger; Neamati, Nouri

    2008-08-01

    HIV-1 integrase (IN) is an essential enzyme for viral infection. Here, we report an extensive {pi} electron orbital interaction between four amino acids, W132, M178, F181 and F185, located at the dimeric interface of IN that is critical for the strand transfer activity alone. Catalysis of nine different mutant IN proteins at these positions were evaluated. Whereas the 3'-processing activity is predominantly strong, the strand transfer activity of each enzyme was completely dependent on an intact {pi} electron orbital interaction at the dimeric interface. Four representative IN mutants were constructed in the context of the infectious NL4.3 HIV-1 viral clone. Whereas viruses with an intact {pi} electron orbital interaction at the IN dimeric interface replicated comparable to wild type, viruses containing an abolished {pi} interaction were non-infectious. Q-PCR analysis of viral DNA forms during viral replication revealed pleiotropic effects of most mutations. We hypothesize that the {pi} interaction is a critical contact point for the assembly of functional IN multimeric complexes, and that IN multimerization is required for a functional pre-integration complex. The rational design of small molecule inhibitors targeting the disruption of this {pi}-{pi} interaction should lead to powerful anti-retroviral drugs.

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

    PubMed

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

    2004-08-01

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

  15. Soluble multi-trimeric TNF superfamily ligand adjuvants enhance immune responses to a HIV-1 Gag DNA vaccine

    PubMed Central

    Kanagavelu, Saravana K.; Snarsky, Victoria; Termini, James M.; Gupta, Sachin; Barzee, Suzanne; Wright, Jacqueline A.; Khan, Wasif N.; Kornbluth, Richard S.; Stone, Geoffrey W.

    2011-01-01

    Background DNA vaccines remain an important component of HIV vaccination strategies, typically as part of a prime/boost vaccination strategy with viral vector or protein boost. A number of DNA prime/viral vector boost vaccines are currently being evaluated for both preclinical studies and in Phase I and Phase II clinical trials. These vaccines would benefit from molecular adjuvants that increase correlates of immunity during the DNA prime. While HIV vaccine immune correlates are still not well defined, there are a number of immune assays that have been shown to correlate with protection from viral challenge including CD8+ T cell avidity, antigen-specific proliferation, and polyfunctional cytokine secretion. Methodology and Principal Findings Recombinant DNA vaccine adjuvants composed of a fusion between Surfactant Protein D (SP-D) and either CD40 Ligand (CD40L) or GITR Ligand (GITRL) were previously shown to enhance HIV-1 Gag DNA vaccines. Here we show that similar fusion constructs composed of the TNF superfamily ligands (TNFSFL) 4-1BBL, OX40L, RANKL, LIGHT, CD70, and BAFF can also enhanced immune responses to a HIV-1 Gag DNA vaccine. BALB/c mice were vaccinated intramuscularly with plasmids expressing secreted Gag and SP-D-TNFSFL fusions. Initially, mice were analyzed 2 weeks or 7 weeks following vaccination to evaluate the relative efficacy of each SP-D-TNFSFL construct. All SP-D-TNFSFL constructs enhanced at least one Gag-specific immune response compared to the parent vaccine. Importantly, the constructs SP-D-4-1BBL, SP-D-OX40L, and SP-D-LIGHT enhanced CD8+ T cell avidity and CD8+/CD4+ T cell proliferation 7 weeks post vaccination. These avidity and proliferation data suggest that 4-1BBL, OX40L, and LIGHT fusion constructs may be particularly effective as vaccine adjuvants. Constructs SP-D-OX40L, SP-D-LIGHT, and SP-D-BAFF enhanced Gag-specific IL-2 secretion in memory T cells, suggesting these adjuvants can increase the number of self-renewing Gag-specific CD8

  16. Cholera toxin B subunit acts as a potent systemic adjuvant for HIV-1 DNA vaccination intramuscularly in mice

    PubMed Central

    Hou, Jue; Liu, Ying; Hsi, Jenny; Wang, Hongzhi; Tao, Ran; Shao, Yiming

    2014-01-01

    Cholera toxin B subunit (CTB) was investigated as a classical mucosal adjuvant that can increase vaccine immunogenicity. In this study, we found out the in vitro efficacy of cholera toxin B subunit (CTB) in activating mice bone marrow-derived dendritic cells (BMDCs) through Toll-like receptor signaling pathways. In vitro RNA and transcriptional level profiling arrays revealed that CTB guides high levels of Th1 and Th2 type cytokines, inflammatory cytokines, and chemokines. Based on the robustness of these profiling results, we examined the induction of HIV Env-specific immunity by CTB co-inoculated with HIV Env DNA vaccine intramuscularly in vivo. CTB enhanced HIV-Env specific cellular immune responses in Env-specific IFN-γ ELISPOT, compared with DNA vaccine alone. Moreover, CTB induced high levels of Env specific humoral response and promoted antibody maturation after the third round of vaccination. This combination immunization strategy induced a Th2-type bias response which is indicative of a high ratio of IgG1/IgG2a. This study reports that CTB as a classical mucosal adjuvant could enhance HIV-1 DNA-based vaccine immunogenicity intramuscularly; therefore, these findings suggest that CTB could serve as an effective candidate adjuvant for DNA vaccination. PMID:24633335

  17. HIV-1 nucleocapsid and ESCRT-component Tsg101 interplay prevents HIV from turning into a DNA-containing virus.

    PubMed

    Chamontin, Célia; Rassam, Patrice; Ferrer, Mireia; Racine, Pierre-Jean; Neyret, Aymeric; Lainé, Sébastien; Milhiet, Pierre-Emmanuel; Mougel, Marylène

    2015-01-01

    HIV-1, the agent of the AIDS pandemic, is an RNA virus that reverse transcribes its RNA genome (gRNA) into DNA, shortly after its entry into cells. Within cells, retroviral assembly requires thousands of structural Gag proteins and two copies of gRNA as well as cellular factors, which converge to the plasma membrane in a finely regulated timeline. In this process, the nucleocapsid domain of Gag (GagNC) ensures gRNA selection and packaging into virions. Subsequent budding and virus release require the recruitment of the cellular ESCRT machinery. Interestingly, mutating GagNC results into the release of DNA-containing viruses, by promo-ting reverse transcription (RTion) prior to virus release, through an unknown mechanism. Therefore, we explored the biogenesis of these DNA-containing particles, combining live-cell total internal-reflection fluorescent microscopy, electron microscopy, trans-complementation assays and biochemical characterization of viral particles. Our results reveal that DNA virus production is the consequence of budding defects associated with Gag aggregation at the plasma membrane and deficiency in the recruitment of Tsg101, a key ESCRT-I component. Indeed, targeting Tsg101 to virus assembly sites restores budding, restricts RTion and favors RNA packaging into viruses. Altogether, our results highlight the role of GagNC in the spatiotemporal control of RTion, via an ESCRT-I-dependent mechanism. PMID:25488808

  18. HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis.

    PubMed

    Jacques, David A; McEwan, William A; Hilditch, Laura; Price, Amanda J; Towers, Greg J; James, Leo C

    2016-08-18

    During the early stages of infection, the HIV-1 capsid protects viral components from cytosolic sensors and nucleases such as cGAS and TREX, respectively, while allowing access to nucleotides for efficient reverse transcription. Here we show that each capsid hexamer has a size-selective pore bound by a ring of six arginine residues and a 'molecular iris' formed by the amino-terminal β-hairpin. The arginine ring creates a strongly positively charged channel that recruits the four nucleotides with on-rates that approach diffusion limits. Progressive removal of pore arginines results in a dose-dependent and concomitant decrease in nucleotide affinity, reverse transcription and infectivity. This positively charged channel is universally conserved in lentiviral capsids despite the fact that it is strongly destabilizing without nucleotides to counteract charge repulsion. We also describe a channel inhibitor, hexacarboxybenzene, which competes for nucleotide binding and efficiently blocks encapsidated reverse transcription, demonstrating the tractability of the pore as a novel drug target. PMID:27509857

  19. Source identification in two criminal cases using phylogenetic analysis of HIV-1 DNA sequences.

    PubMed

    Scaduto, Diane I; Brown, Jeremy M; Haaland, Wade C; Zwickl, Derrick J; Hillis, David M; Metzker, Michael L

    2010-12-14

    Phylogenetic analysis has been widely used to test the a priori hypothesis of epidemiological clustering in suspected transmission chains of HIV-1. Among studies showing strong support for relatedness between HIV samples obtained from infected individuals, evidence for the direction of transmission between epidemiologically related pairs has been lacking. During transmission of HIV, a genetic bottleneck occurs, resulting in the paraphyly of source viruses with respect to those of the recipient. This paraphyly establishes the direction of transmission, from which the source can then be inferred. Here, we present methods and results from two criminal cases, State of Washington v Anthony Eugene Whitfield, case number 04-1-0617-5 (Superior Court of the State of Washington, Thurston County, 2004) and State of Texas v Philippe Padieu, case numbers 219-82276-07, 219-82277-07, 219-82278-07, 219-82279-07, 219-82280-07, and 219-82705-07 (219th Judicial District Court, Collin County, TX, 2009), which provided evidence that direction can be established from blinded case samples. The observed paraphyly from each case study led to the identification of an inferred source (i.e., index case), whose identity was revealed at trial to be that of the defendant. PMID:21078965

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

    PubMed Central

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

    2009-01-01

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

  1. Clustered epitopes within a new poly-epitopic HIV-1 DNA vaccine shows immunogenicity in BALB/c mice.

    PubMed

    Jafarpour, Nazli; Memarnejadian, Arash; Aghasadeghi, Mohammad Reza; Kohram, Fatemeh; Aghababa, Haniyeh; Khoramabadi, Nima; Mahdavi, Mehdi

    2014-08-01

    Despite a huge number of studies towards vaccine development against human immunodeficiency virus-1, no effective vaccine has been approved yet. Thus, new vaccines should be provided with new formulations. Herein, a new DNA vaccine candidate encoding conserved and immunogenic epitopes from HIV-1 antigens of tat, pol, gag and env is designed and constructed. After bioinformatics analyses to find the best epitopes and their tandem, nucleotide sequence corresponding to the designed multiepitope was synthesized and cloned into pcDNA3.1+ vector. Expression of pcDNA3.1-tat/pol/gag/env plasmid was evaluated in HEK293T cells by RT-PCR and western-blotting. Seven groups of BALB/c mice were intramuscularly immunized three times either with 50, 100, 200 µg of plasmid in 2-week intervals or with similar doses of insert-free plasmid. Two weeks after the last injection, proliferation of T cells and secretion of IL4 and IFN-γ cytokines were evaluated using Brdu and ELISA methods, respectively. Results showed the proper expression of the plasmid in protein and mRNA levels. Moreover, the designed multiepitope plasmid was capable of induction of both proliferation responses as well as IFN-γ and IL-4 cytokine production in a considerable level compared to the control groups. Overall, our primary data warranted further detailed studies on the potency of this vaccine. PMID:24842263

  2. Mitochondrial DNA Haplogroup A Decreases the Risk of Drug Addiction but Conversely Increases the Risk of HIV-1 Infection in Chinese Addicts.

    PubMed

    Zhang, A-Mei; Hu, Qiu-Xiang; Liu, Feng-Liang; Bi, Rui; Yang, Bi-Qing; Zhang, Wen; Guo, Hao; Logan, Ian; Zheng, Yong-Tang; Yao, Yong-Gang

    2016-08-01

    Drug addiction is one of the most serious social problems in the world today and addicts are always at a high risk of acquiring HIV infection. Mitochondrial impairment has been reported in both drug addicts and in HIV patients undergoing treatment. In this study, we aimed to investigate whether mitochondrial DNA (mtDNA) haplogroup could affect the risk of drug addiction and HIV-1 infection in Chinese. We analyzed mtDNA sequence variations of 577 Chinese intravenous drug addicts (289 with HIV-1 infection and 288 without) and compared with 2 control populations (n = 362 and n = 850). We quantified the viral load in HIV-1-infected patients with and without haplogroup A status and investigated the potential effect of haplogroup A defining variants m.4824A > G and m.8794C > T on the cellular reactive oxygen species (ROS) levels by using an allotopic expression assay. mtDNA haplogroup A had a protective effect against drug addiction but appeared to confer an increased risk of HIV infection in addicts. HIV-1-infected addicts with haplogroup A had a trend for a higher viral load, although the mean viral load was similar between carriers of haplogroup A and those with other haplogroup. Hela cells overexpressing allele m.8794 T showed significantly decreased ROS levels as compared to cells with the allele m.8794C (P = 0.03). Our results suggested that mtDNA haplogroup A might protect against drug addiction but increase the risk of HIV-1 infection. The contradictory role of haplogroup A might be caused by an alteration in mitochondrial function due to a particular mtDNA ancestral variant. PMID:26162319

  3. U3 Region in the HIV-1 Genome Adopts a G-Quadruplex Structure in Its RNA and DNA Sequence

    PubMed Central

    2015-01-01

    Genomic regions rich in G residues are prone to adopt G-quadruplex structure. Multiple Sp1-binding motifs arranged in tandem have been suggested to form this structure in promoters of cancer-related genes. Here, we demonstrate that the G-rich proviral DNA sequence of the HIV-1 U3 region, which serves as a promoter of viral transcription, adopts a G-quadruplex structure. The sequence contains three binding elements for transcription factor Sp1, which is involved in the regulation of HIV-1 latency, reactivation, and high-level virus expression. We show that the three Sp1 binding motifs can adopt different forms of G-quadruplex structure and that the Sp1 protein can recognize and bind to its site folded into a G-quadruplex. In addition, a c-kit2 specific antibody, designated hf2, binds to two different G-quadruplexes formed in Sp1 sites. Since U3 is encoded at both viral genomic ends, the G-rich sequence is also present in the RNA genome. We demonstrate that the RNA sequence of U3 forms dimers with characteristics known for intermolecular G-quadruplexes. Together with previous reports showing G-quadruplex dimers in the gag and cPPT regions, these results suggest that integrity of the two viral genomes is maintained through numerous intermolecular G-quadruplexes formed in different RNA genome locations. Reconstituted reverse transcription shows that the potassium-dependent structure formed in U3 RNA facilitates RT template switching, suggesting that the G-quadruplex contributes to recombination in U3. PMID:24735378

  4. HIV-1 Integrase Strand Transfer Inhibitors Stabilize an Integrase-Single Blunt-Ended DNA Complex

    PubMed Central

    Bera, Sibes; Pandey, Krishan K.; Vora, Ajaykumar C.; Grandgenett, Duane P.

    2011-01-01

    Summary Integration of HIV (human immunodeficiency virus) cDNA ends by integrase (IN) into host chromosomes involves a concerted integration mechanism. IN juxtaposes two DNA blunt-ends to form the synaptic complex (SC) which is the intermediate in the concerted integration pathway. SC is inactivated by strand transfer inhibitors (STI) with IC50 values of ~20 nM for inhibition of concerted integration. We detected a new nucleoprotein complex on native agarose that was produced in the presence of STI >200 nM, termed IN-single DNA (ISD) complex. Two IN dimers appear to bind in a parallel fashion at the DNA terminus producing a ~32 bp DNaseI protective footprint. In the presence of Raltegravir, MK-2048 and L-841,411, IN incorporated ~20 to 25% of the input blunt-ended DNA substrate into the stabilized ISD complex. Seven other STI also produced the ISD complex (≤ 5% of input DNA). The formation of the ISD complex was not dependent upon 3’ OH processing and the DNA was predominately blunt-ended in the complex. Raltegravir-resistant IN mutant N155H weakly form the ISD complex in the presence of Raltegravir at ~25% level of wild type IN. In contrast, MK-2048 and L-841,411 produced ~3 to 5-fold more ISD than Raltegravir with N155H IN, which is susceptible to these two inhibitors. The results suggest STI are slow binding inhibitors and the potency to form and stabilize the ISD complex is not always related to inhibition of concerted integration. Rather, the apparent binding and dissociation properties of each STI influenced the production of the ISD complex. PMID:21295584

  5. Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin.

    PubMed

    Zargarian, Loussiné; Kanevsky, Igor; Bazzi, Ali; Boynard, Jonathan; Chaminade, Françoise; Fossé, Philippe; Mauffret, Olivier

    2009-07-01

    First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop-loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction. PMID:19417069

  6. Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

    PubMed Central

    Zargarian, Loussiné; Kanevsky, Igor; Bazzi, Ali; Boynard, Jonathan; Chaminade, Françoise; Fossé, Philippe; Mauffret, Olivier

    2009-01-01

    First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction. PMID:19417069

  7. Effects of recombinant human growth hormone on HIV-1-specific T-cell responses, thymic output and proviral DNA in patients on HAART: 48-week follow-up

    PubMed Central

    Herasimtschuk, Anna A; Westrop, Samantha J; Moyle, Graeme J; Downey, Jocelyn S; Imami, Nesrina

    2008-01-01

    Background Efficacious immune-based therapy in treated chronic HIV-1 infection requires the induction of virus-specific CD4+ T cells and subsequent maturation and maintenance of specific memory CD8+ T cells. Concomitant daily administration of recombinant human growth hormone (rhGH) with highly active antiretroviral therapy (HAART) was used in chronically infected patients with lipodystrophy in an attempt to reconstitute these virus-specific T-cell responses. Methods Individuals with chronic HIV-1 infection on HAART were enrolled on a randomized, double-blinded, placebo-controlled study to receive rhGH therapy. We assessed HIV-1-specific proliferative CD4+ and interferon-gamma (IFN-γ)-producing CD8+ T-cell responses, quantified thymic output and proviral HIV-1 DNA at the following time points: baseline; after 12 weeks of rhGH therapy; at 24 weeks, after randomization into three groups [placebo weeks 12–24 (Group A), alternate-day dosing weeks 12–24 (Group B), and twice-per-week dosing weeks 12–24 (Group C)]; and at 48 weeks after all patients had received HAART alone for the final 24 weeks. Results We found significant increases in both proliferative CD4+ and IFN-γ-producing CD8+ HIV-1-specific T-cell responses after daily administration of rhGH. This increase was focused on HIV-1 Gag-specific T-cell responses. Following subsequent randomisation into different dosing regimens, HIV-1-specific proliferative CD4+ T-cell responses declined in patients receiving less frequent dosing of rhGH, while virus-specific IFN-γ-producing CD8+ T-cell responses were maintained for longer periods of time. There was no significant change in thymic output and the cell-associated HIV-1 DNA remained stable in most patients. An increased anti-HIV-1 Nef-specific CD4+ T-cell proliferative response was correlated to a decrease in proviral load, and an increased HIV-1 Gag-specific IFN-γ-producing CD8+ T-cell response correlated with an increase in proviral load. Conclusion The

  8. Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine.

    PubMed

    Yan, Jian; Corbitt, Natasha; Pankhong, Panyupa; Shin, Thomas; Khan, Amir; Sardesai, Niranjan Y; Weiner, David B

    2011-09-22

    DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding

  9. In Vitro HIV-1 LTR Integration into T-Cell Activation Gene CD27 Segment and the Decoy Effect of Modified-Sequence DNA

    PubMed Central

    Ohmori, Rei; Tsuruyama, Tatsuaki

    2012-01-01

    Integration into the host genome is an essential step in the HIV-1 life cycle. However, the host genome sequence that is favored by HIV-1 during integration has never been documented. Here, we report that CD27, a T cell activation gene, includes a sequence that is a target for in vitro HIV-1 cDNA integration. This sequence has a high affinity for integrase, and the target nucleotides responsible for this higher affinity were identified using a crystal microbalance assay. In experiments involving a segment of the CD27 gene, integration converged in the target nucleotides and flanking sequence DNA, indicating that integration is probably dependent upon the secondary structure of the substrate DNA. Notably, decoy modified CD27 sequence DNAs in which the target nucleotides were replaced suppressed integration when accompanying the original CD27 sequence DNA. Our identified CD27 sequence DNA is useful for investigating the biochemistry of integrase and for in vitro assessment of integrase-binding inhibitors. PMID:23209625

  10. Priming with a Simplified Intradermal HIV-1 DNA Vaccine Regimen followed by Boosting with Recombinant HIV-1 MVA Vaccine Is Safe and Immunogenic: A Phase IIa Randomized Clinical Trial

    PubMed Central

    Nilsson, Charlotta; Joachim, Agricola; Geldmacher, Christof; Mann, Philipp; Moshiro, Candida; Aboud, Said; Lyamuya, Eligius; Maboko, Leonard; Missanga, Marco; Kaluwa, Bahati; Mfinanga, Sayoki; Podola, Lilly; Bauer, Asli; Godoy-Ramirez, Karina; Marovich, Mary; Moss, Bernard; Hoelscher, Michael; Gotch, Frances; Stöhr, Wolfgang; Stout, Richard; McCormack, Sheena; Wahren, Britta; Mhalu, Fred; Robb, Merlin L.; Biberfeld, Gunnel; Sandström, Eric; Bakari, Muhammad

    2015-01-01

    Background Intradermal priming with HIV-1 DNA plasmids followed by HIV-1MVA boosting induces strong and broad cellular and humoral immune responses. In our previous HIVIS-03 trial, we used 5 injections with 2 pools of HIV-DNA at separate sites for each priming immunization. The present study explores whether HIV-DNA priming can be simplified by reducing the number of DNA injections and administration of combined versus separated plasmid pools. Methods In this phase IIa, randomized trial, priming was performed using 5 injections of HIV-DNA, 1000 μg total dose, (3 Env and 2 Gag encoding plasmids) compared to two “simplified” regimens of 2 injections of HIV-DNA, 600 μg total dose, of Env- and Gag-encoding plasmid pools with each pool either administered separately or combined. HIV-DNA immunizations were given intradermally at weeks 0, 4, and 12. Boosting was performed intramuscularly with 108 pfu HIV-MVA at weeks 30 and 46. Results 129 healthy Tanzanian participants were enrolled. There were no differences in adverse events between the groups. The proportion of IFN-γ ELISpot responders to Gag and/or Env peptides after the second HIV-MVA boost did not differ significantly between the groups primed with 2 injections of combined HIV-DNA pools, 2 injections with separated pools, and 5 injections with separated pools (90%, 97% and 97%). There were no significant differences in the magnitude of Gag and/or Env IFN-γ ELISpot responses, in CD4+ and CD8+ T cell responses measured as IFN-γ/IL-2 production by intracellular cytokine staining (ICS) or in response rates and median titers for binding antibodies to Env gp160 between study groups. Conclusions A simplified intradermal vaccination regimen with 2 injections of a total of 600 μg with combined HIV-DNA plasmids primed cellular responses as efficiently as the standard regimen of 5 injections of a total of 1000 μg with separated plasmid pools after boosting twice with HIV-MVA. Trial Registration World Health

  11. HIV-DNA Given with or without Intradermal Electroporation Is Safe and Highly Immunogenic in Healthy Swedish HIV-1 DNA/MVA Vaccinees: A Phase I Randomized Trial

    PubMed Central

    Nilsson, Charlotta; Hejdeman, Bo; Godoy-Ramirez, Karina; Tecleab, Teghesti; Scarlatti, Gabriella; Bråve, Andreas; Earl, Patricia L.; Stout, Richard R.; Robb, Merlin L.; Shattock, Robin J.; Biberfeld, Gunnel; Sandström, Eric; Wahren, Britta

    2015-01-01

    Background We compared safety and immunogenicity of intradermal (ID) vaccination with and without electroporation (EP) in a phase I randomized placebo-controlled trial of an HIV-DNA prime HIV-MVA boost vaccine in healthy Swedish volunteers. Methods HIV-DNA plasmids encoding HIV-1 genes gp160 subtypes A, B and C; Rev B; Gag A and B and RTmut B were given ID at weeks 0, 6 and 12 in a dose of 0.6 mg. Twenty-five volunteers received vaccine using a needle-free device (ZetaJet) with (n=16) or without (n=9) ID EP (Dermavax). Five volunteers were placebo recipients. Boosting with recombinant MVA-CMDR expressing HIV-1 Env, Gag, Pol of CRF01_AE (HIV-MVA) or placebo was performed at weeks 24 and 40. Nine of the vaccinees received a subtype C CN54 gp140 protein boost together with HIV-MVA. Results The ID/EP delivery was very well tolerated. After three HIV-DNA immunizations, no statistically significant difference was seen in the IFN-γ ELISpot response rate to Gag between HIV-DNA ID/EP recipients (5/15, 33%) and HIV-DNA ID recipients (1/7, 14%, p=0.6158). The first HIV-MVA or HIV-MVA+gp140 vaccination increased the IFN-γ ELISpot response rate to 18/19 (95%). CD4+ and/or CD8+ T cell responses to Gag or Env were demonstrable in 94% of vaccinees. A balanced CD4+ and CD8+ T cell response was noted, with 78% and 71% responders, respectively. IFN-γ and IL-2 dominated the CD4+ T cell response to Gag and Env. The CD8+ response to Gag was broader with expression of IFN-γ, IL-2, MIP-1β and/or CD107. No differences were seen between DNA vaccine groups. Binding antibodies were induced after the second HIV-MVA+/-gp140 in 93% of vaccinees to subtype C Env, with the highest titers among EP/gp140 recipients. Conclusion Intradermal electroporation of HIV-DNA was well tolerated. Strong cell- and antibody-mediated immune responses were elicited by the HIV-DNA prime and HIV-MVA boosting regimen, with or without intradermal electroporation use. Trial Registration International Standard

  12. Safety and Comparative Immunogenicity of an HIV-1 DNA Vaccine in Combination with Plasmid Interleukin 12 and Impact of Intramuscular Electroporation for Delivery

    PubMed Central

    Kalams, Spyros A.; Parker, Scott D.; Elizaga, Marnie; Metch, Barbara; Edupuganti, Srilatha; Hural, John; De Rosa, Stephen; Carter, Donald K.; Rybczyk, Kyle; Frank, Ian; Fuchs, Jonathan; Koblin, Beryl; Kim, Denny H.; Joseph, Patrice; Keefer, Michael C.; Baden, Lindsey R.; Eldridge, John; Boyer, Jean; Sherwat, Adam; Cardinali, Massimo; Allen, Mary; Pensiero, Michael; Butler, Chris; Khan, Amir S.; Yan, Jian; Sardesai, Niranjan Y.; Kublin, James G.; Weiner, David B.

    2013-01-01

    Background. DNA vaccines have been very poorly immunogenic in humans but have been an effective priming modality in prime-boost regimens. Methods to increase the immunogenicity of DNA vaccines are needed. Methods. HIV Vaccine Trials Network (HVTN) studies 070 and 080 were multicenter, randomized, clinical trials. The human immunodeficiency virus type 1 (HIV-1) PENNVAX®-B DNA vaccine (PV) is a mixture of 3 expression plasmids encoding HIV-1 Clade B Env, Gag, and Pol. The interleukin 12 (IL-12) DNA plasmid expresses human IL-12 proteins p35 and p40. Study subjects were healthy HIV-1–uninfected adults 18–50 years old. Four intramuscular vaccinations were given in HVTN 070, and 3 intramuscular vaccinations were followed by electroporation in HVTN 080. Cellular immune responses were measured by intracellular cytokine staining after stimulation with HIV-1 peptide pools. Results. Vaccination was safe and well tolerated. Administration of PV plus IL-12 with electroporation had a significant dose-sparing effect and provided immunogenicity superior to that observed in the trial without electroporation, despite fewer vaccinations. A total of 71.4% of individuals vaccinated with PV plus IL-12 plasmid with electroporation developed either a CD4+ or CD8+ T-cell response after the second vaccination, and 88.9% developed a CD4+ or CD8+ T-cell response after the third vaccination. Conclusions. Use of electroporation after PV administration provided superior immunogenicity than delivery without electroporation. This study illustrates the power of combined DNA approaches to generate impressive immune responses in humans. PMID:23840043

  13. Membrane bound Indian clade C HIV-1 envelope antigen induces antibodies to diverse and conserved epitopes upon DNA prime/protein boost in rabbits.

    PubMed

    Rangasamy, Sneha Priya; Menon, Veena; Dhopeshwarkar, Priyanka; Pal, Ranajit; Vaniambadi, Kalyanaraman S; Mahalingam, Sundarasamy

    2016-05-01

    The partial success of RV144 human clinical trial demonstrated that ALVAC prime/envelope protein boost vaccine regimen may represent a promising strategy for the development of an effective HIV-1 vaccine. Our earlier study demonstrated that a trimeric HIV-1 envelope gp145 from an Indian clade C isolate elicited cross clade neutralizing antibodies primarily towards Tier 1 isolates. In the present study, we examined the immunogenicity of DNA prime/envelope protein boost vaccine in rabbits using gp160 DNA of the Indian clade C isolate with various cytoplasmic tail truncations and trimeric gp145 protein. Cytoplasmic tail mutants of gp160 exposed epitopes that reacted strongly with a number of broadly neutralizing human monoclonal antibodies against HIV-1. Overall, envelope specific titers were found to be similar in all rabbit groups with higher pseudovirus neutralization in protein only immunized rabbits. The complete linear epitope mapping of rabbit immune sera revealed strong binding to C1, C2, V3, C3 and C4 domains of gp145. Importantly, reactivity of gp41 ecto-domain peptides was observed in DNA prime/protein boost sera but not in the sera of rabbits immunized with protein alone. Moreover, membrane anchored but not soluble envelope encoding DNA immunization elicited antibodies against linear epitopes on the conserved gp41 ecto-domain. Together, these results suggest that priming with DNA encoding cytoplasmic domains of Env alters the quality of antibodies elicited following protein boost and hence may be utilized to generate protective immunity by HIV-1 vaccine. PMID:27032514

  14. Ribonuclease H/DNA Polymerase HIV-1 Reverse Transcriptase Dual Inhibitor: Mechanistic Studies on the Allosteric Mode of Action of Isatin-Based Compound RMNC6

    PubMed Central

    Corona, Angela; Meleddu, Rita; Esposito, Francesca; Distinto, Simona; Bianco, Giulia; Masaoka, Takashi; Maccioni, Elias; Menéndez-Arias, Luis; Alcaro, Stefano; Le Grice, Stuart F. J.; Tramontano, Enzo

    2016-01-01

    The DNA polymerase and ribonuclease H (RNase H) activities of human immunodeficiency virus type 1 (HIV-1) are needed for the replication of the viral genome and are validated drug targets. However, there are no approved drugs inhibiting RNase H and the efficiency of DNA polymerase inhibitors can be diminished by the presence of drug resistance mutations. In this context, drugs inhibiting both activities could represent a significant advance towards better anti-HIV therapies. We report on the mechanisms of allosteric inhibition of a newly synthesized isatin-based compound designated as RMNC6 that showed IC50 values of 1.4 and 9.8 μM on HIV-1 RT-associated RNase H and polymerase activities, respectively. Blind docking studies predict that RMNC6 could bind two different pockets in the RT: one in the DNA polymerase domain (partially overlapping the non-nucleoside RT inhibitor [NNRTI] binding pocket), and a second one close to the RNase H active site. Enzymatic studies showed that RMNC6 interferes with efavirenz (an approved NNRTI) in its binding to the RT polymerase domain, although NNRTI resistance-associated mutations such as K103N, Y181C and Y188L had a minor impact on RT susceptibility to RMNC6. In addition, despite being naturally resistant to NNRTIs, the polymerase activity of HIV-1 group O RT was efficiently inhibited by RMNC6. The compound was also an inhibitor of the RNase H activity of wild-type HIV-1 group O RT, although we observed a 6.5-fold increase in the IC50 in comparison with the prototypic HIV-1 group M subtype B enzyme. Mutagenesis studies showed that RT RNase H domain residues Asn474 and Tyr501, and in a lesser extent Ala502 and Ala508, are critical for RMNC6 inhibition of the endonuclease activity of the RT, without affecting its DNA polymerization activity. Our results show that RMNC6 acts as a dual inhibitor with allosteric sites in the DNA polymerase and the RNase H domains of HIV-1 RT. PMID:26800261

  15. In situ detection of PCR-amplified HIV-1 nucleic acids and tumor necrosis factor cDNA in cervical tissues.

    PubMed Central

    Nuovo, G. J.; Forde, A.; MacConnell, P.; Fahrenwald, R.

    1993-01-01

    This study determined the histological distribution of polymerase chain reaction-amplified human immunodeficiency virus 1 (HIV-1) DNA and RNA in cervical tissues. Amplified HIV-1 DNA and complementary DNA were detected in each of 21 cervical biopsies from women with acquired immunodeficiency syndrome. The viral nucleic acids were most abundant in the endocervical aspect of the transformation zone at the interface of the glandular epithelium and the submucosa and in the deep submucosa around microvessels. Many virally infected cells colabeled with leukocyte common antigen, Mac387, and polymerase chain reaction-amplified tumor necrosis factor complementary DNA, demonstrating that they were activated macrophages. Virally amplified nucleic acids were not detected in 10 controls and in only one of eight cervical tissues from children less than 3 years of age who died due to immunodeficiency syndrome acquired in utero. Determining whether the HIV-1-infected macrophages consistently present in the cervix of adult seropositive women may represent primary infection and, if so, whether they can transport the virus to regional lymph nodes and thus initiate systemic infection requires further study. Images Figure 1 Figure 2 Figure 3 PMID:8317555

  16. Mode of action of SDZ NIM 811, a nonimmunosuppressive cyclosporin A analog with activity against human immunodeficiency virus type 1 (HIV-1): interference with early and late events in HIV-1 replication.

    PubMed

    Steinkasserer, A; Harrison, R; Billich, A; Hammerschmid, F; Werner, G; Wolff, B; Peichl, P; Palfi, G; Schnitzel, W; Mlynar, E

    1995-02-01

    SDZ NIM 811 is a cyclosporin A analog that is completely devoid of immunosuppressive capacity but exhibits potent and selective anti-human immunodeficiency virus type 1 (HIV-1) activity. The mechanism of action of SDZ NIM 811 is clearly different from those of all other anti-HIV agents described so far. In cell-free assays, it is not an inhibitor of reverse transcriptase, protease, integrase, and it does not interfere with Rev or Tat function. SDZ NIM 811 does not down-regulate CD4 or inhibit fusion between infected and uninfected, CD4-expressing cells. p24 production from chronically HIV-infected cells is not impaired either. To elucidate the mode of action of SDZ NIM 811, we performed DNA PCR analysis in HIV-1 IIIB-infected MT4 cells in one cycle of virus replication. The effects of SDZ NIM 811 on the kinetics of viral DNA synthesis, appearance of two-long terminal repeat circles (2-LTR circles), and integration of DNA were studied. SDZ NIM 811 inhibited 2-LTR circle formation in a concentration-dependent manner, which is indicative of nuclear localization of preintegration complexes. Half-maximal inhibition was achieved at 0.17 microgram/ml; this concentration is close to the 50% inhibitory concentrations (0.01 to 0.2 microgram/ml) for viral growth inhibition. As expected, integration of proviral DNA into cellular DNA was also inhibited by SDZ NIM 811. Analysis of the viral particles produced by SDZ NIM 811-treated, chronically infected cells revealed amounts of capsid proteins, reverse transcriptase activity, and viral RNA comparable to those of the untreated control. However, these particles showed a dose-dependent reduction in infectivity (50% inhibitory concentration of 0.028 microgram/ml) which indicates that the assembly process is also impaired by SDZ NIM 811. Gag proteins are postulated to play a role not only in assembly but also in early steps of viral replication, e.g., nuclear localization of the preintegration complex. Recently, it was reported that

  17. Simultaneous runs of the Bayer VERSANT HIV-1 version 3.0 and HCV bDNA version 3.0 quantitative assays on the system 340 platform provide reliable quantitation and improved work flow.

    PubMed

    Elbeik, Tarek; Markowitz, Norman; Nassos, Patricia; Kumar, Uday; Beringer, Scott; Haller, Barbara; Ng, Valerie

    2004-07-01

    Branched DNA (bDNA) assays to quantify human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) consist of three distinct steps, including sample processing, hybridization, and detection, and utilize the System 340 platform for plate incubation and washing. Sample processing differs: HIV-1 from 1 ml of plasma is concentrated by high-speed centrifugation, whereas HCV plasma or serum samples are used without concentration. The first step of hybridization involves viral lysis at 63 degrees C: HIV-1 is performed in a heat block, whereas HCV is performed in System 340. The remaining hybridization and detection steps are similar for HIV-1 and HCV and executed on System 340. In the present study, the HIV-1 bDNA assay was adapted for viral lysis in the System 340 platform. The adaptation, test method 2, includes a 20-s vortex of concentrated viral pellet and lysis working solution, transfer of viral lysate to the 96-well capture plate, and transfer to System 340 programmed for HCV assay specifications. With test method 2, specificity and quantification were within assay specifications. HCV bDNA methodology remains unchanged. Hence, an HIV-1 and an HCV bDNA can be run simultaneously on System 340. With simultaneous testing, laboratories can run full plates, as well as combinations of full and partial plates. Also, simultaneous HIV-1 and HCV bDNA permits labor consolidation and improved workflow while maintaining multitasking and rapid patient result turnaround. PMID:15243070

  18. Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

    PubMed Central

    2011-01-01

    Background There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. Methods Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl β-galactosidase assay with primary isolates of HIV-1. Results This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody

  19. Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments

    NASA Technical Reports Server (NTRS)

    Ojha, R. P.; Dhingra, M. M.; Sarma, M. H.; Myer, Y. P.; Setlik, R. F.; Shibata, M.; Kazim, A. L.; Ornstein, R. L.; Rein, R.; Turner, C. J.; Sarma, R. H.

    1997-01-01

    The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex

  20. Structure of an anti-HIV-1 hammerhead ribozyme complex with a 17-mer DNA substrate analog of HIV-1 gag RNA and a mechanism for the cleavage reaction: 750 MHz NMR and computer experiments.

    PubMed

    Ojha, R P; Dhingra, M M; Sarma, M H; Myer, Y P; Setlik, R F; Shibata, M; Kazim, A L; Ornstein, R L; Rein, R; Turner, C J; Sarma, R H

    1997-10-01

    The structure of an anti-HIV-1 ribozyme-DNA abortive substrate complex was investigated by 750 MHz NMR and computer modeling experiments. The ribozyme was a chimeric molecule with 30 residues-18 DNA nucleotides, and 12 RNA residues in the conserved core. The DNA substrate analog had 17 residues. The chimeric ribozyme and the DNA substrate formed a shortened ribozyme-abortive substrate complex of 47 nucleotides with two DNA stems (stems I and III) and a loop consisting of the conserved core residues. Circular dichroism spectra showed that the DNA stems assume A-family conformation at the NMR concentration and a temperature of 15 degrees C, contrary to the conventional wisdom that DNA duplexes in aqueous solution populate entirely in the B-form. It is proposed that the A-family RNA residues at the core expand the A-family initiated at the core into the DNA stems because of the large free energy requirement for the formation of A/B junctions. Assignments of the base H8/H6 protons and H1' of the 47 residues were made by a NOESY walk. In addition to the methyl groups of all T's, the imino resonances of stems I and III and AH2's were assigned from appropriate NOESY walks. The extracted NMR data along with available crystallographic data, were used to derive a structural model of the complex. Stems I and III of the final model displayed a remarkable similarity to the A form of DNA; in stem III, a GC base pair was found to be moving into the floor of the minor groove defined by flanking AT pairs; data suggest the formation of a buckled rhombic structure with the adjacent pair; in addition, the base pair at the interface of stem III and the loop region displayed deformed geometry. The loop with the catalytic core, and the immediate region of the stems displayed conformational multiplicity within the NMR time scale. A catalytic mechanism for ribozyme action based on the derived structure, and consistent with biochemical data in the literature, is proposed. The complex

  1. Increased Generation of HIV-1 gp120-Reactive CD8+ T Cells by a DNA Vaccine Construct Encoding the Chemokine CCL3

    PubMed Central

    Øynebråten, Inger; Hinkula, Jorma; Fredriksen, Agnete B.; Bogen, Bjarne

    2014-01-01

    DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV-1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8+ T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation. PMID:25122197

  2. DNA vaccines expressing soluble CD4-envelope proteins fused to C3d elicit cross-reactive neutralizing antibodies to HIV-1

    SciTech Connect

    Bower, Joseph F.; Green, Thomas D.; Ross, Ted M. . E-mail: tmr15@pitt.edu

    2004-10-25

    DNA vaccines expressing the envelope (Env) of the human immunodeficiency virus type 1 (HIV-1) have been relatively ineffective at generating high-titer, long-lasting, neutralizing antibodies in a variety of animal models. In this study, DNA vaccines were constructed to express a fusion protein of the soluble human CD4 (sCD4) and the gp120 subunit of the HIV-1 envelope. To enhance the immunogenicity of the expressed fusion protein, three copies of the murine C3d (mC3d{sub 3}) were added to the carboxyl terminus of the complex. Monoclonal antibodies that recognize CD4-induced epitopes on gp120 efficiently bound to sCD4-gp120 or sCD4-gp120-mC3d{sub 3}. In addition, both sCD4-gp120 and sCD4-gp120-mC3d{sub 3} bound to cells expressing appropriate coreceptors in the absence of cell surface hCD4. Mice (BALB/c) vaccinated with DNA vaccines expressing either gp120-mC3d{sub 3} or sCD4-gp120-mC3d{sub 3} elicited antibodies that neutralized homologous virus infection. However, the use of sCD4-gp120-mC3d{sub 3}-DNA elicited the highest titers of neutralizing antibodies that persisted after depletion of anti-hCD4 antibodies. Interestingly, only mice vaccinated with DNA expressing sCD4-gp120-mC3d{sub 3} had antibodies that elicited cross-protective neutralizing antibodies. The fusion of sCD4 to the HIV-1 envelope exposes neutralizing epitopes that elicit broad protective immunity when the fusion complex is coupled with the molecular adjuvant, C3d.

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

    PubMed Central

    Savarino, Andrea

    2007-01-01

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

  4. Mode of inhibition of HIV-1 reverse transcriptase by polyacetylenetriol, a novel inhibitor of RNA- and DNA-directed DNA polymerases.

    PubMed Central

    Loya, Shoshana; Rudi, Amira; Kashman, Yoel; Hizi, Amnon

    2002-01-01

    Polyacetylenetriol (PAT), a natural marine product from the Mediterranean sea sponge Petrosia sp., was found to be a novel general potent inhibitor of DNA polymerases. It inhibits equally well the RNA- and DNA-dependent DNA polymerase activities of retroviral reverse transcriptases (RTs) (i.e. of HIV, murine leukaemia virus and mouse mammary tumour virus) as well as cellular DNA polymerases (i.e. DNA polymerases alpha and beta and Escherichia coli polymerase I). A study of the mode and mechanism of the polymerase inhibition by PAT has been conducted with HIV-1 RT. PAT was shown to be a reversible non-competitive inhibitor. PAT binds RT independently and at a site different from that of the primer-template and dNTP substrates with high affinity (K(i)=0.51 microM and K(i)=0.53 microM with dTTP and with dGTP as the variable substrates respectively). Blocking the polar hydroxy groups of PAT has only a marginal effect on the inhibitory capacity, thus hydrophobic interactions are likely to play a major role in inhibiting RT. Preincubation of RT with the primer-template substrate prior to the interaction with PAT reduces substantially the inhibition capacity, probably by preventing these contacts. PAT does not interfere with the first step of polymerization, the binding of RT to DNA, nor does the inhibitor interfere with the binding of dNTP to RT/DNA complex, as evident from the steady-state kinetic study, whereby K(m) remains unchanged. We assume, therefore, that PAT interferes with subsequent catalytic steps of DNA polymerization. The inhibitor may alter the optimal stereochemistry of the polymerase active site relative to the primer terminus, bound dNTP and the metal ions that are crucial for efficient catalysis or, alternatively, may interfere with the thumb sub-domain movement and, thus, with the translocation of the primer-template following nucleotide incorporation. PMID:11879196

  5. The Frequency of Cytidine Editing of Viral DNA Is Differentially Influenced by Vpx and Nucleosides during HIV-1 or SIVMAC Infection of Dendritic Cells

    PubMed Central

    Nguyen, Xuan-Nhi; Barateau, Véronique; Wu, Nannan; Berger, Gregory; Cimarelli, Andrea

    2015-01-01

    Two cellular factors are currently known to modulate lentiviral infection specifically in myeloid cells: SAMHD1 and APOBEC3A (A3A). SAMHD1 is a deoxynucleoside triphosphohydrolase that interferes with viral infection mostly by limiting the intracellular concentrations of dNTPs, while A3A is a cytidine deaminase that has been described to edit incoming vDNA. The restrictive phenotype of myeloid cells can be alleviated through the direct degradation of SAMHD1 by the HIV-2/SIVSM Vpx protein or else, at least in the case of HIV-1, by the exogenous supplementation of nucleosides that artificially overcome the catabolic activity of SAMHD1 on dNTPs. Here, we have used Vpx and dNs to explore the relationship existing between vDNA cytidine deamination and SAMHD1 during HIV-1 or SIVMAC infection of primary dendritic cells. Our results reveal an interesting inverse correlation between conditions that promote efficient infection of DCs and the extent of vDNA editing that may reflect the different susceptibility of vDNA to cytoplasmic effectors during the infection of myeloid cells. PMID:26496699

  6. Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC.

    PubMed

    Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; Boudier, Christian; De Rocquigny, Hughes; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2011-05-01

    An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5' thymine interacts with residues of the N-terminal zinc knuckle and the 3' guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA-protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids. PMID:21227929

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

    PubMed Central

    2013-01-01

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

  8. Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo

    PubMed Central

    Fox, James M; Hilburn, Silva; Demontis, Maria-Antonietta; Brighty, David W; Rios Grassi, Maria Fernanda; Galvão-Castro, Bernardo; Taylor, Graham P; Martin, Fabiola

    2016-01-01

    Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the “mitotic” spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset. PMID:26985903

  9. Long Terminal Repeat Circular DNA as Markers of Active Viral Replication of Human T Lymphotropic Virus-1 in Vivo.

    PubMed

    Fox, James M; Hilburn, Silva; Demontis, Maria-Antonietta; Brighty, David W; Rios Grassi, Maria Fernanda; Galvão-Castro, Bernardo; Taylor, Graham P; Martin, Fabiola

    2016-03-01

    Clonal expansion of human T-lymphotropic virus type-1 (HTLV-1) infected cells in vivo is well documented. Unlike human immunodeficiency virus type 1 (HIV-1), HTLV-1 plasma RNA is sparse. The contribution of the "mitotic" spread of HTLV-1 compared with infectious spread of the virus to HTLV-1 viral burden in established infection is uncertain. Since extrachromosomal long terminal repeat (LTR) DNA circles are indicators of viral replication in HIV-1 carriers with undetectable plasma HIV RNA, we hypothesised that HTLV-1 LTR circles could indicate reverse transcriptase (RT) usage and infectious activity. 1LTR and 2LTR DNA circles were measured in HTLV-1 cell lines and peripheral blood mononuclear cells (PBMC) of asymptomatic carriers (ACs) and patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukaemia/lymphoma (ATLL). 1LTR DNA circles were detected in 14/20 patients at a mean of 1.38/100 PBMC but did not differentiate disease status nor correlate with HTLV-1 DNA copies. 2LTR DNA circles were detected in 30/31 patients and at higher concentrations in patients with HTLV-1-associated diseases, independent of HTLV-1 DNA load. In an incident case the 2LTR DNA circle concentration increased 2.1 fold at the onset of HAM/TSP compared to baseline. Detectable and fluctuating levels of HTLV-1 DNA circles in patients indicate viral RT usage and virus replication. Our results indicate HTLV-1 viral replication capacity is maintained in chronic infection and may be associated with disease onset. PMID:26985903

  10. Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

    PubMed Central

    Bazzi, Ali; Zargarian, Loussiné; Chaminade, Françoise; Boudier, Christian; De Rocquigny, Hughes; René, Brigitte; Mély, Yves; Fossé, Philippe; Mauffret, Olivier

    2011-01-01

    An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids. PMID:21227929

  11. Impact of the terminal bulges of HIV-1 cTAR DNA on its stability and the destabilizing activity of the nucleocapsid protein NCp7.

    PubMed

    Beltz, Hervé; Azoulay, Joel; Bernacchi, Serena; Clamme, Jean-Pierre; Ficheux, Damien; Roques, Bernard; Darlix, Jean-Luc; Mély, Yves

    2003-04-18

    Reverse transcription of HIV-1 genomic RNA to double-stranded DNA by reverse transcriptase (RT) is a critical step in HIV-1 replication. This process relies on two viral proteins, the RT enzyme and nucleocapsid protein NCp7 that has well documented nucleic acid chaperone properties. At the beginning of the linear DNA synthesis, the newly made minus-strand strong-stop DNA ((-)ssDNA) is transferred to the 3'end of the genomic RNA by means of an hybridization reaction between transactivation response element (TAR) RNA and cTAR DNA sequences. Since both TAR sequences exhibit stable hairpin structures, NCp7 needs to destabilize the TAR structures in order to chaperone their hybridization. To further characterize the relationships between TAR stability and NC-mediated destabilization, the role of the A(49) and G(52) bulged residues in cTAR DNA stability was investigated. The stability of cTAR and mutants where one or the two terminal bulges were replaced by base-pairs as well as the NCp7-mediated destabilization of these cTAR sequences were examined. Thermodynamic data indicate that the two bulges cooperatively destabilize cTAR by reducing the stacking interactions between the bases. This causes a free energy change of about 6.4 kcal/mol and seems to be critical for NC activity. Time-resolved fluorescence data of doubly labelled cTAR derivatives suggest that NC-mediated melting of cTAR ends propagates up to the 10C.A(44) mismatch or T(40) bulge. Fluorescence correlation spectroscopy using two-photon excitation was also used to monitor cTAR ends fraying by NC. Results show that NC causes a very significant increase of cTAR ends fraying, probably limited to the terminal base-pair in the case of cTAR mutants. Since the TAR RNA and cTAR DNA bulges or mismatches appear well conserved among all HIV-1 strains, the present data support the notion of a co-evolutionary relationship between TAR and NC activity. PMID:12684000

  12. HIV-1 Env DNA vaccine plus protein boost delivered by EP expands B- and T-cell responses and neutralizing phenotype in vivo.

    PubMed

    Muthumani, Kar; Wise, Megan C; Broderick, Kate E; Hutnick, Natalie; Goodman, Jonathan; Flingai, Seleeke; Yan, Jian; Bian, Chaoran B; Mendoza, Janess; Tingey, Colleen; Wilson, Christine; Wojtak, Krzysztof; Sardesai, Niranjan Y; Weiner, David B

    2013-01-01

    An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted. PMID:24391921

  13. HIV-1 Env DNA Vaccine plus Protein Boost Delivered by EP Expands B- and T-Cell Responses and Neutralizing Phenotype In Vivo

    PubMed Central

    Muthumani, Kar; Wise, Megan C.; Broderick, Kate E.; Hutnick, Natalie; Goodman, Jonathan; Flingai, Seleeke; Yan, Jian; Bian, Chaoran B.; Mendoza, Janess; Tingey, Colleen; Wilson, Christine; Wojtak, Krzysztof; Sardesai, Niranjan Y.; Weiner, David B.

    2013-01-01

    An effective HIV vaccine will most likely require the induction of strong T-cell responses, broadly neutralizing antibodies (bNAbs), and the elicitation of antibody-dependent cellular cytotoxicity (ADCC). Previously, we demonstrated the induction of strong HIV/SIV cellular immune responses in macaques and humans using synthetic consensus DNA immunogens delivered via adaptive electroporation (EP). However, the ability of this improved DNA approach to prime for relevant antibody responses has not been previously studied. Here, we investigate the immunogenicity of consensus DNA constructs encoding gp140 sequences from HIV-1 subtypes A, B, C and D in a DNA prime-protein boost vaccine regimen. Mice and guinea pigs were primed with single- and multi-clade DNA via EP and boosted with recombinant gp120 protein. Sera were analyzed for gp120 binding and induction of neutralizing antibody activity. Immunization with recombinant Env protein alone induced low-titer binding antibodies with limited neutralization breath. In contrast, the synthetic DNA prime-protein boost protocol induced significantly higher antibody binding titers. Furthermore, sera from DNA prime-protein boost groups were able to neutralize a broader range of viruses in a panel of tier 1 clade B viruses as well as multiple tier 1 clade A and clade C viruses. Further investigation of synthetic DNA prime plus adaptive EP plus protein boost appears warranted. PMID:24391921

  14. GADD45 proteins inhibit HIV-1 replication through specific suppression of HIV-1 transcription.

    PubMed

    Liang, Zhibin; Liu, Ruikang; Zhang, Hui; Zhang, Suzhen; Hu, Xiaomei; Tan, Juan; Liang, Chen; Qiao, Wentao

    2016-06-01

    GADD45 proteins are a group of stress-induced proteins and participate in various cellular pathways including cell cycle regulation, cell survival and death, DNA repair and demethylation. It was recently shown that HIV-1 infection induces the expression of GADD45 proteins. However, the effect of GADD45 on HIV-1 replication has not been studied. Here, we report that overexpression of GADD45 proteins reduces HIV-1 production through suppressing transcription from the HIV-1 LTR promoter. This inhibitory effect is specific to HIV-1, since GADD45 proteins neither inhibit the LTR promoters from other retroviruses nor reduce the production of these viruses. Knockdown of endogenous GADD45 modestly activates HIV-1 in the J-Lat A72 latency cell line, which suggests GADD45 proteins might play a role in maintaining HIV-1 latency. PMID:26994425

  15. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects.

    PubMed

    Gach, Johannes S; Gorlani, Andrea; Dotsey, Emmanuel Y; Becerra, Juan C; Anderson, Chase T M; Berzins, Baiba; Felgner, Philip L; Forthal, Donald N; Deeks, Steven G; Wilkin, Timothy J; Casazza, Joseph P; Koup, Richard A; Katlama, Christine; Autran, Brigitte; Murphy, Robert L; Achenbach, Chad J

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

  16. HIV-1-Specific Antibody Response and Function after DNA Prime and Recombinant Adenovirus 5 Boost HIV Vaccine in HIV-Infected Subjects

    PubMed Central

    Gach, Johannes S.; Gorlani, Andrea; Dotsey, Emmanuel Y.; Becerra, Juan C.; Anderson, Chase T. M.; Berzins, Baiba; Felgner, Philip L.; Forthal, Donald N.; Deeks, Steven G.; Wilkin, Timothy J.; Casazza, Joseph P.; Koup, Richard A.; Katlama, Christine; Autran, Brigitte; Murphy, Robert L.; Achenbach, Chad J.

    2016-01-01

    Little is known about the humoral immune response against DNA prime-recombinant adenovirus 5 (rAd5) boost HIV vaccine among HIV-infected patients on long-term suppressive antiretroviral therapy (ART). Previous studies emphasized cellular immune responses; however, current research suggests both cellular and humoral responses are likely required for a successful therapeutic vaccine. Thus, we aimed to understand antibody response and function induced by vaccination of ART-treated HIV-1-infected patients with immune recovery. All subjects participated in EraMune 02, an open-label randomized clinical trial of ART intensification followed by a six plasmid DNA prime (envA, envB, envC, gagB, polB, nefB) and rAd5 boost HIV vaccine with matching inserts. Antibody binding levels were determined with a recently developed microarray approach. We also analyzed neutralization efficiency and antibody-dependent cellular cytotoxicity (ADCC). We found that the DNA prime-rAd5 boost vaccine induced a significant cross-clade HIV-specific antibody response, which correlated with antibody neutralization efficiency. However, despite the increase in antibody binding levels, the vaccine did not significantly stimulate neutralization or ADCC responses. This finding was also reflected by a lack of change in total CD4+ cell associated HIV DNA in those who received the vaccine. Our results have important implications for further therapeutic vaccine design and administration, especially in HIV-1 infected patients, as boosting of preexisting antibody responses are unlikely to lead to clearance of latent proviruses in the HIV reservoir. PMID:27500639

  17. Multimeric scaffolds displaying the HIV-1 envelope MPER induce MPER-specific antibodies and cross-neutralizing antibodies when co-immunized with gp160 DNA.

    PubMed

    Krebs, Shelly J; McBurney, Sean P; Kovarik, Dina N; Waddell, Chelsea D; Jaworski, J Pablo; Sutton, William F; Gomes, Michelle M; Trovato, Maria; Waagmeester, Garret; Barnett, Susan J; DeBerardinis, Piergiuseppe; Haigwood, Nancy L

    2014-01-01

    Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env. PMID:25514675

  18. Phase 1 Safety and Immunogenicity Evaluation of ADVAX, a Multigenic, DNA-Based Clade C/B' HIV-1 Candidate Vaccine

    PubMed Central

    Vasan, Sandhya; Schlesinger, Sarah J.; Huang, Yaoxing; Hurley, Arlene; Lombardo, Angela; Chen, Zhiwei; Than, Soe; Adesanya, Phumla; Bunce, Catherine; Boaz, Mark; Boyle, Rosanne; Sayeed, Eddy; Clark, Lorna; Dugin, Daniel; Schmidt, Claudia; Song, Yang; Seamons, Laura; Dally, Len; Ho, Martin; Smith, Carol; Markowitz, Martin; Cox, Josephine; Gill, Dilbinder K.; Gilmour, Jill; Keefer, Michael C.; Fast, Patricia; Ho, David D.

    2010-01-01

    Background We conducted a Phase I dose escalation trial of ADVAX, a DNA-based candidate HIV-1 vaccine expressing Clade C/B' env, gag, pol, nef, and tat genes. Sequences were derived from a prevalent circulating recombinant form in Yunnan, China, an area of high HIV-1 incidence. The objective was to evaluate the safety and immunogenicity of ADVAX in human volunteers. Methodology/Principal Findings ADVAX or placebo was administered intramuscularly at months 0, 1 and 3 to 45 healthy volunteers not at high risk for HIV-1. Three dosage levels [0.2 mg (low), 1.0 mg (mid), and 4.0 mg (high)] were tested. Twelve volunteers in each dosage group were assigned to receive ADVAX and three to receive placebo in a double-blind design. Subjects were followed for local and systemic reactogenicity, adverse events, and clinical laboratory parameters. Study follow up was 18 months. Humoral immunogenicity was evaluated by anti-gp120 binding ELISA. Cellular immunogenicity was assessed by a validated IFNγ ELISpot assay and intracellular cytokine staining. ADVAX was safe and well-tolerated, with no vaccine-related serious adverse events. Local and systemic reactogenicity events were reported by 64% and 42% of vaccine recipients, respectively. The majority of events were mild. The IFNγ ELISpot response rates to any HIV antigen were 0/9 (0%) in the placebo group, 3/12 (25%) in the low-dosage group, 4/12 (33%) in the mid-dosage group, and 2/12 (17%) in the high-dosage group. Overall, responses were generally transient and occurred to each gene product, although volunteers responded to single antigens only. Binding antibodies to gp120 were not detected in any volunteers, and HIV seroconversion did not occur. Conclusions/Significance ADVAX delivered intramuscularly is safe, well-tolerated, and elicits modest but transient cellular immune responses. Trial Registration Clinicaltrials.gov NCT00249106 PMID:20111582

  19. Herpes simplex virus type-2 stimulates HIV-1 replication in cervical tissues: implications for HIV-1 transmission and efficacy of anti-HIV-1 microbicides

    PubMed Central

    Rollenhagen, C; Lathrop, M J; Macura, S L; Doncel, G F; Asin, S N

    2014-01-01

    Herpes Simplex virus Type-2 (HSV-2) increases the risk of HIV-1 acquisition, yet the mechanism for this viral pathogen to regulate the susceptibility of the cervicovaginal mucosa to HIV-1 is virtually unknown. Using ex vivo human ectocervical tissue models, we report greater levels of HIV-1 reverse transcription, DNA integration, RNA expression, and virions release in HIV-1/HSV-2 co-infected tissues compared with HIV-1 only infected tissues (P<0.05). Enhanced HIV-1 replication was associated with increased CD4, CCR5, and CD38 transcription (P<0.05) and increased number of CD4+/CCR5+/CD38+ T cells in HIV-1/HSV-2 co-infected tissues compared with tissues infected with HIV-1 alone. Tenofovir (TFV) 1% gel, the leading microbicide candidate, demonstrated only partial protection against HIV-1, when applied vaginally before and after sexual intercourse. It is possible that mucosal inflammation, in particular that induced by HSV-2 infection, may have decreased TFV efficacy. HSV-2 upregulated the number of HIV-1-infected cells and elevated the concentration of TFV needed to decrease HIV-1 infection. Similarly, only high concentrations of TFV inhibited HSV-2 replication in HIV-1/HSV-2-infected tissues. Thus, HSV-2 co-infection and mucosal immune cell activation should be taken into consideration when designing preventative strategies for sexual transmission of HIV-1. PMID:24496317

  20. Persistent Production of an Integrase-Deleted HIV-1 Variant with No Resistance Mutation and Wild-Type Proviral DNA in a Treated Patient

    PubMed Central

    Cotte, Laurent; Saison, Julien; Ramière, Christophe; Ronfort, Corinne; Venet, Fabienne; Tardy, Jean-Claude; Monneret, Guillaume; André, Patrice

    2015-01-01

    Abstract An HIV-infected patient presenting an unexpected viral escape under combined antiretroviral treatment is described. The virus isolated from plasma contained a large deletion in the HIV-1 integrase gene but no known resistance mutation. Nested polymerase chain reactions (PCRs) with patient virus integrase-specific primers and probes were developed and used to detect the mutant from plasma, blood, rectal biopsies, and sperm. The variant progressively emerged during a period of therapy-induced virosuppression, and persisted at a low but detectable level for at least 5 years. Surprisingly, proviral DNA from lymphocytes, rectal cells, and sperm cells was, and remained, mainly wild type. Cellular HIV RNA with the deletion was detected only once from the rectum. The origin and mechanisms underlying this so far not described production at a detectable level are largely hypothetical. This observation raised concern about the ability of defective viruses to spread. PMID:25333615

  1. HIV-1 protease-induced apoptosis

    PubMed Central

    2014-01-01

    Background Apoptosis is one of the presumptive causes of CD4+ T cell depletion during HIV infection and progression to AIDS. However, the precise role of HIV-1 in this process remains unexplained. HIV-1 protease (PR) has been suggested as a possible factor, but a direct link between HIV-1 PR enzymatic activity and apoptosis has not been established. Results Here, we show that expression of active HIV-1 PR induces death in HeLa and HEK-293 cells via the mitochondrial apoptotic pathway. This conclusion is based on in vivo observations of the direct localization of HIV-1 PR in mitochondria, a key player in triggering apoptosis. Moreover, we observed an HIV-1 PR concentration-dependent decrease in mitochondrial membrane potential and the role of HIV-1 PR in activation of caspase 9, PARP cleavage and DNA fragmentation. In addition, in vitro data demonstrated that HIV-1 PR mediates cleavage of mitochondrial proteins Tom22, VDAC and ANT, leading to release of AIF and Hsp60 proteins. By using yeast two-hybrid screening, we also identified a new HIV-1 PR interaction partner, breast carcinoma-associated protein 3 (BCA3). We found that BCA3 accelerates p53 transcriptional activity on the bax promoter, thus elevating the cellular level of pro-apoptotic Bax protein. Conclusion In summary, our results describe the involvement of HIV-1 PR in apoptosis, which is caused either by a direct effect of HIV-1 PR on mitochondrial membrane integrity or by its interaction with cellular protein BCA3. PMID:24886575

  2. Polyvalent HIV-1 Env vaccine formulations delivered by the DNA priming plus protein boosting approach are effective in generating neutralizing antibodies against primary human immunodeficiency virus type 1 isolates from subtypes A, B, C, D and E.

    PubMed

    Wang, Shixia; Pal, Ranajit; Mascola, John R; Chou, Te-Hui W; Mboudjeka, Innocent; Shen, Siyuan; Liu, Qin; Whitney, Stephen; Keen, Timothy; Nair, B C; Kalyanaraman, V S; Markham, Philip; Lu, Shan

    2006-06-20

    A major challenge in developing an HIV-1 vaccine is to identify immunogens and their delivery methods that can elicit broad neutralizing antibodies against primary isolates of different genetic subtypes. Recently, we demonstrated that priming with DNA vaccines expressing primary HIV-1 envelope glycoprotein (Env) followed by recombinant Env protein boosting was successful in generating positive neutralizing antibody responses against a clade B primary HIV-1 isolate, JR-FL, that was not easily neutralized. In the current study, we examined whether the DNA priming plus recombinant protein boosting approach delivering a polyvalent primary Env formulation was able to generate neutralizing antibodies against primary HIV-1 viral isolates from various genetic subtypes. New Zealand White rabbits were first immunized with DNA vaccines expressing one, three or eight primary HIV-1 gp120 antigens delivered by a gene gun followed by recombinant gp120 protein boosting. Neutralizing antibody responses were examined by two independently executed neutralization assays: the first one was a single round infection neutralization assay against a panel of 10 primary HIV-1 isolates of subtypes A, B, C and E and the second one used the PhenoSense assay against a panel of 12 pseudovirues expressing primary HIV-1 Env antigens from subtypes A, B, C, D and E as well as 2 pseudoviruses expressing the Env antigens from MN and NL4-3 viruses. Rabbit sera immunized with the DNA priming plus protein boosting approach, but not DNA vaccine alone or Env protein alone, were capable of neutralizing 7 of 10 viruses in the first assay and 12 of 14 viruses in the second assay. More importantly, sera immunized with the polyvalent Env antigens were able to neutralize a significantly higher percentage of viruses than the sera immunized with the monovalent antigens. Our results suggest that DNA priming followed by recombinant Env protein boosting can be used to deliver polyvalent Env-antigen-based HIV-1

  3. Psychoneuroimmunology and HIV-1.

    ERIC Educational Resources Information Center

    Antoni, Michael H.; And Others

    1990-01-01

    Presents evidence describing benefits of behavioral interventions such as aerobic exercise training on both psychological and immunological functioning among high risk human immunodeficiency virus-Type 1 (HIV-1) seronegative and very early stage seropositive homosexual men. HIV-1 infection is cast as chronic disease for which early…

  4. Altered Response Hierarchy and Increased T-Cell Breadth upon HIV-1 Conserved Element DNA Vaccination in Macaques

    PubMed Central

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K.; Jalah, Rashmi; Broderick, Kate E.; Sardesai, Niranjan Y.; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I.; Pavlakis, George N.; Felber, Barbara K.

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24gag elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55gag increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  5. Altered response hierarchy and increased T-cell breadth upon HIV-1 conserved element DNA vaccination in macaques.

    PubMed

    Kulkarni, Viraj; Valentin, Antonio; Rosati, Margherita; Alicea, Candido; Singh, Ashish K; Jalah, Rashmi; Broderick, Kate E; Sardesai, Niranjan Y; Le Gall, Sylvie; Mothe, Beatriz; Brander, Christian; Rolland, Morgane; Mullins, James I; Pavlakis, George N; Felber, Barbara K

    2014-01-01

    HIV sequence diversity and potential decoy epitopes are hurdles in the development of an effective AIDS vaccine. A DNA vaccine candidate comprising of highly conserved p24(gag) elements (CE) induced robust immunity in all 10 vaccinated macaques, whereas full-length gag DNA vaccination elicited responses to these conserved elements in only 5 of 11 animals, targeting fewer CE per animal. Importantly, boosting CE-primed macaques with DNA expressing full-length p55(gag) increased both magnitude of CE responses and breadth of Gag immunity, demonstrating alteration of the hierarchy of epitope recognition in the presence of pre-existing CE-specific responses. Inclusion of a conserved element immunogen provides a novel and effective strategy to broaden responses against highly diverse pathogens by avoiding decoy epitopes, while focusing responses to critical viral elements for which few escape pathways exist. PMID:24465991

  6. Structural Studies of the HIV-1 Integrase Protein: Compound Screening and Characterization of a DNA-Binding Inhibitor

    PubMed Central

    Hassounah, Said; Mesplède, Thibault; Wainberg, Mark A.

    2015-01-01

    Understanding the HIV integrase protein and mechanisms of resistance to HIV integrase inhibitors is complicated by the lack of a full length HIV integrase crystal structure. Moreover, a lentiviral integrase structure with co-crystallised DNA has not been described. For these reasons, we have developed a structural method that utilizes free software to create quaternary HIV integrase homology models, based partially on available full-length prototype foamy virus integrase structures as well as several structures of truncated HIV integrase. We have tested the utility of these models in screening of small anti-integrase compounds using randomly selected molecules from the ZINC database as well as a well characterized IN:DNA binding inhibitor, FZ41, and a putative IN:DNA binding inhibitor, HDS1. Docking studies showed that the ZINC compounds that had the best binding energies bound at the IN:IN dimer interface and that the FZ41 and HDS1 compounds docked at approximately the same location in integrase, i.e. behind the DNA binding domain, although there is some overlap with the IN:IN dimer interface to which the ZINC compounds bind. Thus, we have revealed two possible locations in integrase that could potentially be targeted by allosteric integrase inhibitors, that are distinct from the binding sites of other allosteric molecules such as LEDGF inhibitors. Virological and biochemical studies confirmed that HDS1 and FZ41 share a similar activity profile and that both can inhibit each of integrase and reverse transcriptase activities. The inhibitory mechanism of HDS1 for HIV integrase seems to be at the DNA binding step and not at either of the strand transfer or 3' processing steps of the integrase reaction. Furthermore, HDS1 does not directly interact with DNA. The modeling and docking methodology described here will be useful for future screening of integrase inhibitors as well as for the generation of models for the study of integrase drug resistance. PMID:26046987

  7. Dolutegravir (S/GSK1349572) exhibits significantly slower dissociation than raltegravir and elvitegravir from wild-type and integrase inhibitor-resistant HIV-1 integrase-DNA complexes.

    PubMed

    Hightower, Kendra E; Wang, Ruolan; Deanda, Felix; Johns, Brian A; Weaver, Kurt; Shen, Yingnian; Tomberlin, Ginger H; Carter, H Luke; Broderick, Timothy; Sigethy, Scott; Seki, Takahiro; Kobayashi, Masanori; Underwood, Mark R

    2011-10-01

    The integrase inhibitor (INI) dolutegravir (DTG; S/GSK1349572) has significant activity against HIV-1 isolates with raltegravir (RAL)- and elvitegravir (ELV)-associated resistance mutations. As an initial step in characterizing the different resistance profiles of DTG, RAL, and ELV, we determined the dissociation rates of these INIs with integrase (IN)-DNA complexes containing a broad panel of IN proteins, including IN substitutions corresponding to signature RAL and ELV resistance mutations. DTG dissociates slowly from a wild-type IN-DNA complex at 37°C with an off-rate of 2.7 × 10(-6) s(-1) and a dissociative half-life (t(1/2)) of 71 h, significantly longer than the half-lives for RAL (8.8 h) and ELV (2.7 h). Prolonged binding (t(1/2), at least 5 h) was observed for DTG with IN-DNA complexes containing E92, Y143, Q148, and N155 substitutions. The addition of a second substitution to either Q148 or N155 typically resulted in an increase in the off-rate compared to that with the single substitution. For all of the IN substitutions tested, the off-rate of DTG from IN-DNA complexes was significantly slower (from 5 to 40 times slower) than the off-rate of RAL or ELV. These data are consistent with the potential for DTG to have a higher genetic barrier to resistance, provide evidence that the INI off-rate may be an important component of the mechanism of INI resistance, and suggest that the slow dissociation of DTG may contribute to its distinctive resistance profile. PMID:21807982

  8. Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase

    PubMed Central

    Renisio, Jean-Guillaume; Cosquer, Sylvain; Cherrak, Ilham; Antri, Saïd El; Mauffret, Olivier; Fermandjian, Serge

    2005-01-01

    The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3′-processing [dinucleotide released from each 3′ end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the 13C/15N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN. PMID:15814814

  9. Uneven Genetic Robustness of HIV-1 Integrase

    PubMed Central

    Rihn, Suzannah J.; Hughes, Joseph; Wilson, Sam J.

    2014-01-01

    -amino-acid IN mutations that could mimic the types of mutations that naturally occur during HIV-1 infection. Previously, we measured the robustness of HIV-1 capsid in this manner and determined that it is extremely intolerant of mutation. In contrast to CA, HIV-1 IN proved relatively robust, with far fewer mutations causing lethal defects. However, when we subsequently mapped the lethal mutations onto a model of the structure of the multisubunit IN-viral DNA complex, we found the lethal mutations that caused virus morphogenesis defects tended to be highly localized at subunit interfaces. This discovery of vulnerable regions of HIV-1 IN could inform development of novel therapeutics. PMID:25339768

  10. Investigation by two-photon fluorescence correlation spectroscopy of the interaction of the nucleocapsid protein of HIV-1 with hairpin loop DNA sequences

    NASA Astrophysics Data System (ADS)

    Mely, Yves; Azoulay, Joel; Beltz, Herve; Clamme, Jean-Pierre; Bernacchi, Serena; Ficheux, Damien; Roques, Bernard P.; Darlix, Jean-Luc

    2004-09-01

    The nucleocapsid protein NCp7 of HIV-1 possesses nucleic acid chaperone properties that are critical for the two strand transfer reactions required during reverse transcription. The first DNA strand transfer relies on the destabilization by NCp7 of double-stranded segments of the transactivation response element, TAR sequence, at the 3' end of the genomic RNA and the complementary sequence cTAR at the 3" terminus of the early product of reverse transcription. To characterize NCp7-mediated nucleic acid destabilization, we investigated by steady-state and time-resolved fluorescence spectroscopy and two photon fluorescence correlation spectroscopy, the interaction of a doubly-labelled cTAR sequence with NCp7. The conformational fluctuations observed in the absence of NCp7 were associated with the rapid opening and closing (fraying) of the double stranded terminal segment of cTAR. NCp7 destabilizes cTAR mainly through a large increase of the opening rate constant. Additionally, the various destabilizing structures (bulges, internal loop, mismatches) spread all over cTAR secondary structure were found to be critical for NCp7 chaperone activity. Taken together, our data enabled us to propose a molecular mechanism for the destabilizing activity of NCp7 on cTAR which is crucial for the formation of the cTAR-TAR complex during the first strand transfer reaction.

  11. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab.

    PubMed

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-10-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an "enhanced and optimized" DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This "enhanced" DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of "adaptive" in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection, against diverse

  12. Optimized and enhanced DNA plasmid vector based in vivo construction of a neutralizing anti-HIV-1 envelope glycoprotein Fab

    PubMed Central

    Muthumani, Kar; Flingai, Seleeke; Wise, Megan; Tingey, Colleen; Ugen, Kenneth E; Weiner, David B

    2013-01-01

    Monoclonal antibody preparations have demonstrated considerable clinical utility in the treatment of specific malignancies, as well as inflammatory and infectious diseases. Antibodies are conventionally delivered by passive administration, typically requiring costly large-scale laboratory development and production. Additional limitations include the necessity for repeat administrations, and the length of in vivo potency. Therefore, the development of methods to generate therapeutic antibodies and antibody like molecules in vivo, distinct from an active antigen-based immunization strategy, would have considerable clinical utility. In fact, adeno-associated viral (AAV) vector mediated delivery of immunoglobulin genes with subsequent generation of functional antibodies has recently been developed. As well, anon-viral vector mediated nucleic acid based delivery technology could permit the generation of therapeutic/prophylactic antibodies in vivo, obviating potential safety issues associated with viral vector based gene delivery. This delivery strategy has limitations as well, mainly due to very low in vivo production and expression of protein from the delivered gene. In the study reported here we have constructed an “enhanced and optimized” DNA plasmid technology to generate immunoglobulin heavy and light chains (i.e., Fab fragments) from an established neutralizing anti-HIV envelope glycoprotein monoclonal antibody (VRC01). This “enhanced” DNA (E-DNA) plasmid technology includes codon/RNA optimization, leader sequence utilization, as well as targeted potentiation of delivery and expression of the Fab immunoglobulin genes through use of “adaptive” in vivo electroporation. The results demonstrate that delivery by this method of a single administration of the optimized Fab expressing constructs resulted in generation of Fab molecules in mouse sera possessing high antigen specific binding and HIV neutralization activity for at least 7 d after injection

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

    PubMed

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

    2016-06-01

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

  14. Synthetic Consensus HIV-1 DNA Induces Potent Cellular Immune Responses and Synthesis of Granzyme B, Perforin in HIV Infected Individuals

    PubMed Central

    Morrow, Matthew P; Tebas, Pablo; Yan, Jian; Ramirez, Lorenzo; Slager, Anna; Kraynyak, Kim; Diehl, Malissa; Shah, Divya; Khan, Amir; Lee, Jessica; Boyer, Jean; Kim, J Joseph; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L

    2015-01-01

    This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection. PMID:25531694

  15. Synthetic consensus HIV-1 DNA induces potent cellular immune responses and synthesis of granzyme B, perforin in HIV infected individuals.

    PubMed

    Morrow, Matthew P; Tebas, Pablo; Yan, Jian; Ramirez, Lorenzo; Slager, Anna; Kraynyak, Kim; Diehl, Malissa; Shah, Divya; Khan, Amir; Lee, Jessica; Boyer, Jean; Kim, J Joseph; Sardesai, Niranjan Y; Weiner, David B; Bagarazzi, Mark L

    2015-03-01

    This study evaluated the safety and immunogenicity of PENNVAX-B in 12 HIV infected individuals. PENNVAX-B is a combination of three optimized synthetic plasmids encoding for multiclade HIV Gag and Pol and a consensus CladeB Env delivered by electroporation. HIV infected individuals whose virus was effectively suppressed using highly active antiretroviral therapy (HAART) received PENNVAX-B DNA followed by electroporation with CELLECTRA-5P at study weeks 0, 4, 8, and 16. Local administration site and systemic reactions to PENNVAX-B were recorded after each treatment along with any adverse events. Pain of the treatment procedure was assessed using a Visual Analog Scale. Whole PBMCs were isolated for use in IFN ELISpot and Flow Cytometric assays. PENNVAX-B was generally safe and well tolerated. Overall, the four dose regimen was not associated with any serious adverse events or severe local or systemic reactions. A rise in antigen-specific SFU was detected in the INFγ ELISpot assay in all 12 participants. T cells from 8/12 participants loaded with both granzyme B and perforin in response to HIV antigen, an immune finding characteristic of long-term nonprogressors (LTNPs) and elite controllers (ECs). Thus administration of PENNVAX-B may prove useful adjunctive therapy to ART for treatment and control of HIV infection. PMID:25531694

  16. Fetal Cord Blood Mononuclear Cells Collected At Term from HIV-1 Infected Women Harbor Transcriptionally Active Integrated Proviral DNA

    PubMed Central

    ELLIS, Jane E.; HAIR, Greg A.; LINDSAY, Michael K.; ANSARI, Aftab A.; SUNDSTROM, J. Bruce

    2007-01-01

    Objective To determine levels of intrauterine infection and transcriptional activity in cord blood mononuclear cells collected at term from fetuses born to HIV-infected pregnant women on highly active anti-retroviral therapy (HAART). Methods RNA and DNA were isolated from maternal placental tissues and fetal cord blood specimens obtained at term from HIV-infected pregnant women on HAART. Levels of integrated HIV provirus and mRNA transcripts were determined by real-time PCR. Results Detectable levels of transcriptionally active integrated provirus were present in approximately 27% of cord blood samples (n=22) collected from fetuses, born to HIV-positive mothers on HAART. Levels of HIV-p24 antigen in cultures detected in randomly selected cord blood samples confirmed the presence of inducible infectious virus. Conclusions These findings suggest that some fetuses from HIV-infected mothers on HAART who may present as HIV-negative infants postpartum, can harbor circulating leukocytes that are productively infected by intrauterine transmission (IUT) of HIV. PMID:17904964

  17. C/EBP- and Tat-mediated activation of the HIV-1 LTR in CD34+ hematopoietic progenitor cells.

    PubMed

    Quiterio, Shane; Grant, Christian; Hogan, Tricia H; Krebs, Fred C; Wigdahl, Brian

    2003-01-01

    Human immunodeficiency virus type 1 (HIV-1) infection of cells of the monocyte/macrophage lineage within the bone marrow and peripheral blood plays an important role in the pathologic events leading to the development of the acquired immune deficiency syndrome (AIDS) as well as HIV-1 dementia (HIVD). The TF-1 erythro-myeloid cell line is being utilized as a model cellular phenotype to examine HIV-1 infection of a hematopoietic progenitor cell population. Expression of TF-1 cell surface marker RNAs and proteins was characterized by RT-PCR and FACS, respectively, and compared to those of the well characterized U-937 monocytic cell line. Transcription factors in TF-1 and U-937 cells that have been shown to be important for sustaining the expression of HIV-1 LTR activity were also examined. TF-1 cells were shown to contain the transcription factors C/EBP, Sp1, and NF-kappaB. C/EBP- and Tat-mediated induction of the YU-2 LTR was examined. Relative C/EBP induction of the HIV-1 strain YU-2 LTR was greater in TF-1 cells than in U-937 cells. When the C/EBP sites I and II were mutated to sequences with a low relative affinity for C/EBP factors, there was a reduction of Tat-mediated trans-activation in TF-1 cells, but not in U-937 cells. These studies form the foundation for investigations into the relationship between HIV-1 infection of bone marrow and peripheral blood precursor cells of the monocyte/macrophage lineage and pathogenesis associated with HIV-1 infection of the immune and central nervous system (CNS). PMID:12642037

  18. The hunt for HIV-1 integrase inhibitors.

    PubMed

    Lataillade, Max; Kozal, Michael J

    2006-07-01

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

  19. A Motif Unique to the Human Dead-Box Protein DDX3 Is Important for Nucleic Acid Binding, ATP Hydrolysis, RNA/DNA Unwinding and HIV-1 Replication

    PubMed Central

    Di Cicco, Giulia; Dietrich, Ursula; Maga, Giovanni

    2011-01-01

    DEAD-box proteins are enzymes endowed with nucleic acid-dependent ATPase, RNA translocase and unwinding activities. The human DEAD-box protein DDX3 has been shown to play important roles in tumor proliferation and viral infections. In particular, DDX3 has been identified as an essential cofactor for HIV-1 replication. Here we characterized a set of DDX3 mutants biochemically with respect to nucleic acid binding, ATPase and helicase activity. In particular, we addressed the functional role of a unique insertion between motifs I and Ia of DDX3 and provide evidence for its implication in nucleic acid binding and HIV-1 replication. We show that human DDX3 lacking this domain binds HIV-1 RNA with lower affinity. Furthermore, a specific peptide ligand for this insertion selected by phage display interferes with HIV-1 replication after transduction into HelaP4 cells. Besides broadening our understanding of the structure-function relationships of this important protein, our results identify a specific domain of DDX3 which may be suited as target for antiviral drugs designed to inhibit cellular cofactors for HIV-1 replication. PMID:21589879

  20. Genome editing strategies: potential tools for eradicating HIV-1/AIDS

    PubMed Central

    Khalili, Kamel; Gordon, Jennifer; Cosentino, Laura; Hu, Wenhui

    2015-01-01

    Current therapy for controlling HIV-1 infection and preventing AIDS progression has profoundly decreased viral replication in cells susceptible to HIV-1 infection, but it does not eliminate the low level of viral replication in latently infected cells which contain integrated copies of HIV-1 proviral DNA. There is an urgent need for the development of HIV-1 genome eradication strategies that will lead to a permanent or “sterile” cure of HIV-1/AIDS. In the past few years, novel nuclease-initiated genome editing tools have been developing rapidly, including ZFNs, TALENs, and the CRISPR/Cas9 system. These surgical knives, which can excise any genome, provide a great opportunity to eradicate the HIV-1 genome by targeting highly conserved regions of the HIV-1 long terminal repeats or essential viral genes. Given the time consuming and costly engineering of target-specific ZFNs and TALENs, the RNA-guided endonuclease Cas9 technology has emerged as a simpler and more versatile technology to allow permanent removal of integrated HIV-1 proviral DNA in eukaryotic cells, and hopefully animal models or human patients. The major unmet challenges of this approach at present include inefficient nuclease gene delivery, potential off-target cleavage, and cell-specific genome targeting. Nanoparticle or lentivirus-mediated delivery of next generation Cas9 technologies including nickase or RNA-guided FokI nuclease (RFN) will further improve the potential for genome editing to become a promising approach for curing HIV-1/AIDS. PMID:25716921

  1. Non-instrumented incubation of a recombinase polymerase amplification assay for the rapid and sensitive detection of proviral HIV-1 DNA.

    PubMed

    Lillis, Lorraine; Lehman, Dara; Singhal, Mitra C; Cantera, Jason; Singleton, Jered; Labarre, Paul; Toyama, Anthony; Piepenburg, Olaf; Parker, Mathew; Wood, Robert; Overbaugh, Julie; Boyle, David S

    2014-01-01

    Sensitive diagnostic tests for infectious diseases often employ nucleic acid amplification technologies (NAATs). However, most NAAT assays, including many isothermal amplification methods, require power-dependent instrumentation for incubation. For use in low resource settings (LRS), diagnostics that do not require consistent electricity supply would be ideal. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that has been shown to typically work at temperatures ranging from 25-43°C, and does not require a stringent incubation temperature for optimal performance. Here we evaluate the ability to incubate an HIV-1 RPA assay, intended for use as an infant HIV diagnostic in LRS, at ambient temperatures or with a simple non-instrumented heat source. To determine the range of expected ambient temperatures in settings where an HIV-1 infant diagnostic would be of most use, a dataset of the seasonal range of daily temperatures in sub Saharan Africa was analyzed and revealed ambient temperatures as low as 10°C and rarely above 43°C. All 24 of 24 (100%) HIV-1 RPA reactions amplified when incubated for 20 minutes between 31°C and 43°C. The amplification from the HIV-1 RPA assay under investigation at temperatures was less consistent below 30°C. Thus, we developed a chemical heater to incubate HIV-1 RPA assays when ambient temperatures are between 10°C and 30°C. All 12/12 (100%) reactions amplified with chemical heat incubation from ambient temperatures of 15°C, 20°C, 25°C and 30°C. We also observed that incubation at 30 minutes improved assay performance at lower temperatures where detection was sporadic using 20 minutes incubation. We have demonstrated that incubation of the RPA HIV-1 assay via ambient temperatures or using chemical heaters yields similar results to using electrically powered devices. We propose that this RPA HIV-1 assay may not need dedicated equipment to be a highly sensitive tool to diagnose infant HIV-1 in

  2. 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

  3. HIV-1 Fusion Assay

    PubMed Central

    Cavrois, Marielle; Neidleman, Jason; Greene, Warner C.

    2016-01-01

    The HIV-1 fusion assay measures all steps in the HIV-1 life cycle up to and including viral fusion. It relies on the incorporation of a β-lactamase Vpr (BlaM-Vpr) protein chimera into the virion and the subsequent transfer of this chimera into the target cell by fusion (Figure 1). The transfer is monitored by the enzymatic cleavage of CCF2, a fluorescent dye substrate of β-lactamase, loaded into the target cells. Cleavage of the β-lactam ring in CCF2 by β-lactamase changes the fluorescence emission spectrum of the dye from green (520 nm) to blue (447 nm). This change reflects virion fusion and can be detected by flow cytometry (Figure 2).

  4. Enhancement of the basal-level activity of HIV-1 long terminal repeat by HIV-1 nucleocapsid protein.

    PubMed

    Zhang, J L; Sharma, P L; Crumpacker, C S

    2000-03-15

    Two HIV-1 proteins, Tat and NCp7 (NC), have zinc finger-like structures. NC is a virion protein and has been shown to accumulate in the nucleus 8 h postinfection. Since transcription factors with zinc fingers assist the transcriptional activity of both RNA polymerases II and III, we examined the effect of NC on HIV-1 LTR-directed gene expression. The HIV-1 NC binds to the HIV-1 LTR and results in a mobility shift in polyacrylamide gel electrophoresis. Competition assays with cold probes revealed that the binding of NC and formation of a DNA-protein complex could be prevented by the addition of excess unlabeled LTR self-probe, but not the HIV-1 V3 envelope gene. The DNase I footprint analysis showed that NC binds to six regions within HIV-1 LTR, four of which are near the transcription start site. The NC alone enhances LTR basal-level activity in RNA runoff experiments. When the general transcription factors (GTFs) were added in the assay, NC enhances NF-kappaB, Sp1, and TFIIB-induced HIV-1 LTR-directed RNA transcription. RNA transcription directed by the adenovirus major late promoter, however, is not significantly affected by NC in the cell-free system. Transient transfection of human T lymphocytes with the plasmids containing HIV-1 nc or gag showed enhancement of LTR-CAT activity. Moreover, transfection of HIV-1 provirus containing mutations in NC zinc-finger domains dramatically decreases the enhancement activity in human T cells, in which HIV-1 LTR is stably integrated into the cellular genome. These observations show that NC binds to HIV-1 LTR and cooperatively enhances GTFs and NF-kappaB induced HIV-1 LTR basal-level activity. NC may play the role of a nucleation protein, which binds to LTR and enhances basal-level transcription by recruiting cellular transcription factors to the HIV-1 promoter in competition with cellular promoters. PMID:10704334

  5. Neuropathology of early HIV-1 infection.

    PubMed

    Gray, F; Scaravilli, F; Everall, I; Chretien, F; An, S; Boche, D; Adle-Biassette, H; Wingertsmann, L; Durigon, M; Hurtrel, B; Chiodi, F; Bell, J; Lantos, P

    1996-01-01

    Early HIV-1 invasion of the central nervous system has been demonstrated by many cerebrospinal fluid studies; however, most HIV-1 carriers remain neurologically unimpaired during the so called "asymptomatic" period lasting from seroconversion to symptomatic AIDS. Therefore, neuropathological studies in the early pre-AIDS stages are very few, and the natural history of central nervous system changes in HIV-1 infection remains poorly understood. Examination of brains of asymptomatic HIV-1 positive individuals who died accidentally and of rare cases with acute fatal encephalopathy revealing HIV infection, and comparison with experimental simian immunodeficiency virus and feline immunodeficiency virus infections suggest that, invasion of the CNS by HIV-1 occurs at the time of primary infection and induces an immunological process in the central nervous system. This includes an inflammatory T-cell reaction with vasculitis and leptomeningitis, and immune activation of brain parenchyma with increased number of microglial cells, upregulation of major histocompatibility complex class II antigens and local production of cytokines. Myelin pallor and gliosis of the white matter are usually found and are likely to be the consequence of opening of the blood brain barrier due to vasculitis; direct damage to oligodendrocytes by cytokines may also interfere. These white matter changes may explain, at least partly, the early cerebral atrophy observed, by magnetic resonance imaging, in asymptomatic HIV-1 carriers. In contrast, cortical damage seems to be a late event in the course of HIV-1 infection. There is no significant neuronal loss at the early stages of the disease, no accompanying increase in glial fibrillary acid protein staining in the cortex, and only exceptional neuronal apoptosis. Although HIV-1 proviral DNA may be demonstrated in a number of brains, viral replication remains very low during the asymptomatic stage of HIV-1 infection. This makes it likely that, although

  6. Broad activation of latent HIV-1 in vivo.

    PubMed

    Barton, Kirston; Hiener, Bonnie; Winckelmann, Anni; Rasmussen, Thomas Aagaard; Shao, Wei; Byth, Karen; Lanfear, Robert; Solomon, Ajantha; McMahon, James; Harrington, Sean; Buzon, Maria; Lichterfeld, Mathias; Denton, Paul W; Olesen, Rikke; Østergaard, Lars; Tolstrup, Martin; Lewin, Sharon R; Søgaard, Ole Schmeltz; Palmer, Sarah

    2016-01-01

    The 'shock and kill' approach to cure human immunodeficiency virus (HIV) includes transcriptional induction of latent HIV-1 proviruses using latency-reversing agents (LRAs) with targeted immunotherapy to purge infected cells. The administration of LRAs (panobinostat or vorinostat) to HIV-1-infected individuals on antiretroviral therapy induces a significant increase in cell-associated unspliced (CA-US) HIV-1 RNA from CD4(+) T cells. However, it is important to discern whether the increases in CA-US HIV-1 RNA are due to limited or broad activation of HIV-1 proviruses. Here we use single-genome sequencing to find that the RNA transcripts observed following LRA administration are genetically diverse, indicating activation of transcription from an extensive range of proviruses. Defective sequences are more frequently found in CA HIV-1 RNA than in HIV-1 DNA, which has implications for developing an accurate measure of HIV-1 reservoir size. Our findings provide insights into the effects of panobinostat and vorinostat as LRAs for latent HIV-1. PMID:27605062

  7. Selectivity for strand-transfer over 3'-processing and susceptibility to clinical resistance of HIV-1 integrase inhibitors are driven by key enzyme-DNA interactions in the active site.

    PubMed

    Métifiot, Mathieu; Johnson, Barry C; Kiselev, Evgeny; Marler, Laura; Zhao, Xue Zhi; Burke, Terrence R; Marchand, Christophe; Hughes, Stephen H; Pommier, Yves

    2016-08-19

    Integrase strand transfer inhibitors (INSTIs) are highly effective against HIV infections. Co-crystal structures of the prototype foamy virus intasome have shown that all three FDA-approved drugs, raltegravir (RAL), elvitegravir and dolutegravir (DTG), act as interfacial inhibitors during the strand transfer (ST) integration step. However, these structures give only a partial sense for the limited inhibition of the 3'-processing reaction by INSTIs and how INSTIs can be modified to overcome drug resistance, notably against the G140S-Q148H double mutation. Based on biochemical experiments with modified oligonucleotides, we demonstrate that both the viral DNA +1 and -1 bases, which flank the 3'-processing site, play a critical role for 3'-processing efficiency and inhibition by RAL and DTG. In addition, the G140S-Q148H (SH) mutant integrase, which has a reduced 3'-processing activity, becomes more active and more resistant to inhibition of 3'-processing by RAL and DTG in the absence of the -1 and +1 bases. Molecular modeling of HIV-1 integrase, together with biochemical data, indicate that the conserved residue Q146 in the flexible loop of HIV-1 integrase is critical for productive viral DNA binding through specific contacts with the virus DNA ends in the 3'-processing and ST reactions. The potency of integrase inhibitors against 3'-processing and their ability to overcome resistance is discussed. PMID:27369381

  8. Comparison of Immunogenicity in Rhesus Macaques of Transmitted-Founder, HIV-1 Group M Consensus, and Trivalent Mosaic Envelope Vaccines Formulated as a DNA Prime, NYVAC, and Envelope Protein Boost

    PubMed Central

    Hulot, Sandrine L.; Korber, Bette; Giorgi, Elena E.; Vandergrift, Nathan; Saunders, Kevin O.; Balachandran, Harikrishnan; Mach, Linh V.; Lifton, Michelle A.; Pantaleo, Giuseppe; Tartaglia, Jim; Phogat, Sanjay; Jacobs, Bertram; Kibler, Karen; Perdiguero, Beatriz; Gomez, Carmen E.; Esteban, Mariano; Rosati, Margherita; Felber, Barbara K.; Pavlakis, George N.; Parks, Robert; Lloyd, Krissey; Sutherland, Laura; Scearce, Richard; Letvin, Norman L.; Seaman, Michael S.; Alam, S. Munir; Montefiori, David; Liao, Hua-Xin; Haynes, Barton F.

    2015-01-01

    ABSTRACT An effective human immunodeficiency virus type 1 (HIV-1) vaccine must induce protective antibody responses, as well as CD4+ and CD8+ T cell responses, that can be effective despite extraordinary diversity of HIV-1. The consensus and mosaic immunogens are complete but artificial proteins, computationally designed to elicit immune responses with improved cross-reactive breadth, to attempt to overcome the challenge of global HIV diversity. In this study, we have compared the immunogenicity of a transmitted-founder (T/F) B clade Env (B.1059), a global group M consensus Env (Con-S), and a global trivalent mosaic Env protein in rhesus macaques. These antigens were delivered using a DNA prime-recombinant NYVAC (rNYVAC) vector and Env protein boost vaccination strategy. While Con-S Env was a single sequence, mosaic immunogens were a set of three Envs optimized to include the most common forms of potential T cell epitopes. Both Con-S and mosaic sequences retained common amino acids encompassed by both antibody and T cell epitopes and were central to globally circulating strains. Mosaics and Con-S Envs expressed as full-length proteins bound well to a number of neutralizing antibodies with discontinuous epitopes. Also, both consensus and mosaic immunogens induced significantly higher gamma interferon (IFN-γ) enzyme-linked immunosorbent spot assay (ELISpot) responses than B.1059 immunogen. Immunization with these proteins, particularly Con-S, also induced significantly higher neutralizing antibodies to viruses than B.1059 Env, primarily to tier 1 viruses. Both Con-S and mosaics stimulated more potent CD8-T cell responses against heterologous Envs than did B.1059. Both antibody and cellular data from this study strengthen the concept of using in silico-designed centralized immunogens for global HIV-1 vaccine development strategies. IMPORTANCE There is an increasing appreciation for the importance of vaccine-induced anti-Env antibody responses for preventing HIV-1

  9. An autoreactive antibody from an SLE/HIV-1 individual broadly neutralizes HIV-1

    PubMed Central

    Bonsignori, Mattia; Wiehe, Kevin; Grimm, Sebastian K.; Lynch, Rebecca; Yang, Guang; Kozink, Daniel M.; Perrin, Florence; Cooper, Abby J.; Hwang, Kwan-Ki; Chen, Xi; Liu, Mengfei; McKee, Krisha; Parks, Robert J.; Eudailey, Joshua; Wang, Minyue; Clowse, Megan; Criscione-Schreiber, Lisa G.; Moody, M. Anthony; Ackerman, Margaret E.; Boyd, Scott D.; Gao, Feng; Kelsoe, Garnett; Verkoczy, Laurent; Tomaras, Georgia D.; Liao, Hua-Xin; Kepler, Thomas B.; Montefiori, David C.; Mascola, John R.; Haynes, Barton F.

    2014-01-01

    Broadly HIV-1–neutralizing antibodies (BnAbs) display one or more unusual traits, including a long heavy chain complementarity-determining region 3 (HCDR3), polyreactivity, and high levels of somatic mutations. These shared characteristics suggest that BnAb development might be limited by immune tolerance controls. It has been postulated that HIV-1–infected individuals with autoimmune disease and defective immune tolerance mechanisms may produce BnAbs more readily than those without autoimmune diseases. In this study, we identified an HIV-1–infected individual with SLE who exhibited controlled viral load (<5,000 copies/ml) in the absence of controlling HLA phenotypes and developed plasma HIV-1 neutralization breadth. We collected memory B cells from this individual and isolated a BnAb, CH98, that targets the CD4 binding site (CD4bs) of HIV-1 envelope glycoprotein 120 (gp120). CH98 bound to human antigens including dsDNA, which is specifically associated with SLE. Anti-dsDNA reactivity was also present in the patient’s plasma. CH98 had a mutation frequency of 25% and 15% nt somatic mutations in the heavy and light chain variable domains, respectively, a long HCDR3, and a deletion in the light chain CDR1. The occurrence of anti-dsDNA reactivity by a HIV-1 CD4bs BnAb in an individual with SLE raises the possibility that some BnAbs and SLE-associated autoantibodies arise from similar pools of B cells. PMID:24614107

  10. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy.

    PubMed

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-Bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  11. Negative Feedback Regulation of HIV-1 by Gene Editing Strategy

    PubMed Central

    Kaminski, Rafal; Chen, Yilan; Salkind, Julian; Bella, Ramona; Young, Won-bin; Ferrante, Pasquale; Karn, Jonathan; Malcolm, Thomas; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    The CRISPR/Cas9 gene editing method is comprised of the guide RNA (gRNA) to target a specific DNA sequence for cleavage and the Cas9 endonuclease for introducing breaks in the double-stranded DNA identified by the gRNA. Co-expression of both a multiplex of HIV-1-specific gRNAs and Cas9 in cells results in the modification and/or excision of the segment of viral DNA, leading to replication-defective virus. In this study, we have personalized the activity of CRISPR/Cas9 by placing the gene encoding Cas9 under the control of a minimal promoter of HIV-1 that is activated by the HIV-1 Tat protein. We demonstrate that functional activation of CRISPR/Cas9 by Tat during the course of viral infection excises the designated segment of the integrated viral DNA and consequently suppresses viral expression. This strategy was also used in a latently infected CD4+ T-cell model after treatment with a variety of HIV-1 stimulating agents including PMA and TSA. Controlled expression of Cas9 by Tat offers a new strategy for safe implementation of the Cas9 technology for ablation of HIV-1 at a very early stage of HIV-1 replication during the course of the acute phase of infection and the reactivation of silent proviral DNA in latently infected cells. PMID:27528385

  12. Potent Cell-Intrinsic Immune Responses in Dendritic Cells Facilitate HIV-1-Specific T Cell Immunity in HIV-1 Elite Controllers

    PubMed Central

    Martin-Gayo, Enrique; Buzon, Maria Jose; Ouyang, Zhengyu; Hickman, Taylor; Cronin, Jacqueline; Pimenova, Dina; Walker, Bruce D.; Lichterfeld, Mathias; Yu, Xu G.

    2015-01-01

    The majority of HIV-1 elite controllers (EC) restrict HIV-1 replication through highly functional HIV-1-specific T cell responses, but mechanisms supporting the evolution of effective HIV-1-specific T cell immunity in these patients remain undefined. Cytosolic immune recognition of HIV-1 in conventional dendritic cells (cDC) can facilitate priming and expansion of HIV-1-specific T cells; however, HIV-1 seems to be able to avoid intracellular immune recognition in cDCs in most infected individuals. Here, we show that exposure of cDCs from EC to HIV-1 leads to a rapid and sustained production of type I interferons and upregulation of several interferon-stimulated effector genes. Emergence of these cell-intrinsic immune responses was associated with a reduced induction of SAMHD1 and LEDGF/p75, and an accumulation of viral reverse transcripts, but inhibited by pharmacological blockade of viral reverse transcription or siRNA-mediated silencing of the cytosolic DNA sensor cGAS. Importantly, improved cell-intrinsic immune recognition of HIV-1 in cDCs from elite controllers translated into stronger abilities to stimulate and expand HIV-1-specific CD8 T cell responses. These data suggest an important role of cell-intrinsic type I interferon secretion in dendritic cells for the induction of effective HIV-1-specific CD8 T cells, and may be helpful for eliciting functional T cell immunity against HIV-1 for preventative or therapeutic clinical purposes. PMID:26067651

  13. Revisiting HIV-1 uncoating.

    PubMed

    Arhel, Nathalie

    2010-01-01

    HIV uncoating is defined as the loss of viral capsid that occurs within the cytoplasm of infected cells before entry of the viral genome into the nucleus. It is an obligatory step of HIV-1 early infection and accompanies the transition between reverse transcription complexes (RTCs), in which reverse transcription occurs, and pre-integration complexes (PICs), which are competent to integrate into the host genome. The study of the nature and timing of HIV-1 uncoating has been paved with difficulties, particularly as a result of the vulnerability of the capsid assembly to experimental manipulation. Nevertheless, recent studies of capsid structure, retroviral restriction and mechanisms of nuclear import, as well as the recent expansion of technical advances in genome-wide studies and cell imagery approaches, have substantially changed our understanding of HIV uncoating. Although early work suggested that uncoating occurs immediately following viral entry in the cell, thus attributing a trivial role for the capsid in infected cells, recent data suggest that uncoating occurs several hours later and that capsid has an all-important role in the cell that it infects: for transport towards the nucleus, reverse transcription and nuclear import. Knowing that uncoating occurs at a later stage suggests that the viral capsid interacts extensively with the cytoskeleton and other cytoplasmic components during its transport to the nucleus, which leads to a considerable reassessment of our efforts to identify potential therapeutic targets for HIV therapy. This review discusses our current understanding of HIV uncoating, the functional interplay between infectivity and timely uncoating, as well as exposing the appropriate methods to study uncoating and addressing the many questions that remain unanswered. PMID:21083892

  14. HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri.

    PubMed

    Ogata, T; Higuchi, H; Mochida, S; Matsumoto, H; Kato, A; Endo, T; Kaji, A; Kaji, H

    1992-11-01

    An aqueous extract of Phyllanthus niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 reverse transcriptase (HIV-1-RT). The inhibitor against HIV-1-RT in this plant was purified by combination of three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus repandus. The 50% inhibitory doses (ID50) of RA on HIV-1-RT and DNA polymerase alpha (from HeLa cells) were 0.05 microM and 0.6 microM, respectively, representing approximately a 10-fold more sensitivity of HIV-1-RT compared with DNA polymerase alpha. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 microM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 microM of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 microM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system. PMID:1283310

  15. HIV-1 Capsid Stabilization Assay.

    PubMed

    Fricke, Thomas; Diaz-Griffero, Felipe

    2016-01-01

    The stability of the HIV-1 core in the cytoplasm is crucial for productive HIV-1 infection. Mutations that stabilize or destabilize the core showed defects in HIV-1 reverse transcription and infection. We developed a novel and simple assay to measure stability of in vitro-assembled HIV-1 CA-NC complexes. This assay allowed us to demonstrate that cytosolic extracts strongly stabilize the HIV-1 core (Fricke et al., J Virol 87:10587-10597, 2013). By using our novel assay, one can measure the ability of different drugs to modulate the stability of in vitro-assembled HIV-1 CA-NC complexes, such as PF74, CAP-1, IXN-053, cyclosporine A, Bi2, and the peptide CAI. We also found that purified CPSF6 (1-321) protein stabilizes in vitro-assembled HIV-1 CA-NC complexes (Fricke et al., J Virol 87:10587-10597, 2013). Here we describe in detail the use of this capsid stability assay. We believe that our assay can be a powerful tool to assess HIV-1 capsid stability in vitro. PMID:26714703

  16. HIV-1 Replication and the Cellular Eukaryotic Translation Apparatus

    PubMed Central

    Guerrero, Santiago; Batisse, Julien; Libre, Camille; Bernacchi, Serena; Marquet, Roland; Paillart, Jean-Christophe

    2015-01-01

    Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication. PMID:25606970

  17. HIV-1 replication and the cellular eukaryotic translation apparatus.

    PubMed

    Guerrero, Santiago; Batisse, Julien; Libre, Camille; Bernacchi, Serena; Marquet, Roland; Paillart, Jean-Christophe

    2015-01-01

    Eukaryotic translation is a complex process composed of three main steps: initiation, elongation, and termination. During infections by RNA- and DNA-viruses, the eukaryotic translation machinery is used to assure optimal viral protein synthesis. Human immunodeficiency virus type I (HIV-1) uses several non-canonical pathways to translate its own proteins, such as leaky scanning, frameshifting, shunt, and cap-independent mechanisms. Moreover, HIV-1 modulates the host translation machinery by targeting key translation factors and overcomes different cellular obstacles that affect protein translation. In this review, we describe how HIV-1 proteins target several components of the eukaryotic translation machinery, which consequently improves viral translation and replication. PMID:25606970

  18. Knockdown of MAP4 and DNAL1 produces a post-fusion and pre-nuclear translocation impairment in HIV-1 replication

    SciTech Connect

    Gallo, Daniel E. Hope, Thomas J.

    2012-01-05

    DNAL1 and MAP4 are both microtubule-associated proteins. These proteins were identified as HIV-1 dependency factors in a screen with wild-type HIV-1. In this study we demonstrate that knockdown using DNAL1 and MAP4 siRNAs and shRNAs inhibits HIV-1 infection regardless of envelope. Using a fusion assay, we show that DNAL1 and MAP4 do not impact fusion. By assaying for late reverse transcripts and 2-LTR circles, we show that DNAL1 and MAP4 inhibit both by approximately 50%. These results demonstrate that DNAL1 and MAP4 impact reverse transcription but not nuclear translocation. DNAL1 and MAP4 knockdown cells do not display cytoskeletal defects. Together these experiments indicate that DNAL1 and MAP4 may exert their functions in the HIV life cycle at reverse transcription, prior to nuclear translocation.

  19. A cooperative and specific DNA-binding mode of HIV-1 integrase depends on the nature of the metallic cofactor and involves the zinc-containing N-terminal domain

    PubMed Central

    Carayon, Kevin; Leh, Hervé; Henry, Etienne; Simon, Françoise; Mouscadet, Jean-François; Deprez, Eric

    2010-01-01

    HIV-1 integrase catalyzes the insertion of the viral genome into chromosomal DNA. We characterized the structural determinants of the 3′-processing reaction specificity—the first reaction of the integration process—at the DNA-binding level. We found that the integrase N-terminal domain, containing a pseudo zinc-finger motif, plays a key role, at least indirectly, in the formation of specific integrase–DNA contacts. This motif mediates a cooperative DNA binding of integrase that occurs only with the cognate/viral DNA sequence and the physiologically relevant Mg2+ cofactor. The DNA-binding was essentially non-cooperative with Mn2+ or using non-specific/random sequences, regardless of the metallic cofactor. 2,2′-Dithiobisbenzamide-1 induced zinc ejection from integrase by covalently targeting the zinc-finger motif, and significantly decreased the Hill coefficient of the Mg2+-mediated integrase–DNA interaction, without affecting the overall affinity. Concomitantly, 2,2′-dithiobisbenzamide-1 severely impaired 3′-processing (IC50 = 11–15 nM), suggesting that zinc ejection primarily perturbs the nature of the active integrase oligomer. A less specific and weaker catalytic effect of 2,2′-dithiobisbenzamide-1 is mediated by Cys 56 in the catalytic core and, notably, accounts for the weaker inhibition of the non-cooperative Mn2+-dependent 3′-processing. Our data show that the cooperative DNA-binding mode is strongly related to the sequence-specific DNA-binding, and depends on the simultaneous presence of the Mg2+ cofactor and the zinc effector. PMID:20164093

  20. Circulating Monocytes Are Not a Major Reservoir of HIV-1 in Elite Suppressors▿

    PubMed Central

    Spivak, Adam M.; Salgado, Maria; Rabi, S. Alireza; O'Connell, Karen A.; Blankson, Joel N.

    2011-01-01

    Circulating HIV-1-infected monocytes have been identified in patients on highly active antiretroviral therapy and may represent an important barrier to viral eradication. The nature of these cells in HIV-1-infected patients who maintain undetectable viral loads and preserved CD4+ T cell counts without antiretroviral therapy (known as elite controllers or elite suppressors [ES]) is unknown. We describe here infrequent recovery of proviral HIV-1 DNA from circulating monocytes relative to CD4+ T cells in ES, despite permissiveness of these cells to HIV-1 viral entry ex vivo. Thus, monocytes do not appear to be a major reservoir of HIV-1 in ES. PMID:21795348

  1. Hyperthermia Stimulates HIV-1 Replication

    PubMed Central

    Roesch, Ferdinand; Meziane, Oussama; Kula, Anna; Nisole, Sébastien; Porrot, Françoise; Anderson, Ian; Mammano, Fabrizio; Fassati, Ariberto; Marcello, Alessandro; Benkirane, Monsef; Schwartz, Olivier

    2012-01-01

    HIV-infected individuals may experience fever episodes. Fever is an elevation of the body temperature accompanied by inflammation. It is usually beneficial for the host through enhancement of immunological defenses. In cultures, transient non-physiological heat shock (42–45°C) and Heat Shock Proteins (HSPs) modulate HIV-1 replication, through poorly defined mechanisms. The effect of physiological hyperthermia (38–40°C) on HIV-1 infection has not been extensively investigated. Here, we show that culturing primary CD4+ T lymphocytes and cell lines at a fever-like temperature (39.5°C) increased the efficiency of HIV-1 replication by 2 to 7 fold. Hyperthermia did not facilitate viral entry nor reverse transcription, but increased Tat transactivation of the LTR viral promoter. Hyperthermia also boosted HIV-1 reactivation in a model of latently-infected cells. By imaging HIV-1 transcription, we further show that Hsp90 co-localized with actively transcribing provirus, and this phenomenon was enhanced at 39.5°C. The Hsp90 inhibitor 17-AAG abrogated the increase of HIV-1 replication in hyperthermic cells. Altogether, our results indicate that fever may directly stimulate HIV-1 replication, in a process involving Hsp90 and facilitation of Tat-mediated LTR activity. PMID:22807676

  2. Correlates of HIV-1 Genital Shedding in Tanzanian Women

    PubMed Central

    Tanton, Clare; Weiss, Helen A.; Le Goff, Jerome; Changalucha, John; Rusizoka, Mary; Baisley, Kathy; Everett, Dean; Ross, David A.; Belec, Laurent; Hayes, Richard J.; Watson-Jones, Deborah

    2011-01-01

    Background Understanding the correlates of HIV shedding is important to inform strategies to reduce HIV infectiousness. We examined correlates of genital HIV-1 RNA in women who were seropositive for both herpes simplex virus (HSV)-2 and HIV-1 and who were enrolled in a randomised controlled trial of HSV suppressive therapy (aciclovir 400 mg b.i.d vs. placebo) in Tanzania. Methodology Samples, including a cervico-vaginal lavage, were collected and tested for genital HIV-1 and HSV and reproductive tract infections (RTIs) at randomisation and 6, 12 and 24 months follow-up. Data from all women at randomisation and women in the placebo arm during follow-up were analysed using generalised estimating equations to determine the correlates of cervico-vaginal HIV-1 RNA detection and load. Principal Findings Cervico-vaginal HIV-1 RNA was detected at 52.0% of 971 visits among 482 women, and was independently associated with plasma viral load, presence of genital ulcers, pregnancy, bloody cervical or vaginal discharge, abnormal vaginal discharge, cervical ectopy, Neisseria gonorrhoeae, Chlamydia trachomatis, Trichomonas vaginalis, an intermediate bacterial vaginosis score and HSV DNA detection. Similar factors were associated with genital HIV-1 RNA load. Conclusions RTIs were associated with increased presence and quantity of genital HIV-1 RNA in this population. These results highlight the importance of integrating effective RTI treatment into HIV care services. PMID:21390251

  3. Antiviral activity of CYC202 in HIV-1-infected cells.

    PubMed

    Agbottah, Emmanuel; de La Fuente, Cynthia; Nekhai, Sergie; Barnett, Anna; Gianella-Borradori, Athos; Pumfery, Anne; Kashanchi, Fatah

    2005-01-28

    There are currently 40 million individuals in the world infected with human immunodeficiency virus (HIV). The introduction of highly active antiretroviral therapy (HAART) has led to a significant reduction in AIDS-related morbidity and mortality. Unfortunately, up to 25% of patients discontinue their initial HAART regimen. Current HIV-1 inhibitors target the fusion of the virus to the cell and two viral proteins, reverse transcriptase and protease. Here, we examined whether other targets, such as an activated transcription factor, could be targeted to block HIV-1 replication. We specifically asked whether we could target a cellular kinase needed for HIV-1 transcription using CYC202 (R-roscovitine), a pharmacological cyclin-dependent kinase inhibitor. We targeted the cdk2-cyclin E complex in HIV-1-infected cells because both cdk2 and cyclin E are nonessential during mammalian development and are likely replaced by other kinases. We found that CYC202 effectively inhibits wild type and resistant HIV-1 mutants in T-cells, monocytes, and peripheral blood mononuclear cells at a low IC(50) and sensitizes these cells to enhanced apoptosis resulting in a dramatic drop in viral titers. Interestingly, the effect of CYC202 is independent of cell cycle stage and more specific for the cdk2-cyclin E complex. Finally, we show that cdk2-cyclin E is loaded onto the HIV-1 genome in vivo and that CYC202 is able to inhibit the uploading of this cdk-cyclin complex onto HIV-1 DNA. Therefore, targeting cellular enzymes necessary for HIV-1 transcription, which are not needed for cell survival, is a compelling strategy to inhibit wild type and mutant HIV-1 strains. PMID:15531588

  4. Eradicating HIV-1 infection: seeking to clear a persistent pathogen

    PubMed Central

    Archin, Nancie M.; Sung, Julia Marsh; Garrido, Carolina; Soriano-Sarabia, Natalia; Margolis, David M.

    2015-01-01

    Effective antiretroviral therapy (ART) blunts viraemia, which enables HIV-1-infected individuals to control infection and live long, productive lives. However, HIV-1 infection remains incurable owing to the persistence of a viral reservoir that harbours integrated provirus within host cellular DNA. This latent infection is unaffected by ART and hidden from the immune system. Recent studies have focused on the development of therapies to disrupt latency. These efforts unmasked residual viral genomes and highlighted the need to enable the clearance of latently infected cells, perhaps via old and new strategies that improve the HIV-1-specific immune response. In this Review, we explore new approaches to eradicate established HIV-1 infection and avoid the burden of lifelong ART. PMID:25402363

  5. Imperfect DNA mirror repeats in the gag gene of HIV-1 (HXB2) identify key functional domains and coincide with protein structural elements in each of the mature proteins

    PubMed Central

    Lang, Dorothy M

    2007-01-01

    Background A DNA mirror repeat is a sequence segment delimited on the basis of its containing a center of symmetry on a single strand, e.g. 5'-GCATGGTACG-3'. It is most frequently described in association with a functionally significant site in a genomic sequence, and its occurrence is regarded as noteworthy, if not unusual. However, imperfect mirror repeats (IMRs) having ≥ 50% symmetry are common in the protein coding DNA of monomeric proteins and their distribution has been found to coincide with protein structural elements – helices, β sheets and turns. In this study, the distribution of IMRs is evaluated in a polyprotein – to determine whether IMRs may be related to the position or order of protein cleavage or other hierarchal aspects of protein function. The gag gene of HIV-1 [GenBank:K03455] was selected for the study because its protein motifs and structural components are well documented. Results There is a highly specific relationship between IMRs and structural and functional aspects of the Gag polyprotein. The five longest IMRs in the polyprotein translate a key functional segment in each of the five cleavage products. Throughout the protein, IMRs coincide with functionally significant segments of the protein. A detailed annotation of the protein, which combines structural, functional and IMR data illustrates these associations. There is a significant statistical correlation between the ends of IMRs and the ends of PSEs in each of the mature proteins. Weakly symmetric IMRs (≥ 33%) are related to cleavage positions and processes. Conclusion The frequency and distribution of IMRs in HIV-1 Gag indicates that DNA symmetry is a fundamental property of protein coding DNA and that different levels of symmetry are associated with different functional aspects of the gene and its protein. The interaction between IMRs and protein structure and function is precise and interwoven over the entire length of the polyprotein. The distribution of IMRs and their

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  8. Salmonella typhi Ty21a bacterial ghost vector augments HIV-1 gp140 DNA vaccine-induced peripheral and mucosal antibody responses via TLR4 pathway.

    PubMed

    Wen, Jing; Yang, Yi; Zhao, Guangyu; Tong, Shuang; Yu, Hong; Jin, Xia; Du, Lanying; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2012-08-24

    Because of their stability and ease of manipulation, DNA vaccines have considerable potential for eliciting immune responses. However, they are limited by their weak immunogenicity, especially in humans. To address this challenge, we explored a new strategy of HIV vaccine delivery using Salmonella typhi Ty21a bacterial ghosts (BGs). We found that Ty21a BGs loaded with an HIV gp140 DNA vaccine (Ty21a BG-DNA) were readily taken up by murine macrophage RAW264.7 cells, and gp140 was efficiently expressed in these cells. Peripheral and intestinal mucosal anti-gp120 antibody responses in mice vaccinated with BGs-DNA vaccine were significantly higher than those in mice immunized with naked DNA vaccine. The enhancement of antibody responses was associated with BG-induced production of IL-10 through TLR4 pathway. These results demonstrate that Ty21a BGs is a novel and effective delivery vehicle for DNA vaccines, which could therefore be used as a new strategy for development of HIV vaccines. PMID:22819719

  9. Buried surface analysis of HIV-1 reverse transcriptase p66/p51 heterodimer and its interaction with dsDNA template/primer.

    PubMed

    Ding, J; Jacobo-Molina, A; Tantillo, C; Lu, X; Nanni, R G; Arnold, E

    1994-06-01

    The p66/p51 human immunodeficiency virus type 1 reverse transcriptase is a heterodimer with identical N-terminal amino acid sequences. The enzyme contains two polymerization domains and one RNase H domain, which is located at the C-terminus of the p66 subunit. Both polymerization domains fold into four individual subdomains that are not arranged in a similar fashion, forming an unusually asymmetric dimer. The complexity of the RT p66/p51 heterodimer structure is simplified using solvent-accessibility surface areas to describe the buried surface area of contact among the different subdomains. In addition, the RT/DNA contacts in the recently published RT/DNA/Fab structure [Jacobo-Molina et al., Proc. Natl Acad. Sci. USA, 90, 6320-6324 (1993)] are described using the same approach. Finally, the RT/DNA complex is compared with other dimeric DNA-binding proteins. It was found that the size of the protein and the extent of the dimer interface were not directly related to the extent of contact between the protein and the DNA. Furthermore, RT, the only protein that is not a sequence-specific DNA binding protein in this analysis, had the largest surface of interaction with the nucleic acid. PMID:7530020

  10. HIV-1 RT-dependent DNAzyme expression inhibits HIV-1 replication without the emergence of escape viruses

    PubMed Central

    Sugiyama, Ryuichi; Hayafune, Masaaki; Habu, Yuichiro; Yamamoto, Norio; Takaku, Hiroshi

    2011-01-01

    DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tRMDtRL, or one of two truncated tRNAs (Lys-3), tRMDΔARMtRL or tRMD3′-endtRL. Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations. PMID:20833635

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

    PubMed Central

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

    2015-01-01

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

  12. 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

  13. A zinc(II)-based two-dimensional MOF for sensitive and selective sensing of HIV-1 ds-DNA sequences.

    PubMed

    Zhao, Hai-Qing; Qiu, Gui-Hua; Liang, Zhen; Li, Min-Min; Sun, Bin; Qin, Liang; Yang, Shui-Ping; Chen, Wen-Hua; Chen, Jin-Xiang

    2016-05-30

    Coordination reaction of a known three-dimensional (3D) polymer precursor {Na3[Na9(Cbdcp)6(H2O)18]}n (A, Cbdcp = N-(4-carboxybenzyl)-(3,5-dicarboxyl)pyridinium) with Zn(NO3)2·6H2O in H2O or H2O/DMF at 100 °C and in the presence of aspirin, 5-fluorouracil (5-FU) as modulators, trans-1,2-bis(4-pyridyl)ethylene (bpe) or 1,2-bis(4-pyridyl)ethane (bpea) as ancillary ligands afforded six novel Zn(II)-based metal-organic frameworks (MOFs), that is, {[Zn(Cbdcp)(H2O)3]·H2O}n (1, 1D zigzag chain), {[Zn(HCbdcp)2]·H2O}n (2, 2D sheet), {[Zn(Cbdcp)(bpe)1/2]·2H2O}n (3, 3D polymer), {[Zn(Cbdcp)(bpe)1/2]·2H2O}n (4, 2D network), {[Zn(Cbdcp)(bpea)1/2]·2H2O}n (5, 3D polymer) and {[Zn(Cbdcp)(bpea)1/2]·2H2O}n (6, 2D network). Among them, compound 2 contains aromatic rings, positively charged pyridinium, Zn(2+) cation centers and carboxylic acid groups lined up on the 2D sheet structure with a certain extended surface exposure. The unique structure of 2 facilitates effective association with carboxyfluorescein (FAM) labeled probe single stranded DNA (probe ss-DNA, delineates as P-DNA) to yield a P-DNA@2 system, and leads to fluorescence quenching of FAM via a photoinduced electron transfer process. The P-DNA@2 system is effective and reliable for the detection of human immunodeficiency virus 1 ds-DNA (HIV ds-DNA) sequences and capable of distinguishing complementary HIV ds-DNA from mismatched target sequences with the detection limit as low as 10 pM (S/N = 3). PMID:27154832

  14. Phase I Randomized Clinical Trial of VRC DNA and rAd5 HIV-1 Vaccine Delivery by Intramuscular (IM), Subcutaneous (SC) and Intradermal (ID) Administration (VRC 011)

    PubMed Central

    Enama, Mary E.; Ledgerwood, Julie E.; Novik, Laura; Nason, Martha C.; Gordon, Ingelise J.; Holman, LaSonji; Bailer, Robert T.; Roederer, Mario; Koup, Richard A.; Mascola, John R.; Nabel, Gary J.; Graham, Barney S.

    2014-01-01

    Background Phase 1 evaluation of the VRC HIV DNA and rAd5 vaccines delivered intramuscularly (IM) supported proceeding to a Phase 2 b efficacy study. Here we report comparison of the IM, subcutaneous (SC) and intradermal (ID) routes of administration. Methods Sixty subjects were randomized to 6 schedules to evaluate the IM, SC or ID route for prime injections. Three schedules included DNA primes (Wks 0,4,8) and 3 schedules included rAd5 prime (Wk0); all included rAd5 IM boost (Wk24). DNA vaccine dosage was 4 mg IM or SC, but 0.4 mg ID, while all rAd5 vaccinations were 1010 PU. All injections were administered by needle and syringe. Results Overall, 27/30 subjects completed 3 DNA primes; 30/30 subjects completed rAd5 primes. Mild local pruritus (itchiness), superficial skin lesions and injection site nodules were associated with ID and SC, but not IM injections. All routes induced T-cell and antibody immune responses after rAd5 boosting. Overall, >95% had Env antibody and >80% had Env T-cell responses. Conclusions The pattern of local reactogenicity following ID and SC injections differed from IM injections but all routes were well-tolerated. There was no evidence of an immunogenicity advantage following SC or ID delivery, supporting IM delivery as the preferred route of administration. Trial Registration Clinicaltrials.gov NCT00321061 PMID:24621858

  15. Increased HIV-1 activity in anal high-grade squamous intraepithelial lesions compared with unaffected anal mucosa in men who have sex with men.

    PubMed

    Pollakis, Georgios; Richel, Olivier; Vis, Joost D; Prins, Jan M; Paxton, William A; de Vries, Henry J C

    2014-06-01

    We studied 3 patients with focal intra-anal tissue high-grade squamous intraepithelial lesions (HSILs). All had increased human immunodeficiency virus type 1 (HIV-1) RNA and DNA in lesions compared with that in healthy mucosa. HIV-1 RNA and HIV-1 episomal DNA were indicative of ongoing viral replication, more so in anal HSILs. PMID:24604897

  16. A Case of Seronegative HIV-1 Infection

    PubMed Central

    Spivak, Adam M.; Brennan, Tim; O'Connell, Karen; Sydnor, Emily; Williams, Thomas M.; Siliciano, Robert F.; Gallant, Joel E.; Blankson, Joel N.

    2009-01-01

    Patients infected with HIV-1 typically seroconvert within weeks of primary infection. In rare cases, patients do not develop antibodies against HIV-1 despite demonstrable infection. We describe an HLA-B*5802 positive individual who presented with AIDS despite repeatedly negative HIV-1 antibody screening tests. Phylogenetic analysis of env clones revealed little sequence diversity, and weak HIV-1 specific CD8+ T cell responses were present to Gag epitopes. The patient seroconverted after immune reconstitution on HAART. Lack of an antibody response to HIV-1 is rare and appears to be due to a defect in HIV-1-specific immunity rather than infection with attenuated virus. PMID:20039801

  17. HIV-1 assembly in macrophages

    PubMed Central

    2010-01-01

    The molecular mechanisms involved in the assembly of newly synthesized Human Immunodeficiency Virus (HIV) particles are poorly understood. Most of the work on HIV-1 assembly has been performed in T cells in which viral particle budding and assembly take place at the plasma membrane. In contrast, few studies have been performed on macrophages, the other major target of HIV-1. Infected macrophages represent a viral reservoir and probably play a key role in HIV-1 physiopathology. Indeed macrophages retain infectious particles for long periods of time, keeping them protected from anti-viral immune response or drug treatments. Here, we present an overview of what is known about HIV-1 assembly in macrophages as compared to T lymphocytes or cell lines. Early electron microscopy studies suggested that viral assembly takes place at the limiting membrane of an intracellular compartment in macrophages and not at the plasma membrane as in T cells. This was first considered as a late endosomal compartment in which viral budding seems to be similar to the process of vesicle release into multi-vesicular bodies. This view was notably supported by a large body of evidence involving the ESCRT (Endosomal Sorting Complex Required for Transport) machinery in HIV-1 budding, the observation of viral budding profiles in such compartments by immuno-electron microscopy, and the presence of late endosomal markers associated with macrophage-derived virions. However, this model needs to be revisited as recent data indicate that the viral compartment has a neutral pH and can be connected to the plasma membrane via very thin micro-channels. To date, the exact nature and biogenesis of the HIV assembly compartment in macrophages remains elusive. Many cellular proteins potentially involved in the late phases of HIV-1 cycle have been identified; and, recently, the list has grown rapidly with the publication of four independent genome-wide screens. However, their respective roles in infected cells

  18. Pin1 liberates the human immunodeficiency virus type-1 (HIV-1): Must we stop it?

    PubMed

    Hou, Hai; Wang, Jing-Zhang; Liu, Bao-Guo; Zhang, Ting

    2015-07-01

    Acquired immune deficiency syndrome (AIDS) is mainly caused by the human immunodeficiency virus type-1 (HIV-1). To our knowledge, this is the first review focusing on the vital role of Pin1 in the infection of HIV-1 and the development of AIDS. We and others have demonstrated that Pin1, the only known cis-to-trans isomerase recognizing the pThr/pSer-Pro motifs in proteins, plays striking roles in several human diseases. Interestingly, recent evidence gradually indicates that Pin1 regulates several key steps of the life cycle of HIV-1, including the uncoating of the HIV-1 core, the reverse transcription of the RNA genome of HIV-1, and the integration of the HIV-1 cDNA into human chromosomes. Whereas inhibiting Pin1 suppresses all of these key steps and attenuates the replication of HIV-1, at the same time different PIN1 gene variants are correlated with the susceptibility to HIV-1 infection. Furthermore, Pin1 potentially promotes HIV-1 infection by activating multiple oncogenes and inactivating multiple tumor suppressors, extending the life span of HIV-infected cells. These descriptions suggest Pin1 as a promising therapeutic target for the prevention of HIV-1 and highlight the possibility of blocking the development of AIDS by Pin1 inhibitors. PMID:25913034

  19. X4 and R5 HIV-1 have distinct post-entry requirements for uracil DNA glycosylase during infection of primary cells.

    PubMed

    Jones, Kate L; Roche, Michael; Gantier, Michael P; Begum, Nasim A; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R G; Mak, Johnson

    2010-06-11

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is approximately 10-20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  20. X4 and R5 HIV-1 Have Distinct Post-entry Requirements for Uracil DNA Glycosylase during Infection of Primary Cells

    PubMed Central

    Jones, Kate L.; Roche, Michael; Gantier, Michael P.; Begum, Nasim A.; Honjo, Tasuku; Caradonna, Salvatore; Williams, Bryan R. G.; Mak, Johnson

    2010-01-01

    It has been assumed that R5 and X4 HIV utilize similar strategies to support viral cDNA synthesis post viral entry. In this study, we provide evidence to show that R5 and X4 HIV have distinct requirements for host cell uracil DNA glycosylase (UNG2) during the early stage of infection. UNG2 has been previously implicated in HIV infection, but its precise role remains controversial. In this study we show that, although UNG2 is highly expressed in different cell lines, UNG2 levels are low in the natural host cells of HIV. Short interfering RNA knockdown of endogenous UNG2 in primary cells showed that UNG2 is required for R5 but not X4 HIV infection and that this requirement is bypassed when HIV enters the target cell via vesicular stomatitis virus envelope-glycoprotein-mediated endocytosis. We also show that short interfering RNA knockdown of UNG2 in virus-producing primary cells leads to defective R5 HIV virions that are unable to complete viral cDNA synthesis. Quantitative PCR analysis revealed that endogenous UNG2 levels are transiently up-regulated post HIV infection, and this increase in UNG2 mRNA is ∼10–20 times higher in R5 versus X4 HIV-infected cells. Our data show that both virion-associated UNG2 and HIV infection-induced UNG2 expression are critical for reverse transcription during R5 but not X4 HIV infection. More importantly, we have made the novel observation that R5 and X4 HIV have distinct host cell factor requirements and differential capacities to induce gene expression during the early stages of infection. These differences may result from activation of distinct signaling cascades and/or infection of divergent T-lymphocyte subpopulations. PMID:20371602

  1. Productive Replication of vif-Chimeric HIV-1 in Feline Cells▿

    PubMed Central

    Stern, Melissa A.; Hu, Chunling; Saenz, Dyana T.; Fadel, Hind J.; Sims, Olivia; Peretz, Mary; Poeschla, Eric M.

    2010-01-01

    Nonprimate animal models of HIV-1 infection are prevented by missing cellular cofactors and by antiviral actions of species-specific host defense factors. These blocks are profound in rodents but may be less abundant in certain Carnivora. Here, we enabled productive, spreading replication and passage of HIV-1 in feline cells. Feline fibroblasts, T-cell lines, and primary peripheral blood mononuclear cells supported early and late HIV-1 life cycle phases in a manner equivalent to that of human cells, except that produced virions had low infectivity. Stable expression of feline immunodeficiency virus (FIV) Vif-green fluorescent protein (GFP) in HIV-1 entry receptor-complemented feline (CrFK) cells enabled robust spreading HIV-1 replication. FIV Vif colocalized with feline APOBEC3 (fA3) proteins, targeted them for degradation, and prevented G→A hypermutation of the HIV-1 cDNA by fA3CH and fA3H. HIV-1 Vif was inactive against fA3s as expected and even paradoxically augmented restriction in some assays. In an interesting contrast, simian immunodeficiency virus SIVmac Vif had substantial anti-fA3 activities, which were complete against fA3CH and partial against fA3H. Moreover, both primate lentiviral Vifs colocalized with fA3s and could be pulled down from cell lysates by fA3CH. HIV-1 molecular clones that encode FIV Vif or SIVmac Vif (HIV-1VF and HIV-1VS) were then constructed. These viruses replicated productively in HIV-1 receptor-expressing CrFK cells and could be passaged serially to uninfected cells. Thus, with the exception of entry receptors, the cat genome can supply the dependency factors needed by HIV-1, and a main restriction can be countered by vif chimerism. The results raise the possibility that the domestic cat could yield an animal model of HIV-1 infection. PMID:20463079

  2. HIV-1 evades innate immune recognition through specific cofactor recruitment

    NASA Astrophysics Data System (ADS)

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J.; Price, Amanda J.; Blondeau, Caroline; Hilditch, Laura; Jacques, David A.; Selwood, David L.; James, Leo C.; Noursadeghi, Mahdad; Towers, Greg J.

    2013-11-01

    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages.

  3. HIV-1 evades innate immune recognition through specific cofactor recruitment.

    PubMed

    Rasaiyaah, Jane; Tan, Choon Ping; Fletcher, Adam J; Price, Amanda J; Blondeau, Caroline; Hilditch, Laura; Jacques, David A; Selwood, David L; James, Leo C; Noursadeghi, Mahdad; Towers, Greg J

    2013-11-21

    Human immunodeficiency virus (HIV)-1 is able to replicate in primary human macrophages without stimulating innate immunity despite reverse transcription of genomic RNA into double-stranded DNA, an activity that might be expected to trigger innate pattern recognition receptors. We reasoned that if correctly orchestrated HIV-1 uncoating and nuclear entry is important for evasion of innate sensors then manipulation of specific interactions between HIV-1 capsid and host factors that putatively regulate these processes should trigger pattern recognition receptors and stimulate type 1 interferon (IFN) secretion. Here we show that HIV-1 capsid mutants N74D and P90A, which are impaired for interaction with cofactors cleavage and polyadenylation specificity factor subunit 6 (CPSF6) and cyclophilins (Nup358 and CypA), respectively, cannot replicate in primary human monocyte-derived macrophages because they trigger innate sensors leading to nuclear translocation of NF-κB and IRF3, the production of soluble type 1 IFN and induction of an antiviral state. Depletion of CPSF6 with short hairpin RNA expression allows wild-type virus to trigger innate sensors and IFN production. In each case, suppressed replication is rescued by IFN-receptor blockade, demonstrating a role for IFN in restriction. IFN production is dependent on viral reverse transcription but not integration, indicating that a viral reverse transcription product comprises the HIV-1 pathogen-associated molecular pattern. Finally, we show that we can pharmacologically induce wild-type HIV-1 infection to stimulate IFN secretion and an antiviral state using a non-immunosuppressive cyclosporine analogue. We conclude that HIV-1 has evolved to use CPSF6 and cyclophilins to cloak its replication, allowing evasion of innate immune sensors and induction of a cell-autonomous innate immune response in primary human macrophages. PMID:24196705

  4. Specificity and 6-Month Durability of Immune Responses Induced by DNA and Recombinant Modified Vaccinia Ankara Vaccines Expressing HIV-1 Virus-Like Particles

    PubMed Central

    Goepfert, Paul A.; Elizaga, Marnie L.; Seaton, Kelly; Tomaras, Georgia D.; Montefiori, David C.; Sato, Alicia; Hural, John; DeRosa, Stephen C.; Kalams, Spyros A.; McElrath, M. Juliana; Keefer, Michael C.; Baden, Lindsey R.; Lama, Javier R.; Sanchez, Jorge; Mulligan, Mark J.; Buchbinder, Susan P.; Hammer, Scott M.; Koblin, Beryl A.; Pensiero, Michael; Butler, Chris; Moss, Bernard; Robinson, Harriet L.; Donastorg, Yeycy; Qin, Li; Lawrence, Dale; Cardinali, Massimo; Bae, Jin; Holt, Renée; Redinger, Huguette; Johannessen, Jan; Broder, Gail; Moody-White, Jerri; McKay, Butch; Calazans, Gabriela; Bentley, Carter; Kakinami, Lisa; Skibinski, Katie; Estep, Scharla; Tseng, Jenny; Swenson, Molly; Madenwald, Tamra; Overton, Edgar Turner; Edupuganti, Srilatha; Rouphael, Nadine; Whitaker, Jennifer; Hay, C Mhorag; Bunce, Catherine A; Gonzales, Pedro; Hurtado, Juan Carlos; Dolin, Raphael; Mayer, Ken; Walsh, Steven; Johnson, Jennifer

    2014-01-01

    Background. Clade B DNA and recombinant modified vaccinia Ankara (MVA) vaccines producing virus-like particles displaying trimeric membrane-bound envelope glycoprotein (Env) were tested in a phase 2a trial in human immunodeficiency virus (HIV)–uninfected adults for safety, immunogenicity, and 6-month durability of immune responses. Methods. A total of 299 individuals received 2 doses of JS7 DNA vaccine and 2 doses of MVA/HIV62B at 0, 2, 4, and 6 months, respectively (the DDMM regimen); 3 doses of MVA/HIV62B at 0, 2, and 6 months (the MMM regimen); or placebo injections. Results. At peak response, 93.2% of the DDMM group and 98.4% of the MMM group had binding antibodies for Env. These binding antibodies were more frequent and of higher magnitude for the transmembrane subunit (gp41) than the receptor-binding subunit (gp120) of Env. For both regimens, response rates were higher for CD4+ T cells (66.4% in the DDMM group and 43.1% in the MMM group) than for CD8+ T cells (21.8% in the DDMM group and 14.9% in the MMM group). Responding CD4+ and CD8+ T cells were biased toward Gag, and >70% produced 2 or 3 of the 4 cytokines evaluated (ie, interferon γ, interleukin 2, tumor necrosis factor α, and granzyme B). Six months after vaccination, the magnitudes of antibodies and T-cell responses had decreased by <3-fold. Conclusions. DDMM and MMM vaccinations with virus-like particle–expressing immunogens elicited durable antibody and T-cell responses. PMID:24403557

  5. Rational development of radiopharmaceuticals for HIV-1.

    PubMed

    Lau, Chuen-Yen; Maldarelli, Frank; Eckelman, William C; Neumann, Ronald D

    2014-04-01

    The global battle against HIV-1 would benefit from a sensitive and specific radiopharmaceutical to localize HIV-infected cells. Ideally, this probe would be able to identify latently infected host cells containing replication competent HIV sequences. Clinical and research applications would include assessment of reservoirs, informing clinical management by facilitating assessment of burden of infection in different compartments, monitoring disease progression and monitoring response to therapy. A "rational" development approach could facilitate efficient identification of an appropriate targeted radiopharmaceutical. Rational development starts with understanding characteristics of the disease that can be effectively targeted and then engineering radiopharmaceuticals to hone in on an appropriate target, which in the case of HIV-1 (HIV) might be an HIV-specific product on or in the host cell, a differentially expressed gene product, an integrated DNA sequence specific enzymatic activity, part of the inflammatory response, or a combination of these. This is different from the current approach that starts with a radiopharmaceutical for a target associated with a disease, mostly from autopsy studies, without a strong rationale for the potential to impact patient care. At present, no targeted therapies are available for HIV latency, although a number of approaches are under study. Here we discuss requirements for a radiopharmaceutical useful in strategies targeting persistently infected cells. The radiopharmaceutical for HIV should be developed based on HIV biology, studied in an animal model and then in humans, and ultimately used in clinical and research settings. PMID:24607432

  6. Novel Approaches to Inhibiting HIV-1 Replication

    PubMed Central

    Adamson, Catherine S.; Freed, Eric O.

    2009-01-01

    Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. PMID:19782103

  7. Novel approaches to inhibiting HIV-1 replication.

    PubMed

    Adamson, Catherine S; Freed, Eric O

    2010-01-01

    Considerable success has been achieved in the treatment of HIV-1 infection, and more than two-dozen antiretroviral drugs are available targeting several distinct steps in the viral replication cycle. However, resistance to these compounds emerges readily, even in the context of combination therapy. Drug toxicity, adverse drug-drug interactions, and accompanying poor patient adherence can also lead to treatment failure. These considerations make continued development of novel antiretroviral therapeutics necessary. In this article, we highlight a number of steps in the HIV-1 replication cycle that represent promising targets for drug discovery. These include lipid raft microdomains, the RNase H activity of the viral enzyme reverse transcriptase, uncoating of the viral core, host cell machinery involved in the integration of the viral DNA into host cell chromatin, virus assembly, maturation, and budding, and the functions of several viral accessory proteins. We discuss the relevant molecular and cell biology, and describe progress to date in developing inhibitors against these novel targets. This article forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, Vol 85, issue 1, 2010. PMID:19782103

  8. APOBEC4 Enhances the Replication of HIV-1

    PubMed Central

    Hofmann, Henning; Hanschmann, Kay-Martin; Mühlebach, Michael D.; Schumann, Gerald G.; König, Renate; Cichutek, Klaus; Häussinger, Dieter; Münk, Carsten

    2016-01-01

    APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters. PMID:27249646

  9. Herpes simplex virus type 2 (HSV-2) genital shedding in HSV-2-/HIV-1-co-infected women receiving effective combination antiretroviral therapy.

    PubMed

    Péré, Héléne; Rascanu, Aida; LeGoff, Jérome; Matta, Mathieu; Bois, Frédéric; Lortholary, Olivier; Leroy, Valériane; Launay, Odile; Bélec, Laurent

    2016-03-01

    The dynamics of genital shedding of HSV-2 DNA was assessed in HIV-1-infected women taking combination antiretroviral therapy (cART). HIV-1 RNA, HIV-1 DNA and HSV DNA loads were measured during 12-18 months using frozen plasma, PBMC and cervicovaginal lavage samples from 22 HIV-1-infected women, including 17 women naive for antiretroviral therapy initiating cART and 5 women with virological failure switching to a new regimen. Nineteen (86%) women were HSV-2-seropositive. Among HSV-2-/HIV-1-co-infected women, HIV-1 RNA loads showed a rapid fall from baseline after one month of cART, in parallel in paired plasma and cervicovaginal secretions. In contrast, HIV-1 DNA loads did not show significant variations from baseline up to 18 months of treatment in both systemic and genital compartments. HSV DNA was detected at least once in 12 (63%) of 19 women during follow up: HSV-2 shedding in the genital compartment was observed in 11% of cervicovaginal samples at baseline and in 16% after initiating or switching cART. Cervicovaginal HIV-1 RNA loads were strongly associated with plasma HIV-1 RNA loads over time, but not with cervicovaginal HSV DNA loads. Reactivation of genital HSV-2 replication frequently occurred despite effective cART in HSV-2-/HIV-1-co-infected women. Genital HSV-2 replication under cART does not influence cervicovaginal HIV-1 RNA or DNA shedding. PMID:25769886

  10. Knockdown of the cellular protein LRPPRC attenuates HIV-1 infection.

    PubMed

    Schweitzer, Cameron J; Matthews, John M; Madson, Christian J; Donnellan, Meghan R; Cerny, Ronald L; Belshan, Michael

    2012-01-01

    HIV-1 exploits numerous host cellular pathways for productive infection. To identify novel factors involved in HIV-1 replication, HIV-1 integrase and matrix protein complexes were captured at 4 hours post infection for proteomic analysis using an affinity purification system. Leucine-rich PPR-motif containing (LRPPRC) protein, a cellular protein involved in mitochondrial function, cell metabolism, and cell-cycle progression was identified as one of the candidate HIV-1 factors. Co-immunoprecipitation RT-PCR experiments confirmed that LRPPRC associated with HIV-1 nucleic acids during the early steps of virus infection. To establish if LRPPRC was critical for HIV-1 infection, three independent LRPPRC knockdown cell lines were constructed (2.7, 3.6, and 4.1). Subcellular fractionation of these cell lines revealed differential knockdown of LRPPRC in subcellular compartments. LRPPRC was knocked down in the insoluble/cytoskeletal fractions of all three cell lines, but the 3.6 and 4.1 cells also showed a reduction in nuclear LRPPRC. Additionally, several cellular factors were downregulated and/or disrupted by loss of LRPPRC. HIV-1 infection was reduced in all three cell lines, but virus production and RNA encapsidation were unaffected, suggesting that LRPPRC was critical for the afferent stage of virus replication. Two of the three cell lines (3.6, 4.1) were refractory for murine leukemia virus infection, a virus dependent on cellular proliferation for productive infection. Consistent with this, these two cell lines exhibited reduced cellular growth with no loss of cellular viability or change in cell cycle phenotype. The early steps of virus infection were also differentially affected among the cell lines. A reduced level of preintegration complex formation was observed in all three cell lines, but viral DNA nuclear import was reduced only in the 3.6 and 4.1 cells. Combined, these data identify LRPPRC as a HIV-1 factor that is involved in HIV-1 replication through more

  11. Strategies for Eliciting HIV-1 Inhibitory Antibodies

    PubMed Central

    Tomaras, Georgia D.; Haynes, Barton F.

    2012-01-01

    Purpose of review Major roadblocks persist in the development of vaccines that elicit potent neutralizing antibodies targeting diverse HIV-1 strains, similar to known broadly neutralizing HIV-1 human monoclonal antibodies. Alternatively, other types of anti-HIV-1 envelope antibodies that may not neutralize HIV-1 in traditional neutralization assays but have other anti-HIV-1 activities (hereafter termed HIV-1 inhibitory antibodies) can be elicited by current vaccine strategies, and numerous studies are exploring their roles in preventing HIV-1 acquisition. We review examples of strategies for eliciting potentially protective HIV-1 inhibitory antibodies. Recent Findings Heterologous prime-boost strategies can yield anti-HIV immune responses; although only one (canarypox prime, Env protein boost) has been tested and shown positive results in an efficacy trial (RV144). Although the immune correlates of protection are as yet undefined, the reduced rate of acquisition without a significant effect on initial viral loads or CD4+ T cell counts, have raised the hypothesis of an RV144 vaccine-elicited transient protective B cell response. Summary In light of the RV144 trial, there is a critical need to define the entire functional spectrum of anti-HIV-1 antibodies, how easily each can be elicited, and how effective different types of antibody effector mechanisms can be in prevention of HIV-1 transmission. PMID:20978384

  12. ZASC1 Stimulates HIV-1 Transcription Elongation by Recruiting P-TEFb and TAT to the LTR Promoter

    PubMed Central

    Bruce, James W.; Reddington, Rachel; Mathieu, Elizabeth; Bracken, Megan; Young, John A. T.; Ahlquist, Paul

    2013-01-01

    Transcription from the HIV-1 LTR promoter efficiently initiates but rapidly terminates because of a non-processive form of RNA polymerase II. This premature termination is overcome by assembly of an HIV-1 TAT/P-TEFb complex at the transactivation response region (TAR), a structured RNA element encoded by the first 59 nt of HIV-1 mRNA. Here we have identified a conserved DNA-binding element for the cellular transcription factor, ZASC1, in the HIV-1 core promoter immediately upstream of TAR. We show that ZASC1 interacts with TAT and P-TEFb, co-operating with TAT to regulate HIV-1 gene expression, and promoting HIV-1 transcriptional elongation. The importance of ZASC1 to HIV-1 transcription elongation was confirmed through mutagenesis of the ZASC1 binding sites in the LTR promoter, shRNAs targeting ZASC1 and expression of dominant negative ZASC1. Chromatin immunoprecipitation analysis revealed that ZASC1 recruits Tat and P-TEFb to the HIV-1 core promoter in a TAR-independent manner. Thus, we have identified ZASC1 as novel regulator of HIV-1 gene expression that functions through the DNA-dependent, RNA-independent recruitment of TAT/P-TEFb to the HIV-1 promoter. PMID:24204263

  13. Differentiation between human immunodeficiency virus type 1 (HIV-1) and HIV-2 isolates by nonradioisotopic reverse transcriptase-typing assay.

    PubMed Central

    Urabe, T; Sano, K; Nakano, T; Odawara, F; Lee, M H; Otake, T; Okubo, S; Hayami, M; Misaki, H; Baba, M

    1994-01-01

    We tested whether human immunodeficiency virus type 1 (HIV-1) could be differentiated from HIV-2 by a reverse transcriptase (RT)-typing assay that measured the reduction of enzyme activity owing to specific antibody. RT-inhibiting antibody was examined for HIV type specificity by a new nonradioisotopic RT assay. Antibodies from four rabbits immunized with recombinant HIV-1 RT and from 23 HIV-1-seropositive individuals all specifically inhibited the enzyme activities of two HIV-1 strains (LAV-1 and GH-3), three zidovudine-resistant HIV-1 mutants, and a recombinant HIV-1 RT. However, none of these antisera affected the activities of six HIV-2 strains (GH-1, GH-2, GH-4, GH-5, GH-6, LAV-2ROD), Rous-associated virus type 2, and DNA polymerase I from Escherichia coli. In contrast, HIV-2 antibody from a rabbit immunized with disrupted GH-1 virions blocked the enzyme activities of the six HIV-2 strains but not those of the three HIV-1 strains, Rous-associated virus type 2, or DNA polymerase I. These results indicate that the antigenic domains of HIV-1 and HIV-2 RTs recognized by their inhibiting antibodies are distinct from each other and are highly conserved. Clinical HIV isolates from 18 HIV-1-seropositive individuals and 3 HIV-2-seropositive Ghanaian individuals were identified as HIV-1 and HIV-2, respectively, by the nonradioisotopic RT-typing assay. Images PMID:7527425

  14. Functional organization of human SAMHD1 and mechanisms of HIV-1 restriction

    PubMed Central

    Ahn, Jinwoo

    2016-01-01

    Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) is a triphosphohydrolase that catalyzes the conversion of deoxyribonucleoside triphosphate to deoxyribonucleoside and triphosphate. SAMHD1 has been a recent focus of study since it was identified as a potent human immunodeficiency virus-1 (HIV-1) restriction factor in the intrinsic antiviral immune system. Recent biochemical and biological studies have suggested that SAMHD1 restricts HIV-1 infection in non-cycling cells by limiting the pool of deoxyribonucleoside triphosphates, thereby interfering with HIV-1 reverse transcription. SAMHD1 also possesses single-stranded DNA and RNA binding activity, with reported nuclease activity, conferring additional HIV-1 restriction function. This review summarizes current knowledge regarding the structure of SAMHD1 and the regulation of its function in HIV-1 restriction. PMID:26733158

  15. Functional organization of human SAMHD1 and mechanisms of HIV-1 restriction.

    PubMed

    Ahn, Jinwoo

    2016-04-01

    Sterile alpha motif and histidine-aspartate domain containing protein 1 (SAMHD1) is a triphosphohydrolase that catalyzes the conversion of deoxyribonucleoside triphosphate to deoxyribonucleoside and triphosphate. SAMHD1 has been a recent focus of study since it was identified as a potent human immunodeficiency virus-1 (HIV-1) restriction factor in the intrinsic antiviral immune system. Recent biochemical and biological studies have suggested that SAMHD1 restricts HIV-1 infection in non-cycling cells by limiting the pool of deoxyribonucleoside triphosphates, thereby interfering with HIV-1 reverse transcription. SAMHD1 also possesses single-stranded DNA and RNA binding activity, with reported nuclease activity, conferring additional HIV-1 restriction function. This review summarizes current knowledge regarding the structure of SAMHD1 and the regulation of its function in HIV-1 restriction. PMID:26733158

  16. Regulatory T cells and chronic immune activation in human immunodeficiency virus 1 (HIV-1)-infected children

    PubMed Central

    Freguja, R; Gianesin, K; Mosconi, I; Zanchetta, M; Carmona, F; Rampon, O; Giaquinto, C; De Rossi, A

    2011-01-01

    The function of CD4+ T cells with regulatory activity (Tregs) is the down-regulation of immune responses. This suppressive activity may limit the magnitude of effector responses, resulting in failure to control human immunodeficiency virus 1 (HIV-1) infection, but may also suppress chronic immune activation, a characteristic feature of HIV-1 disease. We evaluated the correlation between viral load, immune activation and Tregs in HIV-1-infected children. Eighty-nine HIV-1-infected children (aged 6–14 years) were included in the study and analysed for HIV-1 plasmaviraemia, HIV-1 DNA load, CD4 and CD8 cell subsets. Treg cells [CD4+ CD25highCD127lowforkhead box P3 (FoxP3high)] and CD8-activated T cells (CD8+CD38+) were determined by flow cytometry. Results showed that the number of activated CD8+CD38+ T cells increased in relation to HIV-1 RNA plasmaviraemia (r = 0·403, P < 0·0001). The proportion of Tregs also correlated positively with HIV-1 plasmaviraemia (r = 0·323, P = 0·002), but correlated inversely with CD4+ cells (r = −0·312, P = 0·004), thus suggesting a selective expansion along with increased viraemia and CD4+ depletion. Interestingly, a positive correlation was found between the levels of Tregs and CD8+CD38+ T cells (r = 0·305, P = 0·005), and the percentage of Tregs tended to correlate with HIV-1 DNA load (r = 0·224, P = 0·062). Overall, these findings suggest that immune activation contributes to the expansion of Treg cells. In turn, the suppressive activity of Tregs may impair effector responses against HIV-1, but appears to be ineffective in limiting immune activation. PMID:21438872

  17. HIV-1 Vpr—a still “enigmatic multitasker”

    PubMed Central

    Guenzel, Carolin A.; Hérate, Cécile; Benichou, Serge

    2014-01-01

    Like other HIV-1 auxiliary proteins, Vpr is conserved within all the human (HIV-1, HIV-2) and simian (SIV) immunodeficiency viruses. However, Vpr and homologous HIV-2, and SIV Vpx are the only viral auxiliary proteins specifically incorporated into virus particles through direct interaction with the Gag precursor, indicating that this presence in the core of the mature virions is mainly required for optimal establishment of the early steps of the virus life cycle in the newly infected cell. In spite of its small size, a plethora of effects and functions have been attributed to Vpr, including induction of cell cycle arrest and apoptosis, modulation of the fidelity of reverse transcription, nuclear import of viral DNA in macrophages and other non-dividing cells, and transcriptional modulation of viral and host cell genes. Even if some more recent studies identified a few cellular targets that HIV-1 Vpr may utilize in order to perform its different tasks, the real role and functions of Vpr during the course of natural infection are still enigmatic. In this review, we will summarize the main reported functions of HIV-1 Vpr and their significance in the context of the viral life cycle. PMID:24744753

  18. High frequency of HIV-1 infections with multiple HIV-1 strains in men having sex with men (MSM) in Senegal.

    PubMed

    Leye, Nafissatou; Vidal, Nicole; Ndiaye, Ousseynou; Diop-Ndiaye, Halimatou; Wade, Abdoulaye Sidibé; Mboup, Souleymane; Delaporte, Eric; Toure-Kane, Coumba; Peeters, Martine

    2013-12-01

    Circulating and unique recombinant HIV-1 strains continue to be identified and their number increases over time, suggesting that co-infection with multiple HIV-1 is frequent. In this study we analyzed to what extent dual infections with different HIV-1 variants occur in a population group with high risk behaviour, high HIV-1 prevalence and in an area where multiple HIV-1 subtypes and Circulating Recombinant Forms (CRFs) co-circulate. We studied 69 MSM with our recently developed multi-region hybridization assay (MHA), based on fluorescent probe detection for eight common variants circulating in West and West Central Africa. At least 11 (15.9%) of the 69 patients were simultaneously infected with two different HIV-1 subtypes and/or CRFs. Among the 29 samples identified as subtype C by MHA in gag, 15 (57.7%) reacted with both C1 and C2 probes. Sequence analysis suggests that the majority of the samples reactive with C1 and C2 probes are most likely infected with two different subtype C clades. Single genome amplification and DNA dilutions confirmed dual infection with subtype D and C for MSM1193, triple infection with two different C subtype strains and one CRF02_AG strain in MSM1157 and showed that MSM3017 is at least co-infected with CRF06_cpx and CRF02_AG and another strain that could not be classified. Comparison of all subtype C sequences from the MSM population and from the general population from this and previous studies confirmed the intermixing of HIV-1 variants between low-risk women and high-risk men as shown by the intermixing of subtype C variants from MSM1157 and a female patient (02SN-HALD478). Comparison of dual infection rates between the general population and MSM in Senegal, show also clearly the importance of high HIV prevalence and high risk behavior in dual infections and subsequent intermixing of HIV-1 variants which can lead to emergence and spread of new recombinants (CRFs). PMID:24035811

  19. Identification of HIV-1 Genitourinary Tract Compartmentalization by Analyzing the env Gene Sequences in Urine

    PubMed Central

    BLASI, Maria; CARPENTER, J. Harris; BALAKUMARAN, Bala; CARA, Andrea; GAO, Feng; KLOTMAN, Mary E.

    2015-01-01

    Objective HIV-1 persists indefinitely in memory CD4+ T cells and other long-lived cellular reservoirs despite antiretroviral therapy (ART). Our group had previously demonstrated that HIV-1 can establish a productive infection in renal epithelial cells and that the kidney represents a separate compartment for HIV-1 replication. Here, to better understand the viruses in this unique site, we genetically characterized and compared the viruses in blood and urine specimens from twenty-four HIV-1 infected subjects with detectable viremia. Design and Methods Blood and urine samples were obtained from 35 HIV-1 positive subjects. Single-genome amplification was performed on HIV-1 env RNA and DNA isolated from urine supernatants and urine derived cell pellets respectively, as well as from plasma and PBMC from the same individuals. Neighbor-joining trees were constructed under the Kimura 2-parameter mode. Results We amplified and sequenced the full-length HIV-1 envelope (env) gene from twelve of the twenty-four individuals, indicating that fifty percent (50%) of the viremic HIV-1 positive patients had viral RNA in their urine. Phylogenetic analysis of the env sequences from four subjects with more than fifteen urine-derived env sequences showed that the majority of the sequences from urine formed distinct cluster(s) independent of those PBMC and plasma-derived sequences, consistent with viral compartmentalization in the urine. Conclusions Our results suggest the presence of a distinct HIV compartment in the genitourinary tract. PMID:26372275

  20. Development of Methods for Coordinate Measurement of Total Cell-Associated and Integrated Human Immunodeficiency Virus Type 1 (HIV-1) DNA Forms in Routine Clinical Samples: Levels Are Not Associated with Clinical Parameters, but Low Levels of Integrated HIV-1 DNA May Be Prognostic for Continued Successful Therapy▿

    PubMed Central

    Carr, J. M.; Cheney, K. M.; Coolen, C.; Davis, A.; Shaw, D.; Ferguson, W.; Chang, G.; Higgins, G.; Burrell, C.; Li, P.

    2007-01-01

    We have adapted our established Alu PCR assay for proviral DNA and PCR for total cellular DNA to a real-time PCR format and applied these to human immunodeficiency virus (HIV)-positive specimens collected for routine determination of the plasma viral load (pVL). In a cohort of five patients, measurements of integrated viral load (iVL) and cell-associated viral load (cVL) in CD4+ cells isolated by a single positive selection step were not indicative of HIV DNA levels in the circulation, and further analysis was performed on peripheral blood mononuclear cells (PBMC). In a cohort of 46 samples total cVL was quantitated in most samples, but iVL could be quantitated in only 47.8%, since in 26% iVL was undetectable and in 21.7% the results were invalid due to high levels of unintegrated HIV DNA. There was no correlation of cVL or iVL with pVL, CD4 count, or duration of successful antiretroviral treatment. Out of 26 patients with undetectable pVL, 4 patients failed therapy within the subsequent 12 months and had higher than average iVL, but this was not the case for cVL. Among nine patients with long-term undetectable pVL, no consistent decline in cVL or iVL was seen with time, and changes in cVL and iVL within a patient could be concordant or discordant. These results show that cVL and iVL can be coordinately measured in PBMC from clinical samples but do not correlate with pVL, CD4 counts, or length of suppressive antiretroviral therapy. Interestingly, a high iVL (but not a high cVL) in patients with undetectable pVL was associated with subsequent treatment failure. PMID:17314225

  1. Antiretroviral-Free HIV-1 Remission and Viral Rebound Following Allogeneic Stem Cell Transplantation: A Report of Two Cases

    PubMed Central

    Henrich, Timothy J.; Hanhauser, Emily; Marty, Francisco M.; Sirignano, Michael N.; Keating, Sheila; Lee, Tzong-Hae; Robles, Yvonne P.; Davis, Benjamin T.; Li, Jonathan Z.; Heisey, Andrea; Hill, Alison L.; Busch, Michael P.; Armand, Philippe; Soiffer, Robert J.; Altfeld, Marcus; Kuritzkes, Daniel R.

    2014-01-01

    Background It is unknown if the reduction in HIV-1 reservoirs observed following allogeneic hematopoietic stem cell transplantation (HSCT) with susceptible donor cells is sufficient to achieve sustained HIV-1 remission. Objective To characterize HIV-1 reservoirs in blood and tissues, and to perform analytical antiretroviral treatment interruptions to determine the potential for allogeneic HSCT to lead to sustained antiretroviral-free HIV-1 remission. Design Characterization of HIV-1 reservoirs and immunity before and after antiretroviral interruption. Setting Tertiary care center. Patients Two HIV-infected men with undetectable HIV-1 following allogeneic HSCT for hematologic malignancies. Measurements Quantification of HIV-1 in various tissues after HSCT and the duration of antiretroviral-free HIV-1 remission after treatment interruption. Results No HIV-1 was detected from peripheral blood or rectal mucosa prior to analytical treatment interruption. Plasma HIV-1 RNA and cell-associated HIV-1 DNA remained undetectable until 12 to 32 weeks after antiretroviral cessation. Both patients experienced rebound viremia with the development of acute retroviral syndrome within one to two weeks of the most recent negative viral load measurement. One patient developed new efavirenz resistance after re-initiation of antiretroviral therapy. Re-initiation of active therapy led to viral decay and resolution of symptoms in both patients. Limitations The study was limited to 2 patients. Conclusions Allogeneic HSCT may lead to loss of detectable HIV-1 from blood and gut tissue and variable periods of antiretroviral-free HIV-1 remission, but viral rebound can occur despite a minimum 3-log10 reduction in reservoir size. Long-lived tissue reservoirs may have contributed to viral persistence. Defining the nature and half-life of such reservoirs is essential in order to achieve durable antiretroviral-free HIV-1 remission. PMID:25047577

  2. 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

  3. Identification of HIV-1 Inhibitors Targeting The Nucleocapsid Protein

    PubMed Central

    Breuer, Sebastian; Chang, Max W.; Yuan, Jinyun; Torbett, Bruce E.

    2012-01-01

    The HIV-1 nucleocapsid (NC) is a RNA/DNA binding protein encoded within the Gag polyprotein, which is critical for the selection and chaperoning of viral genomic RNA during virion assembly. RNA/DNA binding occurs through a highly conserved zinc-knuckle motif present in NC. Given the necessity of NC-viral RNA/DNA interaction for viral replication, identification of compounds that disrupt the NC-RNA/DNA interaction may have value as an anti-viral strategy. To identify small molecules that disrupt NC-viral RNA/DNA binding a high-throughput fluorescence polarization assay was developed and a library of 14,400 diverse, drug-like compounds was screened. Compounds that disrupted NC binding to a fluorescence-labeled DNA tracer were next evaluated by differential scanning fluorimetry to identify compounds that must bind to NC or Gag to impart their effects. Two compounds were identified that inhibited NC-DNA interaction, specifically bound NC with nM affinity, and showed modest anti-HIV-1 activity in ex vivo cell assays. PMID:22587465

  4. Discovery of Novel Small-Molecule HIV-1 Replication Inhibitors That Stabilize Capsid Complexes

    PubMed Central

    Titolo, Steve; Lemke, Christopher T.; Goudreau, Nathalie; Mercier, Jean-François; Wardrop, Elizabeth; Shah, Vaibhav B.; von Schwedler, Uta K.; Langelier, Charles; Banik, Soma S. R.; Aiken, Christopher; Sundquist, Wesley I.

    2013-01-01

    The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTD site 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid. PMID:23817385

  5. Discovery of novel small-molecule HIV-1 replication inhibitors that stabilize capsid complexes.

    PubMed

    Lamorte, Louie; Titolo, Steve; Lemke, Christopher T; Goudreau, Nathalie; Mercier, Jean-François; Wardrop, Elizabeth; Shah, Vaibhav B; von Schwedler, Uta K; Langelier, Charles; Banik, Soma S R; Aiken, Christopher; Sundquist, Wesley I; Mason, Stephen W

    2013-10-01

    The identification of novel antiretroviral agents is required to provide alternative treatment options for HIV-1-infected patients. The screening of a phenotypic cell-based viral replication assay led to the identification of a novel class of 4,5-dihydro-1H-pyrrolo[3,4-c]pyrazol-6-one (pyrrolopyrazolone) HIV-1 inhibitors, exemplified by two compounds: BI-1 and BI-2. These compounds inhibited early postentry stages of viral replication at a step(s) following reverse transcription but prior to 2 long terminal repeat (2-LTR) circle formation, suggesting that they may block nuclear targeting of the preintegration complex. Selection of viruses resistant to BI-2 revealed that substitutions at residues A105 and T107 within the capsid (CA) amino-terminal domain (CANTD) conferred high-level resistance to both compounds, implicating CA as the antiviral target. Direct binding of BI-1 and/or BI-2 to CANTD was demonstrated using isothermal titration calorimetry and nuclear magnetic resonance (NMR) chemical shift titration analyses. A high-resolution crystal structure of the BI-1:CANTD complex revealed that the inhibitor bound within a recently identified inhibitor binding pocket (CANTD site 2) between CA helices 4, 5, and 7, on the surface of the CANTD, that also corresponds to the binding site for the host factor CPSF-6. The functional consequences of BI-1 and BI-2 binding differ from previously characterized inhibitors that bind the same site since the BI compounds did not inhibit reverse transcription but stabilized preassembled CA complexes. Hence, this new class of antiviral compounds binds CA and may inhibit viral replication by stabilizing the viral capsid. PMID:23817385

  6. Nuclear trafficking of the HIV-1 pre-integration complex depends on the ADAM10 intracellular domain

    SciTech Connect

    Endsley, Mark A.; Somasunderam, Anoma D.; Li, Guangyu; Oezguen, Numan; Thiviyanathan, Varatharasa; Murray, James L.; Rubin, Donald H.; Hodge, Thomas W.; and others

    2014-04-15

    Previously, we showed that ADAM10 is necessary for HIV-1 replication in primary human macrophages and immortalized cell lines. Silencing ADAM10 expression interrupted the HIV-1 life cycle prior to nuclear translocation of viral cDNA. Furthermore, our data indicated that HIV-1 replication depends on the expression of ADAM15 and γ-secretase, which proteolytically processes ADAM10. Silencing ADAM15 or γ-secretase expression inhibits HIV-1 replication between reverse transcription and nuclear entry. Here, we show that ADAM10 expression also supports replication in CD4{sup +} T lymphocytes. The intracellular domain (ICD) of ADAM10 associates with the HIV-1 pre-integration complex (PIC) in the cytoplasm and immunoprecipitates and co-localizes with HIV-1 integrase, a key component of PIC. Taken together, our data support a model whereby ADAM15/γ-secretase processing of ADAM10 releases the ICD, which then incorporates into HIV-1 PIC to facilitate nuclear trafficking. Thus, these studies suggest ADAM10 as a novel therapeutic target for inhibiting HIV-1 prior to nuclear entry. - Highlights: • Nuclear trafficking of the HIV-1 pre-integration complex depends on ADAM10. • ADAM10 associates with HIV-1 integrase in the pre-integration complex. • HIV-1 replication depends on the expression of ADAM15 and γ-secretase. • Silencing ADAM15 or γ-secretase expression inhibits nuclear import of viral cDNA. • ADAM10 is important for HIV-1 replication in human macrophages and CD4{sup +} T lymphocytes.

  7. Molecular Dynamics Studies on the HIV-1 Integrase Catalytic Domain

    SciTech Connect

    Lins, Roberto D.; Briggs, J. M.; Straatsma, TP; Carlson, Heather A.; Greenwald, Jason; Choe, Senyon; Mccammon, Andy

    1999-06-30

    The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome, thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. Two 1-ns molecular dynamics simulations have been carried out on completely hydrated models of the HIV-1 integrase catalytic domain, one with no metal ions and another with one magnesium ion in the catalytic site. The simulations predict that the region of the active site that is missing in the published crystal structures has (at the time of this work) more secondary structure than previously thought. The flexibility of this region has been discussed with respect to the mechanistic function of the enzyme. The results of these simulations will be used as part of inhibitor design projects directed against the catalytic domain of the enzyme.

  8. Cytoplasmic dynein promotes HIV-1 uncoating.

    PubMed

    Pawlica, Paulina; Berthoux, Lionel

    2014-11-01

    Retroviral capsid (CA) cores undergo uncoating during their retrograde transport (toward the nucleus), and/or after reaching the nuclear membrane. However, whether HIV-1 CA core uncoating is dependent upon its transport is not understood. There is some evidence that HIV-1 cores retrograde transport involves cytoplasmic dynein complexes translocating on microtubules. Here we investigate the role of dynein-dependent transport in HIV-1 uncoating. To interfere with dynein function, we depleted dynein heavy chain (DHC) using RNA interference, and we over-expressed p50/dynamitin. In immunofluorescence microscopy experiments, DHC depletion caused an accumulation of CA foci in HIV-1 infected cells. Using a biochemical assay to monitor HIV-1 CA core disassembly in infected cells, we observed an increase in amounts of intact (pelletable) CA cores upon DHC depletion or p50 over-expression. Results from these two complementary assays suggest that inhibiting dynein-mediated transport interferes with HIV-1 uncoating in infected cells, indicating the existence of a functional link between HIV-1 transport and uncoating. PMID:25375884

  9. Cyclophilin B enhances HIV-1 infection.

    PubMed

    DeBoer, Jason; Madson, Christian J; Belshan, Michael

    2016-02-01

    Cyclophilin B (CypB) is a member of the immunophilin family and intracellular chaperone. It predominantly localizes to the ER, but also contains a nuclear localization signal and is secreted from cells. CypB has been shown to interact with the Gag protein of human immunodeficiency type 1 (HIV-1). Several proteomic and genetic studies identified it as a potential factor involved in HIV replication. Herein, we show that over-expression of CypB enhances HIV infection by increasing nuclear import of viral DNA. This enhancement was unaffected by cyclosporine treatment and requires the N-terminus of the protein. The N-terminus contains an ER leader sequence, putative nuclear localization signal, and is required for secretion. Deletion of the N-terminus resulted in mislocalization from the ER and suppression of HIV infection. Passive transfer experiments showed that secreted CypB did not impact HIV infection. Combined, these experiments show that intracellular CypB modulates a pathway of HIV nuclear import. PMID:26774171

  10. In vitro Uncoating of HIV-1 Cores

    PubMed Central

    Shah, Vaibhav B.; Aiken, Christopher

    2011-01-01

    The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone1, 2. Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core. Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form3 in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells4. This indicates that the stability of the capsid is critical for HIV-1 infection. HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37°C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability4, 5, 6. It should also be useful for studying the role of cellular factors in HIV-1 uncoating. PMID:22105356

  11. In vitro uncoating of HIV-1 cores.

    PubMed

    Shah, Vaibhav B; Aiken, Christopher

    2011-01-01

    The genome of the retroviruses is encased in a capsid surrounded by a lipid envelope. For lentiviruses, such as HIV-1, the conical capsid shell is composed of CA protein arranged as a lattice of hexagon. The capsid is closed by 7 pentamers at the broad end and 5 at the narrow end of the cone(1, 2). Encased in this capsid shell is the viral ribonucleoprotein complex, and together they comprise the core. Following fusion of the viral membrane with the target cell membrane, the HIV-1 is released into the cytoplasm. The capsid then disassembles releasing free CA in the soluble form(3) in a process referred to as uncoating. The intracellular location and timing of HIV-1 uncoating are poorly understood. Single amino-acid substitutions in CA that alter the stability of the capsid also impair the ability of HIV-1 to infect cells(4). This indicates that the stability of the capsid is critical for HIV-1 infection. HIV-1 uncoating has been difficult to study due to lack of availability of sensitive and reliable assays for this process. Here we describe a quantitative method for studying uncoating in vitro using cores isolated from infectious HIV-1 particles. The approach involves isolation of cores by sedimentation of concentrated virions through a layer of detergent and into a linear sucrose gradient, in the cold. To quantify uncoating, the isolated cores are incubated at 37°C for various timed intervals and subsequently pelleted by ultracentrifugation. The extent of uncoating is analyzed by quantifying the fraction of CA in the supernatant. This approach has been employed to analyze effects of viral mutations on HIV-1 capsid stability(4, 5, 6). It should also be useful for studying the role of cellular factors in HIV-1 uncoating. PMID:22105356

  12. Macrophages and HIV-1: An Unhealthy Constellation.

    PubMed

    Sattentau, Quentin J; Stevenson, Mario

    2016-03-01

    Lentiviruses have a long-documented association with macrophages. Abundant evidence exists for in vitro and, in a tissue-specific manner, in vivo infection of macrophages by the primate lentiviruses HIV-1 and SIV. However, macrophage contribution to aspects of HIV-1 and SIV pathogenesis, and their role in viral persistence in individuals on suppressive antiretroviral therapy, remains unclear. Here we discuss recent evidence implicating macrophages in HIV-1-mediated disease and highlight directions for further investigation. PMID:26962941

  13. HIV-1 Transmission Patterns in Antiretroviral Therapy-Naïve, HIV-Infected North Americans Based on Phylogenetic Analysis by Population Level and Ultra-Deep DNA Sequencing

    PubMed Central

    Ross, Lisa L.; Horton, Joseph; Hasan, Samiul; Brown, James R.; Murphy, Daniel; DeJesus, Edwin; Potter, Martin; LaMarca, Anthony; Melendez-Rivera, Ivan; Ward, Douglas; Uy, Jonathon; Shaefer, Mark S.

    2014-01-01

    Factors that contribute to the transmission of human immunodeficiency virus type 1 (HIV-1), especially drug-resistant HIV-1 variants remain a significant public health concern. In-depth phylogenetic analyses of viral sequences obtained in the screening phase from antiretroviral-naïve HIV-infected patients seeking enrollment in EPZ108859, a large open-label study in the USA, Canada and Puerto Rico (ClinicalTrials.gov NCT00440947) were examined for insights into the roles of drug resistance and epidemiological factors that could impact disease dissemination. Viral transmission clusters (VTCs) were initially predicted from a phylogenetic analysis of population level HIV-1 pol sequences obtained from 690 antiretroviral-naïve subjects in 2007. Subsequently, the predicted VTCs were tested for robustness by ultra deep sequencing (UDS) using pyrosequencing technology and further phylogenetic analyses. The demographic characteristics of clustered and non-clustered subjects were then compared. From 690 subjects, 69 were assigned to 1 of 30 VTCs, each containing 2 to 5 subjects. Race composition of VTCs were significantly more likely to be white (72% vs. 60%; p = 0.04). VTCs had fewer reverse transcriptase and major PI resistance mutations (9% vs. 24%; p = 0.002) than non-clustered sequences. Both men-who-have-sex-with-men (MSM) (68% vs. 48%; p = 0.001) and Canadians (29% vs. 14%; p = 0.03) were significantly more frequent in VTCs than non-clustered sequences. Of the 515 subjects who initiated antiretroviral therapy, 33 experienced confirmed virologic failure through 144 weeks while only 3/33 were from VTCs. Fewer VTCs subjects (as compared to those with non-clustering virus) had HIV-1 with resistance-associated mutations or experienced virologic failure during the course of the study. Our analysis shows specific geographical and drug resistance trends that correlate well with transmission clusters defined by HIV sequences of similarity. Furthermore, our

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

    PubMed Central

    Kline, Erik R.; Sutliff, Roy L.

    2008-01-01

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

  15. Citron kinase enhances ubiquitination of HIV-1 Gag protein and intracellular HIV-1 budding.

    PubMed

    Ding, Jiwei; Zhao, Jianyuan; Sun, Lei; Mi, Zeyun; Cen, Shan

    2016-09-01

    Assembly and budding of human immunodeficiency virus type 1 (HIV-1) particles is a complex process involving a number of host proteins. We have previously reported that the RhoA effector citron kinase enhances HIV-1 production. However, the underlying mechanism is not clear. In this study, we found that citron kinase interacted with HIV-1 Gag protein via its zinc finger and leucine zipper domains. Electron microscopy analysis revealed that citron kinase induced viral particle assembly in multivesicular bodies (MVBs). Citron kinase enhanced ubiquitination of HIV-1 Gag protein. Knockdown of Nedd4L, a member of the HECT ubiquitin E3 ligase family, partly decreased the ability of citron kinase to enhance HIV-1 production and reduced ubiquitination of HIV-1 Gag. Interestingly, the function of citron kinase to promote HIV-1 budding was severely impaired when endogenous ALIX was knocked down. Overexpression of the AAA-type ATPase VPS4 eliminated citron-kinase-mediated enhancement of HIV-1 production. Our results suggest that citron kinase interacts with HIV-1 Gag and enhances HIV-1 production by promoting Gag ubiquitination and inducing viral release via the MVB pathway. PMID:27339686

  16. Recruitment of chromatin-modifying enzymes by CTIP2 promotes HIV-1 transcriptional silencing

    PubMed Central

    Marban, Céline; Suzanne, Stella; Dequiedt, Franck; de Walque, Stéphane; Redel, Laetitia; Van Lint, Carine; Aunis, Dominique; Rohr, Olivier

    2007-01-01

    Following entry and reverse transcription, the HIV-1 genome is integrated into the host genome. In contrast to productively infected cells, latently infected cells frequently harbor HIV-1 genomes integrated in heterochromatic structures, allowing persistence of transcriptionally silent proviruses. Microglial cells are the main HIV-1 target cells in the central nervous system and constitute an important reservoir for viral pathogenesis. In the present work, we show that, in microglial cells, the co-repressor COUP-TF interacting protein 2 (CTIP2) recruits a multienzymatic chromatin-modifying complex and establishes a heterochromatic environment at the HIV-1 promoter. We report that CTIP2 recruits histone deacetylase (HDAC)1 and HDAC2 to promote local histone H3 deacetylation at the HIV-1 promoter region. In addition, DNA-bound CTIP2 also associates with the histone methyltransferase SUV39H1, which increases local histone H3 lysine 9 methylation. This allows concomitant recruitment of HP1 proteins to the viral promoter and formation of local heterochromatin, leading to HIV-1 silencing. Altogether, our findings uncover new therapeutic opportunities for purging latent HIV-1 viruses from their cellular reservoirs. PMID:17245431

  17. Boosting of HIV-1 Neutralizing Antibody Responses by a Distally Related Retroviral Envelope Protein

    PubMed Central

    Uchtenhagen, Hannes; Schiffner, Torben; Bowles, Emma; Heyndrickx, Leo; LaBranche, Celia; Applequist, Steven E.; Jansson, Marianne; De Silva, Thushan; Back, Jaap Willem; Achour, Adnane; Scarlatti, Gabriella; Fomsgaard, Anders; Montefiori, David; Stewart-Jones, Guillaume; Spetz, Anna-Lena

    2014-01-01

    Our knowledge of the binding sites for neutralizing antibodies (NAbs) that recognize a broad range of HIV-1 strains (bNAb) has substantially increased in recent years. However, gaps remain in our understanding of how to focus B-cell responses to vulnerable conserved sites within the HIV-1 envelope glycoprotein (Env). Here we report an immunization strategy composed of a trivalent HIV-1 (clade B envs) DNA prime, followed by a SIVmac239 gp140 Env protein boost that aimed to focus the immune response to structurally conserved parts of the HIV-1 and SIV Envs. Heterologous NAb titres, primarily to tier 1 HIV-1 isolates, elicited during the trivalent HIV-1 env prime, were significantly increased by the SIVmac239 gp140 protein boost in rabbits. Epitope mapping of antibody binding reactivity revealed preferential recognition of the C1, C2, V2, V3 and V5 regions. These results provide a proof of concept that a distally related retroviral SIV Env protein boost can increase pre-existing NAb responses against HIV-1. PMID:24829409

  18. Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing

    PubMed Central

    Kaminski, Rafal; Chen, Yilan; Fischer, Tracy; Tedaldi, Ellen; Napoli, Alessandro; Zhang, Yonggang; Karn, Jonathan; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS. PMID:26939770

  19. Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing.

    PubMed

    Kaminski, Rafal; Chen, Yilan; Fischer, Tracy; Tedaldi, Ellen; Napoli, Alessandro; Zhang, Yonggang; Karn, Jonathan; Hu, Wenhui; Khalili, Kamel

    2016-01-01

    We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5' and 3' LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS. PMID:26939770

  20. 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

  1. AIDS dementia is associated with massive, activated HIV-1 infection and concomitant expression of several cytokines.

    PubMed Central

    Nuovo, G. J.; Alfieri, M. L.

    1996-01-01

    BACKGROUND: We recently showed that acquired immunodeficiency syndrome (AIDS) dementia is associated with activated infection of microglia, neurons, and astrocytes by HIV-1. However, it is doubtful whether infection per se is responsible for the dramatic symptoms associated with AIDS dementia. The purpose of this study was to determine the histologic distribution of messenger RNAs (mRNAs) of several cytokines that have been implicated in AIDS pathogenesis and to correlate this expression pattern with the in situ localization of polymerase chain reaction (PCR)-amplified HIV-1 nucleic acids in the central nervous system (CNS). MATERIALS AND METHODS: HIV-1 DNA was detected by PCR in situ hybridization. HIV-1 RNA and cytokine expression, including tumor necrosis factor alpha (TNF), inducible nitric oxide synthetase (iNOS), and macrophage inflammatory protein alpha (MIP-1 alpha) and MIP-1 beta mRNA were detected by reverse transcriptase (RT) in situ PCR. RESULTS: Amplified viral DNA was detected in each of the seven HIV-1-positive cases and in none of the five negative controls. In people with AIDS dementia, many HIV-1 DNA-positive cells were detected in regions of the CNS that corresponded to clinical symptomatology. In AIDS patients with minimal CNS involvement, rare HIV-1-infected microglial cells were noted. Viral RNA was detected primarily in cases of AIDS dementia. TNF, iNOS, MIP-1 alpha and MIP-1 beta expression localized to tissues from AIDS dementia cases where HIV-1 infected cells were plentiful. Colocalization experiments showed that these cytokines were transcribed mostly by viral-negative cells. CONCLUSIONS: These results suggest that two key elements in AIDS dementia are massive productive viral infection, involving microglia, neurons, and astrocytes, and concomitant stimulation of cytokine transcription in the neighboring uninfected cells. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 PMID:8784788

  2. Cross-subtype detection of HIV-1 using reverse transcription and recombinase polymerase amplification.

    PubMed

    Lillis, Lorraine; Lehman, Dara A; Siverson, Joshua B; Weis, Julie; Cantera, Jason; Parker, Mathew; Piepenburg, Olaf; Overbaugh, Julie; Boyle, David S

    2016-04-01

    A low complexity diagnostic test that rapidly and reliably detects HIV infection in infants at the point of care could facilitate early treatment, improving outcomes. However, many infant HIV diagnostics can only be performed in laboratory settings. Recombinase polymerase amplification (RPA) is an isothermal amplification technology that can rapidly amplify proviral DNA from multiple subtypes of HIV-1 in under twenty minutes without complex equipment. In this study we added reverse transcription (RT) to RPA to allow detection of both HIV-1 RNA and DNA. We show that this RT-RPA HIV-1 assay has a limit of detection of 10-30 copies of an exact sequence matched DNA or RNA, respectively. In addition, at 100 copies of RNA or DNA, the assay detected 171 of 175 (97.7%) sequence variants that represent all the major subtypes and recombinant forms of HIV-1 Groups M and O. This data suggests that the application of RT-RPA for the combined detection of HIV-1 viral RNA and proviral DNA may prove a highly sensitive tool for rapid and accurate diagnosis of infant HIV. PMID:26821087

  3. HIV-1 propagates in human neuroblastoma cells.

    PubMed

    Shapshak, P; Sun, N C; Resnick, L; Thornthwaite, J T; Schiller, P; Yoshioka, M; Svenningsson, A; Tourtellotte, W W; Imagawa, D T

    1991-01-01

    A major question in the pathogenesis of AIDS encephalopathy and dementia is whether HIV-1 directly infects cells of the central nervous system (CNS). The propagation of HIV was attempted in six cell lines: three related and three unrelated to the nervous system. HIV was able to propagate in two human neuroblastoma cell lines and a lymphocytic cell line control but did not result in infections of African green monkey kidney cells, human cervix carcinoma cells, and one human brain astrocytoma cell line. Neuroblastoma cell lines infected with HIV showed peaks of reverse transcriptase activity at 10-14 days postinfection. After prolonged growth in cell cultures, one of the neuroblastoma cell lines showed multiphasic virus production, additional high peaks of reverse transcriptase activity, 20-fold greater than the first, lasting from 36 to 74 days and 110 to 140 days postinfection. The presence of HIV was confirmed by p24 antigen capture. The neuroblastoma cell lines had weak but detectable levels of CD4 immunoreactivity by immunoperoxidase and flow immunocytometric analysis. Although no T4-specific RNA sequences were detected by hybridization of Northern blots of total and poly A-selected RNA extracted from the two neuroblastoma cell lines by using a T4 specific complimentary DNA probe, monoclonal antibodies to the CD4 receptor blocked HIV infection in both neuroblastoma cell lines. Thus, the infection of neuroblastoma cells by HIV occurs in part by a CD4-dependent mechanism. Passaging the neuroblastoma cell lines weekly and bimonthly resulted in similar cell cycle-DNA content patterns for the more permissive cell line and with significant numbers of cells in the S phase. HIV-infected neuroblastoma cell lines provide an in vitro model for the evaluation of virus-host cell interactions and may be useful in addressing the issue of the persistence of HIV in the human CNS. PMID:1704060

  4. 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

  5. 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

  6. HIV-1 Eradication: Early Trials (and Tribulations).

    PubMed

    Spivak, Adam M; Planelles, Vicente

    2016-01-01

    Antiretroviral therapy (ART) has rendered HIV-1 infection a manageable illness for those with access to treatment. However, ART does not lead to viral eradication owing to the persistence of replication-competent, unexpressed proviruses in long-lived cellular reservoirs. The potential for long-term drug toxicities and the lack of access to ART for most people living with HIV-1 infection have fueled scientific interest in understanding the nature of this latent reservoir. Exploration of HIV-1 persistence at the cellular and molecular level in resting memory CD4(+) T cells, the predominant viral reservoir in patients on ART, has uncovered potential strategies to reverse latency. We review recent advances in pharmacologically based 'shock and kill' HIV-1 eradication strategies, including comparative analysis of early clinical trials. PMID:26691297

  7. Development of prophylactic vaccines against HIV-1

    PubMed Central

    2013-01-01

    The focus of most current HIV-1 vaccine development is on antibody-based approaches. This is because certain antibody responses correlated with protection from HIV-1 acquisition in the RV144 phase III trial, and because a series of potent and broad spectrum neutralizing antibodies have been isolated from infected individuals. Taken together, these two findings suggest ways forward to develop a neutralizing antibody-based vaccine. However, understanding of the correlates of protection from disease in HIV-1 and other infections strongly suggests that we should not ignore CTL-based research. Here we review recent progress in the field and highlight the challenges implicit in HIV-1 vaccine design and some potential solutions. PMID:23866844

  8. Signaling through Toll-like receptors triggers HIV-1 replication in latently infected mast cells.

    PubMed

    Sundstrom, J Bruce; Little, Dawn M; Villinger, Francois; Ellis, Jane E; Ansari, Aftab A

    2004-04-01

    Evidence that human progenitor mast cells are susceptible to infection with CCR5-tropic strains of HIV-1 and that circulating HIV-1-infected FcepsilonRIalpha(+) cells with a similar progenitor phenotype have been isolated from AIDS patients has led to speculation that mast cells may serve as a potential reservoir for infectious HIV-1. In this study, progenitor mast cells, developed in vitro from CD34(+) cord blood stem cells, were experimentally infected with the CCR5-tropic strain HIV-1Bal after 28 days in culture as they reached their HIV-1-susceptible progenitor stage. HIV-1 p24 Ag levels were readily detectable by day 7 postinfection (PI), peaked at 2-3 wk PI as mature (tryptase/chymase-positive) HIV-1 infection-resistant mast cells emerged, and then steadily declined to below detectable limits by 10 wk PI, at which point integrated HIV-1 proviral DNA was confirmed by PCR quantitation in ( approximately 34% of) latently infected mast cells. Stimulation by ligands for Toll-like receptor (TLR) 2, TLR4, or TLR9 significantly enhanced viral replication in a dose- and time-dependent manner in both HIV-1-infected progenitor and latently infected mature mast cells, without promoting degranulation, apoptosis, cellular proliferation, or dysregulation of TLR agonist-induced cytokine production in infected mast cells. Limiting dilution analysis of TLR activated, latently infected mature mast cells indicated that one in four was capable of establishing productive infections in A301 sentinel cells. Taken together, these results indicate that mast cells may serve both as a viral reservoir and as a model for studying mechanisms of postintegration latency in HIV infection. PMID:15034054

  9. Phosphodiesterase 8a Supports HIV-1 Replication in Macrophages at the Level of Reverse Transcription

    PubMed Central

    Booiman, Thijs; Cobos Jiménez, Viviana; van Dort, Karel A.; van 't Wout, Angélique B.; Kootstra, Neeltje A.

    2014-01-01

    Background HIV-1 infected macrophages play a key role in HIV-1 infection. Even during anti-retroviral treatment, macrophages keep producing virus due to suboptimal tissue penetration and reduced efficacy of antiretrovirals. It is therefore of major importance to understand which host factors are involved in HIV-1 replication in macrophages. Previously, we have shown that genetic polymorphisms in phosphodiesterase 8a (PDE8A) are strongly associated with HIV-1 replication in these cells. Here we analyzed the mechanism and regulation of PDE8A in HIV-1 replication in macrophages. Results PDE8A mRNA expression strongly increases upon differentiation of monocytes into macrophages, which corresponds to the increased susceptibility of mature macrophages to HIV-1. In parallel, expression of microRNA miR-145-5p, predicted to target PDE8A mRNA, strongly decreased. The interaction of miR-145-5p with the 3′ UTR of PDE8A mRNA could be experimentally validated, suggesting that indeed miR-145-5p can regulate PDE8A expression levels. Knockdown of PDE8A in macrophages resulted in a decrease in total HIV-1 replication and proviral DNA levels. These observations confirm that PDE8A regulates HIV-1 replication in macrophages and that this effect is mediated through early steps in the viral replication cycle. Conclusions PDE8A is highly expressed in macrophages, and its expression is regulated by miR-145-5p. Our findings strongly suggest that PDE8A supports HIV-1 replication in macrophages and that this effect is mediated at the level of reverse transcription. PMID:25295610

  10. 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

  11. The use of HIV-1 integration site analysis information in clinical studies aiming at HIV cure.

    PubMed

    Kiselinova, Maja; De Spiegelaere, Ward; Vandekerckhove, Linos

    2016-01-01

    The mechanisms for the establishment and the persistence of the latent HIV-1 reservoir remain to be completely defined. HIV-1 infection is characterised by the integration of the reverse transcribed proviral DNA into the host's genome. This integrated proviral DNA can remain replication silent, but a small part of it is fully competent to restart viral replication when treatment is interrupted. Hence, this replication-competent provirus is the cause of viral rebound and is called the viral reservoir. The exact site of proviral integration within the host's cellular chromosome may affect the transcriptional activity of HIV. Thanks to recent technological advances, HIV-1 integration site analysis has been used to assess HIV-1 reservoirs in HIV-infected individuals. Analysis of HIV-1 integration sites in infected individuals undergoing suppressive ART led to identification of expanded clonal cell populations, indicating that clonal proliferation of the proviral reservoir may contribute to the long-term persistence of viral reservoirs. Here we describe the findings of several clinical studies, where a comprehensive HIV-1 integration site analysis was performed. PMID:27482458

  12. Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells

    NASA Technical Reports Server (NTRS)

    Plymale, D. R.; Tang, D. S.; Comardelle, A. M.; Fermin, C. D.; Lewis, D. E.; Garry, R. F.

    1999-01-01

    BACKGROUND: Data currently available on HIV-1-induced cytopathology is unclear regarding the mechanism of cell killing. OBJECTIVE: To clarify the extent to which apoptosis or necrosis is involved in HIV-1-induced cell death in view of conflicting existing data. METHODS: T lymphoblastoid cells or peripheral blood mononuclear cells were infected by various strains of HIV-1 and the numbers of apoptotic or necrotic cells were quantified at various times after infection using video-image analysis techniques; the results were compared with the amount of fragmented DNA using a quantitative method. Measurement of mitochondrial transmembrane potential (deltapsi(m)) and intracellular calcium concentrations [Ca2+]i was performed with fluorescent probes and fluorescence concentration analysis (FCA). RESULTS: Although lymphoblastoid and monocytoid cells acutely infected by HIV-1 had increased levels of fragmented DNA, a marker of apoptotic cell death, few (<12%) had condensed chromatin and fragmented nuclei, the morphological features of apoptosis. The predominant alterations in acutely infected cells were distended endoplasmic reticulum and abnormal mitochondria; these ultrastructural changes are consistent with necrosis, although some infected cells simultaneously displayed features of both necrosis and apoptosis. Viability of cells persistently infected by HIV-1 was only minimally reduced from that of uninfected cells. This reduction was accounted for by an increased propensity of the persistently infected cells to die by apoptosis. Alterations in [Ca2+]i and deltapsi(m) occurred in both acutely and persistently infected cells. CONCLUSION: Both necrosis and apoptosis contribute to HIV-1-induced killing of CD4 cells.

  13. HIV-1 transmission linkage in an HIV-1 prevention clinical trial

    SciTech Connect

    Leitner, Thomas; Campbell, Mary S; Mullins, James I; Hughes, James P; Wong, Kim G; Raugi, Dana N; Scrensen, Stefanie

    2009-01-01

    HIV-1 sequencing has been used extensively in epidemiologic and forensic studies to investigate patterns of HIV-1 transmission. However, the criteria for establishing genetic linkage between HIV-1 strains in HIV-1 prevention trials have not been formalized. The Partners in Prevention HSV/HIV Transmission Study (ClinicaITrials.gov NCT00194519) enrolled 3408 HIV-1 serodiscordant heterosexual African couples to determine the efficacy of genital herpes suppression with acyclovir in reducing HIV-1 transmission. The trial analysis required laboratory confirmation of HIV-1 linkage between enrolled partners in couples in which seroconversion occurred. Here we describe the process and results from HIV-1 sequencing studies used to perform transmission linkage determination in this clinical trial. Consensus Sanger sequencing of env (C2-V3-C3) and gag (p17-p24) genes was performed on plasma HIV-1 RNA from both partners within 3 months of seroconversion; env single molecule or pyrosequencing was also performed in some cases. For linkage, we required monophyletic clustering between HIV-1 sequences in the transmitting and seroconverting partners, and developed a Bayesian algorithm using genetic distances to evaluate the posterior probability of linkage of participants sequences. Adjudicators classified transmissions as linked, unlinked, or indeterminate. Among 151 seroconversion events, we found 108 (71.5%) linked, 40 (26.5%) unlinked, and 3 (2.0%) to have indeterminate transmissions. Nine (8.3%) were linked by consensus gag sequencing only and 8 (7.4%) required deep sequencing of env. In this first use of HIV-1 sequencing to establish endpoints in a large clinical trial, more than one-fourth of transmissions were unlinked to the enrolled partner, illustrating the relevance of these methods in the design of future HIV-1 prevention trials in serodiscordant couples. A hierarchy of sequencing techniques, analysis methods, and expert adjudication contributed to the linkage

  14. Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo.

    PubMed

    Simonetti, Francesco R; Sobolewski, Michele D; Fyne, Elizabeth; Shao, Wei; Spindler, Jonathan; Hattori, Junko; Anderson, Elizabeth M; Watters, Sarah A; Hill, Shawn; Wu, Xiaolin; Wells, David; Su, Li; Luke, Brian T; Halvas, Elias K; Besson, Guillaume; Penrose, Kerri J; Yang, Zhiming; Kwan, Richard W; Van Waes, Carter; Uldrick, Thomas; Citrin, Deborah E; Kovacs, Joseph; Polis, Michael A; Rehm, Catherine A; Gorelick, Robert; Piatak, Michael; Keele, Brandon F; Kearney, Mary F; Coffin, John M; Hughes, Stephen H; Mellors, John W; Maldarelli, Frank

    2016-02-16

    Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4(+)T cells. Some of these CD4(+)T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4(+) T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1-infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4(+)T cells can be a reservoir of infectious HIV-1. PMID:26858442

  15. Clonally expanded CD4+ T cells can produce infectious HIV-1 in vivo

    PubMed Central

    Simonetti, Francesco R.; Sobolewski, Michele D.; Fyne, Elizabeth; Shao, Wei; Spindler, Jonathan; Hattori, Junko; Anderson, Elizabeth M.; Watters, Sarah A.; Hill, Shawn; Wu, Xiaolin; Wells, David; Su, Li; Luke, Brian T.; Halvas, Elias K.; Besson, Guillaume; Penrose, Kerri J.; Yang, Zhiming; Kwan, Richard W.; Van Waes, Carter; Uldrick, Thomas; Citrin, Deborah E.; Kovacs, Joseph; Polis, Michael A.; Rehm, Catherine A.; Gorelick, Robert; Piatak, Michael; Keele, Brandon F.; Kearney, Mary F.; Coffin, John M.; Hughes, Stephen H.; Mellors, John W.; Maldarelli, Frank

    2016-01-01

    Reservoirs of infectious HIV-1 persist despite years of combination antiretroviral therapy and make curing HIV-1 infections a major challenge. Most of the proviral DNA resides in CD4+T cells. Some of these CD4+T cells are clonally expanded; most of the proviruses are defective. It is not known if any of the clonally expanded cells carry replication-competent proviruses. We report that a highly expanded CD4+ T-cell clone contains an intact provirus. The highly expanded clone produced infectious virus that was detected as persistent plasma viremia during cART in an HIV-1–infected patient who had squamous cell cancer. Cells containing the intact provirus were widely distributed and significantly enriched in cancer metastases. These results show that clonally expanded CD4+T cells can be a reservoir of infectious HIV-1. PMID:26858442

  16. Roles of HIV-1 capsid in viral replication and immune evasion.

    PubMed

    Le Sage, Valerie; Mouland, Andrew J; Valiente-Echeverría, Fernando

    2014-11-26

    The primary roles of the human immunodeficiency virus type 1 (HIV-1) capsid (CA) protein are to encapsidate and protect the viral RNA genome. It is becoming increasing apparent that HIV-1 CA is a multifunctional protein that acts early during infection to coordinate uncoating, reverse transcription, nuclear import of the pre-integration complex and integration of double stranded viral DNA into the host genome. Additionally, numerous recent studies indicate that CA is playing a crucial function in HIV-1 immune evasion. Here we summarize the current knowledge on HIV-1 CA and its interactions with the host cell to promote infection. The fact that CA engages in a number of different protein-protein interactions with the host makes it an interesting target for the development of new potent antiviral agents. PMID:25036886

  17. Exosomes: Implications in HIV-1 Pathogenesis

    PubMed Central

    Madison, Marisa N.; Okeoma, Chioma M.

    2015-01-01

    Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. The role of exosomes in viral pathogenesis, especially human immunodeficiency virus type 1 [HIV-1] is beginning to unravel. Recent research reports suggest that exosomes from various sources play important but different roles in the pathogenesis of HIV-1. From these reports, it appears that the source of exosomes is the defining factor for the exosomal effect on HIV-1. In this review, we will describe how HIV-1 infection is modulated by exosomes and in turn how exosomes are targeted by HIV-1 factors. Finally, we will discuss potentially emerging therapeutic options based on exosomal cargos that may have promise in preventing HIV-1 transmission. PMID:26205405

  18. The Host Proteins Transportin SR2/TNPO3 and Cyclophilin A Exert Opposing Effects on HIV-1 Uncoating

    PubMed Central

    Shah, Vaibhav B.; Shi, Jiong; Hout, David R.; Oztop, Ilker; Krishnan, Lavanya; Ahn, Jinwoo; Shotwell, Matthew S.; Engelman, Alan

    2013-01-01

    Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells. PMID:23097435

  19. The host proteins transportin SR2/TNPO3 and cyclophilin A exert opposing effects on HIV-1 uncoating.

    PubMed

    Shah, Vaibhav B; Shi, Jiong; Hout, David R; Oztop, Ilker; Krishnan, Lavanya; Ahn, Jinwoo; Shotwell, Matthew S; Engelman, Alan; Aiken, Christopher

    2013-01-01

    Following entry of the HIV-1 core into target cells, productive infection depends on the proper disassembly of the viral capsid (uncoating). Although much is known regarding HIV-1 entry, the actions of host cell proteins that HIV-1 utilizes during early postentry steps are poorly understood. One such factor, transportin SR2 (TRN-SR2)/transportin 3 (TNPO3), promotes infection by HIV-1 and some other lentiviruses, and recent studies have genetically linked TNPO3 dependence of infection to the viral capsid protein (CA). Here we report that purified recombinant TNPO3 stimulates the uncoating of HIV-1 cores in vitro. The stimulatory effect was reduced by RanGTP, a known ligand for transportin family members. Depletion of TNPO3 in target cells rendered HIV-1 less susceptible to inhibition by PF74, a small-molecule HIV-1 inhibitor that induces premature uncoating. In contrast to the case for TNPO3, addition of the CA-binding host protein cyclophilin A (CypA) inhibited HIV-1 uncoating and reduced the stimulatory effect of TNPO3 on uncoating in vitro. In cells in which TNPO3 was depleted, HIV-1 infection was enhanced 4-fold by addition of cyclosporine, indicating that the requirement for TNPO3 in HIV-1 infection is modulated by CypA-CA interactions. Although TNPO3 was localized primarily to the cytoplasm, depletion of TNPO3 from target cells inhibited HIV-1 infection without reducing the accumulation of nuclear proviral DNA, suggesting that TNPO3 facilitates a stage of the virus life cycle subsequent to nuclear entry. Our results suggest that TNPO3 and cyclophilin A facilitate HIV-1 infection by coordinating proper uncoating of the core in target cells. PMID:23097435

  20. Progress on the induction of neutralizing antibodies against HIV-1

    PubMed Central

    Vaine, Michael; Lu, Shan; Wang, Shixia

    2013-01-01

    The Human Immunodeficiency Virus Type -1 (HIV-1), the causative agent of AIDS in humans, is one of the most catastrophic pandemics to affect human health care in the latter 20th century. The best hope of controlling this pandemic is the development of a successful prophylactic vaccine. However, to date, this goal has proven to be exceptionally elusive. The recent failure of an experimental AIDS vaccine in a phase IIb study named the STEP trial, intended to solely elicit cell mediated immune responses against HIV-1, has highlighted the need for a balanced immune response consisting of not only cellular immunity but also a broad and potent antibody response which can prevent the infection of HIV-1. This article will review the efforts being made up to this point to elicit such antibody responses, especially with regards to the use of a DNA prime-protein boost regimen which has been proven to be a highly effective platform for the induction of neutralizing antibodies in both animal and early phase human studies. PMID:19627166

  1. Molecular and pathologic insights from latent HIV-1 infection in the human brain

    PubMed Central

    Desplats, Paula; Dumaop, Wilmar; Smith, David; Adame, Anthony; Everall, Ian; Letendre, Scott; Ellis, Ronald; Cherner, Mariana; Grant, Igor

    2013-01-01

    Objective: We aimed to investigate whether HIV latency in the CNS might have adverse molecular, pathologic, and clinical consequences. Methods: This was a case-control comparison of HIV-1 seropositive (HIV+) patients with clinical and neuropathologic examination. Based on the levels of HIV-1 DNA, RNA, and p24 in the brain, cases were classified as controls, latent HIV CNS infection, and HIV encephalitis (HIVE). Analysis of epigenetic markers including BCL11B, neurodegeneration, and neuroinflammation was performed utilizing immunoblot, confocal microscopy, immunochemistry/image analysis, and qPCR. Detailed antemortem neurocognitive data were available for 23 out of the 32 cases. Results: HIV+ controls (n = 12) had no detectable HIV-1 DNA, RNA, or p24 in the CNS; latent HIV+ cases (n = 10) showed high levels of HIV-1 DNA but no HIV RNA or p24; and HIVE cases (n = 10) had high levels of HIV-1 DNA, RNA, and p24. Compared to HIV+ controls, the HIV+ latent cases displayed moderate cognitive impairment with neurodegenerative and neuroinflammatory alterations, although to a lesser extent than HIVE cases. Remarkably, HIV+ latent cases showed higher levels of BCL11B and other chromatin modifiers involved in silencing. Increased BCL11B was associated with deregulation of proinflammatory genes like interleukin-6, tumor necrosis factor–α, and CD74. Conclusion: Persistence of latent HIV-1 infection in the CNS was associated with increased levels of chromatin modifiers, including BCL11B. Alteration of these epigenetic factors might result in abnormal transcriptomes, leading to inflammation, neurodegeneration, and neurocognitive impairment. BCL11B and other epigenetic factors involved in silencing might represent potential targets for HIV-1 involvement of the CNS. PMID:23486877

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

    PubMed Central

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

    2013-01-01

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

  3. Identification of cellular factors binding to acetylated HIV-1 integrase.

    PubMed

    Allouch, Awatef; Cereseto, Anna

    2011-11-01

    The viral protein integrase (IN) catalyzes the integration of the HIV-1 cDNA into the host cellular genome. We have recently demonstrated that IN is acetylated by a cellular histone acetyltransferase, p300, which modifies three lysines located in the C-terminus of the viral factor (Cereseto et al. in EMBO J 24:3070-3081, 2005). This modification enhances IN catalytic activity, as demonstrated by in vitro assays. Consistently, mutations introduced in the targeted lysines greatly decrease the efficiency of HIV-1 integration. Acetylation was proven to regulate protein functions by modulating protein-protein interactions. HIV-1 to efficiently complete its replication steps, including the integration reaction, requires interacting with numerous cellular factors. Therefore, we sought to investigate whether acetylation might modulate the interaction between IN and the cellular factors. To this aim we performed a yeast two-hybrid screening that differs from the screenings so far performed (Rain et al. in Methods 47:291-297, 2009; Studamire and Goff in Retrovirology 5:48, 2008) for using as bait IN constitutively acetylated. From this analysis we have identified thirteen cellular factors involved in transcription, chromatin remodeling, nuclear transport, RNA binding, protein synthesis regulation and microtubule organization. To validate these interactions, binding assays were performed showing that acetylation increases the affinity of IN with specific factors. Nevertheless, few two-hybrid hits bind with the same affinity the acetylated and the unmodified IN. These results further underlie the relevance of IN post-translational modification by acetylation in HIV-1 replication cycle. PMID:20016921

  4. Crystal Structure of NFAT Bound to the HIV-1 LTR Tandem κB Enhancer Element

    SciTech Connect

    Bates, Darren L.; Barthel, Kristen K.B.; Wu, Yongqing; Kalhor, Reza; Stroud, James C.; Giffin, Michael J.; Chen, Lin

    2008-05-27

    Here, we have determined the crystal structure of the DNA binding domain of NFAT bound to the HIV-1 long terminal repeat (LTR) tandem {kappa}B enhancer element of 3.05 {angstrom} resolution. NFAT binds as a dimer to the upstream {kappa}B site (Core II), but as a monomer to the 3' end of the downstream {kappa}B site (Core I). The DNA shows a significant bend near the 5' end of Core I, where a lysine residue from NFAT bound to the 3' end of Core II inserts into the minor groove and seems to cause DNA bases to flip out. Consistent with this structural feature, the 5' end of Core I become hypersensitive to dimethylsulfate in the in vivo footprinting upon transcriptional activation of the HIV-1 LTR. Our studies provide a basis for futher investigating the functional mechanism of NFAT in HIV-1 transcription and replication.

  5. Frequent Incorporation of Ribonucleotides during HIV-1 Reverse Transcription and Their Attenuated Repair in Macrophages*

    PubMed Central

    Kennedy, Edward M.; Amie, Sarah M.; Bambara, Robert A.; Kim, Baek

    2012-01-01

    Macrophages are well known long-lived reservoirs of HIV-1. Unlike activated CD4+ T cells, this nondividing HIV-1 target cell type contains a very low level of the deoxynucleoside triphosphates (dNTPs) required for proviral DNA synthesis whereas the ribonucleoside triphosphate (rNTP) levels remain in the millimolar range, resulting in an extremely low dNTP/rNTP ratio. Biochemical simulations demonstrate that HIV-1 reverse transcriptase (RT) efficiently incorporates ribonucleoside monophosphates (rNMPs) during DNA synthesis at this ratio, predicting frequent rNMP incorporation by the virus specifically in macrophages. Indeed, HIV-1 RT incorporates rNMPs at a remarkable rate of 1/146 nucleotides during macrophage infection. This greatly exceeds known rates for cellular replicative polymerases. In contrast, little or no rNMP incorporation is detected in CD4+ T cells. Repair of these rNMP lesions is also substantially delayed in macrophages compared with CD4+ T cells. Single rNMPs embedded in a DNA template are known to induce cellular DNA polymerase pausing, which mechanistically contributes to mutation synthesis. Indeed, we also observed that embedded rNMPs in a dsDNA template also induce HIV-1 RT DNA synthesis pausing. Moreover, unrepaired rNMPs incorporated into the provirus during HIV-1 reverse transcription would be generally mutagenic as was shown in Saccharomyces cerevisiae. Most importantly, the frequent incorporation of rNMPs makes them an ideal candidate for development of a new class of HIV RT inhibitors. PMID:22383524

  6. Envelope gene evolution and HIV-1 neuropathogenesis

    PubMed Central

    Vázquez-Santiago, Fabián J.; Rivera-Amill, Vanessa

    2016-01-01

    In the era of combined antiretroviral therapy (cART), HIV-associated neurocognitive disorders (HAND) account for 40 to 56% of all HIV+ cases. During the acute stage of HIV-1 infection (<6 months), the virus invades and replicates within the central nervous system (CNS). Compared to peripheral tissues, the local CNS cell population expresses distinct levels of chemokine receptors, which levels exert selective pressure on the invading virus. HIV-1 envelope (env) sequences recovered from the brains and cerebrospinal fluid (CSF) of neurocognitively impaired HIV+ subjects often display higher nucleotide variability as compared to non-impaired HIV+ subjects. Specifically, env evolution provides HIV-1 with the strategies to evade host immune response, to reduce chemokine receptor dependence, to increase co-receptor binding efficiency, and to potentiate neurotoxicity. The evolution of env within the CNS leads to changes that may result in the emergence of novel isolates with neurotoxic and neurovirulent features. However, whether specific factors of HIV-1 evolution lead to the emergence of neurovirulent and neurotropic isolates remains ill-defined. HIV-1 env evolution is an ongoing phenomenon that occurs independently of neurological and neurocognitive disease severity; thus HIV env evolution may play a pivotal and reciprocal role in the etiology of HAND. Despite the use of cART, the reactivation of latent viral reservoirs represents a clinical challenge because of the replenishment of the viral pool that may subsequently lead to persistent infection. Therefore, gaining a more complete understanding of how HIV-1 env evolves over the course of the disease should be considered for the development of future therapies aimed at controlling CNS burden, diminishing persistent viremia, and eradicating viral reservoirs. Here we review the current literature on the role of HIV-1 env evolution in the setting of HAND disease progression and on the impact of cART on the dynamics of

  7. Persistent HIV-1 replication during antiretroviral therapy

    PubMed Central

    Martinez-Picado, Javier; Deeks, Steven G.

    2016-01-01

    Purpose of review The present review will highlight some of the recent findings regarding the capacity of HIV-1 to replicate during antiretroviral therapy (ART). Recent findings Although ART is highly effective at inhibiting HIV replication, it is not curative. Several mechanisms contribute to HIV persistence during ART, including HIV latency, immune dysfunction, and perhaps persistent low-level spread of the virus to uninfected cells (replication). The success in curing HIV will depend on efficiently targeting these three aspects. The degree to which HIV replicates during ART remains controversial. Most studies have failed to find any evidence of HIV evolution in blood, even with samples collected over many years, although a recent very intensive study of three individuals suggested that the virus population does shift, at least during the first few months of therapy. Stronger but still not definitive evidence for replication comes from a series of studies in which standard regimens were intensified with an integration inhibitor, resulting in changes in episomal DNA (blood) and cell-associated RNA (tissue). Limited drug penetration within tissues and the presence of immune sanctuaries have been argued as potential mechanisms allowing HIV to spread during ART. Mathematical models suggest that HIV replication and evolution is possible even without the selection of fully drug-resistant variants. As persistent HIV replication could have clinical consequences and might limit the efficacy of curative interventions, determining if HIV replicates during ART and why, should remain a key focus of the HIV research community. Summary Residual viral replication likely persists in lymphoid tissues, at least in a subset of individuals. Abnormal levels of immune activation might contribute to sustain virus replication. PMID:27078619

  8. Th1/17 Polarization of CD4 T Cells Supports HIV-1 Persistence during Antiretroviral Therapy

    PubMed Central

    Sun, Hong; Kim, Dhohyung; Li, Xiaodong; Kiselinova, Maja; Ouyang, Zhengyu; Vandekerckhove, Linos; Shang, Hong; Rosenberg, Eric S.; Yu, Xu G.

    2015-01-01

    ABSTRACT The ability to persist long term in latently infected CD4 T cells represents a characteristic feature of HIV-1 infection and the predominant barrier to efforts aiming at viral eradication and cure. Yet, increasing evidence suggests that only small subsets of CD4 T cells with specific developmental and maturational profiles are able to effectively support HIV-1 long-term persistence. Here, we analyzed how the functional polarization of CD4 T cells shapes and structures the reservoirs of HIV-1-infected cells. We found that CD4 T cells enriched for a Th1/17 polarization had elevated susceptibilities to HIV-1 infection in ex vivo assays, harbored high levels of HIV-1 DNA in persons treated with antiretroviral therapy, and made a disproportionately increased contribution to the viral reservoir relative to their contribution to the CD4 T memory cell pool. Moreover, HIV-1 DNA levels in Th1/17 cells remained stable over many years of antiretroviral therapy, resulting in a progressively increasing contribution of these cells to the viral reservoir, and phylogenetic studies suggested preferential long-term persistence of identical viral sequences during prolonged antiretroviral treatment in this cell compartment. Together, these data suggest that Th1/17 CD4 T cells represent a preferred site for HIV-1 DNA long-term persistence in patients receiving antiretroviral therapy. IMPORTANCE Current antiretroviral therapy is very effective in suppressing active HIV-1 replication but does not fully eliminate virally infected cells. The ability of HIV-1 to persist long term despite suppressive antiretroviral combination therapy represents a perplexing aspect of HIV-1 disease pathogenesis, since most HIV-1 target cells are activated, short-lived CD4 T cells. This study suggests that CD4 T helper cells with Th1/17 polarization have a preferential role as a long-term reservoir for HIV-1 infection during antiretroviral therapy, possibly because these cells may imitate some of the

  9. 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

  10. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules

    PubMed Central

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S. Y.

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This “shock” approach is then followed by “kill” of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  11. Specific Elimination of Latently HIV-1 Infected Cells Using HIV-1 Protease-Sensitive Toxin Nanocapsules.

    PubMed

    Wen, Jing; Yan, Ming; Liu, Yang; Li, Jie; Xie, Yiming; Lu, Yunfeng; Kamata, Masakazu; Chen, Irvin S Y

    2016-01-01

    Anti-retroviral drugs suppress HIV-1 plasma viremia to undetectable levels; however, latent HIV-1 persists in reservoirs within HIV-1-infected patients. The silent provirus can be activated through the use of drugs, including protein kinase C activators and histone deacetylase inhibitors. This "shock" approach is then followed by "kill" of the producing cells either through direct HIV-1-induced cell death or natural immune mechanisms. However, these mechanisms are relatively slow and effectiveness is unclear. Here, we develop an approach to specifically target and kill cells that are activated early in the process of virus production. We utilize a novel nanocapsule technology whereby the ricin A chain is encapsulated in an inactive form within a polymer shell. Specificity for release of the ricin A toxin is conferred by peptide crosslinkers that are sensitive to cleavage by HIV-1 protease. By using well-established latent infection models, J-Lat and U1 cells, we demonstrate that only within an HIV-1-producing cell expressing functional HIV-1 protease will the nanocapsule release its ricin A cargo, shutting down viral and cellular protein synthesis, and ultimately leading to rapid death of the producer cell. Thus, we provide proof of principle for a novel technology to kill HIV-1-producing cells without effects on non-target cells. PMID:27049645

  12. 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

  13. TopoisomeraseIIβ in HIV-1 transactivation.

    PubMed

    Chekuri, Anil; Bhaskar, C; Bollimpelli, V Satish; Kondapi, Anand K

    2016-03-01

    TopoisomeraseIIβ, an isoform of type II topoisomerase, was found to be functional in various viral infections. Its plausible role in HIV life cycle was also suggested earlier, but not clearly established. In the present study, we have investigated the role of TopoIIβ in HIV-1 infection by its gain and loss of function. Overexpression of TopoIIβ lead to an increase in viral replication, resulting in enhanced virion production. HIV-1 replication was impaired when TopoIIβ was down regulated by siRNA and inhibited by ICRF-193 and merbarone. The role of TopoIIβ in HIV-1 transcription was shown through its interaction with Tat and recruitement to long terminal repeat (LTR) region by co-immunoprecipitation and ChIP assays. Involvement of TopoIIβ in transactivation of HIV-1 LTR was confirmed by luciferase assay in reporter cell line, TZM bl and also by transfection of reporter exogenously. It was also observed that LTR transactivation commensurated with the expression of TopoIIβ in the presence of Tat. In addition, a decreased viral gene expression on treatment with merbarone exemplifies the importance of catalytic activity of TopoIIβ in viral replication. These observations indicate that TopoIIβ is involved in the cascade of coactivator complexes that are recruited to LTR for regulation of HIV-1 transcription. PMID:26876283

  14. HIV-1 Genetic Variability and Clinical Implications

    PubMed Central

    Santoro, Maria Mercedes; Perno, Carlo Federico

    2013-01-01

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

  15. Multiple proviral integration events after virological synapse-mediated HIV-1 spread

    SciTech Connect

    Russell, Rebecca A.; Martin, Nicola; Mitar, Ivonne; Jones, Emma; Sattentau, Quentin J.

    2013-08-15

    HIV-1 can move directly between T cells via virological synapses (VS). Although aspects of the molecular and cellular mechanisms underlying this mode of spread have been elucidated, the outcomes for infection of the target cell remain incompletely understood. We set out to determine whether HIV-1 transfer via VS results in productive, high-multiplicity HIV-1 infection. We found that HIV-1 cell-to-cell spread resulted in nuclear import of multiple proviruses into target cells as seen by fluorescence in-situ hybridization. Proviral integration into the target cell genome was significantly higher than that seen in a cell-free infection system, and consequent de novo viral DNA and RNA production in the target cell detected by quantitative PCR increased over time. Our data show efficient proviral integration across VS, implying the probability of multiple integration events in target cells that drive productive T cell infection. - Highlights: • Cell-to-cell HIV-1 infection delivers multiple vRNA copies to the target cell. • Cell-to-cell infection results in productive infection of the target cell. • Cell-to-cell transmission is more efficient than cell-free HIV-1 infection. • Suggests a mechanism for recombination in cells infected with multiple viral genomes.

  16. 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

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

    PubMed Central

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

    2008-01-01

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

  18. Blocking CXCL9 Decreases HIV-1 Replication and Enhances the Activity of Prophylactic Antiretrovirals in Human Cervical Tissues

    PubMed Central

    Macura, Sherrill L.; Lathrop, Melissa J.; Gui, Jiang; Doncel, Gustavo F.; Rollenhagen, Christiane

    2016-01-01

    Objectives: The interferon-gamma–induced chemokine CXCL9 is expressed in a wide range of inflammatory conditions including those affecting the female genital tract. CXCL9 promotes immune cell recruitment, activation, and proliferation. The role of CXCL9 in modulating HIV-1 infection of cervicovaginal tissues, a main portal of viral entry, however, has not been established. We report a link between CXCL9 and HIV-1 replication in human cervical tissues and propose CXCL9 as a potential target to enhance the anti–HIV-1 activity of prophylactic antiretrovirals. Design: Using ex vivo infection of human cervical tissues as a model of mucosal HIV-1 acquisition, we described the effect of CXCL9 neutralization on HIV-1 gene expression and mucosal CD4+ T-cell activation. The anti-HIV-1 activity of tenofovir, the leading mucosal pre-exposure prophylactic microbicide, alone or in combination with CXCL9 neutralization was also studied. Methods: HIV-1 replication was evaluated by p24 ELISA. HIV-1 DNA and RNA, and CD4, CCR5, and CD38 transcription were evaluated by quantitative real-time polymerase chain reaction. Frequency of activated cervical CD4+ T cells was quantified using fluorescence-activated cell sorting. Results: Antibody blocking of CXCL9 reduced HIV-1 replication by decreasing mucosal CD4+ T-cell activation. CXCL9 neutralization in combination with suboptimal concentrations of tenofovir, possibly present in the cervicovaginal tissues of women using the drug inconsistently, demonstrated an earlier and greater decrease in HIV-1 replication compared with tissues treated with tenofovir alone. Conclusions: CXCL9 neutralization reduces HIV-1 replication and may be an effective target to enhance the efficacy of prophylactic antiretrovirals. PMID:26545124

  19. HIV-1 Reservoirs During Suppressive Therapy.

    PubMed

    Barton, Kirston; Winckelmann, Anni; Palmer, Sarah

    2016-05-01

    The introduction of antiretroviral therapy (ART) 20 years ago has dramatically reduced morbidity and mortality associated with HIV-1. Initially there was hope that ART would be curative, but it quickly became clear that even though ART was able to restore CD4(+) T cell counts and suppress viral loads below levels of detection, discontinuation of treatment resulted in a rapid rebound of infection. This is due to persistence of a small reservoir of latently infected cells with a long half-life, which necessitates life-long ART. Over the past few years, significant progress has been made in defining and characterizing the latent reservoir of HIV-1, and here we review how understanding the latent reservoir during suppressive therapy will lead to significant advances in curative approaches for HIV-1. PMID:26875617

  20. HIV-1 immunopathogenesis in humanized mouse models

    PubMed Central

    Zhang, Liguo; Su, Lishan

    2012-01-01

    In recent years, the technology of constructing chimeric mice with humanized immune systems has markedly improved. Multiple lineages of human immune cells develop in immunodeficient mice that have been transplanted with human hematopoietic stem cells. More importantly, these mice mount functional humoral and cellular immune responses upon immunization or microbial infection. Human immunodeficiency virus type I (HIV-1) can establish an infection in humanized mice, resulting in CD4+ T-cell depletion and an accompanying nonspecific immune activation, which mimics the immunopathology in HIV-1-infected human patients. This makes humanized mice an optimal model for studying the mechanisms of HIV-1 immunopathogenesis and for developing novel immune-based therapies. PMID:22504952

  1. Population genomics of intrapatient HIV-1 evolution.

    PubMed

    Zanini, Fabio; Brodin, Johanna; Thebo, Lina; Lanz, Christa; Bratt, Göran; Albert, Jan; Neher, Richard A

    2015-01-01

    Many microbial populations rapidly adapt to changing environments with multiple variants competing for survival. To quantify such complex evolutionary dynamics in vivo, time resolved and genome wide data including rare variants are essential. We performed whole-genome deep sequencing of HIV-1 populations in 9 untreated patients, with 6-12 longitudinal samples per patient spanning 5-8 years of infection. The data can be accessed and explored via an interactive web application. We show that patterns of minor diversity are reproducible between patients and mirror global HIV-1 diversity, suggesting a universal landscape of fitness costs that control diversity. Reversions towards the ancestral HIV-1 sequence are observed throughout infection and account for almost one third of all sequence changes. Reversion rates depend strongly on conservation. Frequent recombination limits linkage disequilibrium to about 100 bp in most of the genome, but strong hitch-hiking due to short range linkage limits diversity. PMID:26652000

  2. MAS NMR of HIV-1 protein assemblies

    NASA Astrophysics Data System (ADS)

    Suiter, Christopher L.; Quinn, Caitlin M.; Lu, Manman; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

    2015-04-01

    The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates.

  3. MAS NMR of HIV-1 protein assemblies.

    PubMed

    Suiter, Christopher L; Quinn, Caitlin M; Lu, Manman; Hou, Guangjin; Zhang, Huilan; Polenova, Tatyana

    2015-04-01

    The negative global impact of the AIDS pandemic is well known. In this perspective article, the utility of magic angle spinning (MAS) NMR spectroscopy to answer pressing questions related to the structure and dynamics of HIV-1 protein assemblies is examined. In recent years, MAS NMR has undergone major technological developments enabling studies of large viral assemblies. We discuss some of these evolving methods and technologies and provide a perspective on the current state of MAS NMR as applied to the investigations into structure and dynamics of HIV-1 assemblies of CA capsid protein and of Gag maturation intermediates. PMID:25797001

  4. Novel vaccine vectors for HIV-1

    PubMed Central

    Picker, Louis J.

    2014-01-01

    The ultimate solution to the global HIV-1 epidemic will probably require the development of a safe and effective vaccine. Multiple vaccine platforms have been evaluated in both preclinical and clinical trials, but, given the disappointing results of the clinical efficacy studies so far, novel vaccine approaches are needed. In this Opinion article, we discuss the scientific basis and clinical potential of novel adenovirus and cytomegalovirus vaccine vectors for HIV-1 as two contrasting, but potentially complementary, vector approaches. Both of these vector platforms have demonstrated partial protection against stringent simian immunodeficiency virus challenges in rhesus monkeys using different immunological mechanisms. PMID:25296195

  5. Laparoscopic sterilization in HIV-1-positive women.

    PubMed

    Intaraprasert, S; Taneepanichskul, S; Chaturachinda, K

    1996-11-01

    Laparoscopic sterilizations in HIV-1-positive women were performed. Patients, who were HIV-1-positive, underwent voluntary laparoscopic sterilization. The mean age of patients was 27.5 +/- 3.8 years. Most were of low socioeconomic status. The mean duration of the operation was 14.4 +/- 5.4 min. No accidental injury to the surgical team was recorded, and no complications occurred among the patients. It was concluded that laparoscopic sterilization in HIV-positive patients was safe with low risk of HIV transmission to the surgical team. PMID:8934065

  6. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  7. Inhibition of HIV-1 reactivation by a telomerase-derived peptide in a HSP90-dependent manner

    PubMed Central

    Kim, Hong; Choi, Myung-Soo; Inn, Kyung-Soo; Kim, Bum-Joon

    2016-01-01

    A peptide vaccine designed to induce T-cell immunity to telomerase, GV1001, has been shown to modulate cellular signaling pathways and confer a direct anti-cancer effect through the interaction with heat shock protein (HSP) 90 and 70. Here, we have found that GV1001 can modulate transactivation protein-mediated human immunodeficiency virus (HIV)-1 transactivation in an HSP90-dependent manner. GV1001 treatment resulted in significant suppression of HIV-1 replication and rescue of infected cells from death by HIV-1. Transactivation of HIV-long terminal repeat (LTR) was inhibited by GV1001, indicating that GV1001 suppressed the transcription from proviral HIV DNA. The anti-HIV-1 activity of GV1001 was completely abrogated by an HSP90-neutralizing antibody, indicating that the antiviral activity depends on HSP90. Further mechanistic studies revealed that GV1001 suppresses basal NF-κB activation, which is required for HIV-1 LTR transactivation in an HSP90-dependent manner. Inhibition of LTR transactivation by GV1001 suggests its potential to suppress HIV-1 reactivation from latency. Indeed, PMA-mediated reactivation of HIV-1 from latent infected cells was suppressed by GV1001. The results suggest the potential therapeutic use of GV1001, a peptide proven to be safe for human use, as an anti-HIV-1 agent to suppress the reactivation from latently infected cells. PMID:27363520

  8. An altered intestinal mucosal microbiome in HIV-1 infection is associated with mucosal and systemic immune activation and endotoxemia.

    PubMed

    Dillon, S M; Lee, E J; Kotter, C V; Austin, G L; Dong, Z; Hecht, D K; Gianella, S; Siewe, B; Smith, D M; Landay, A L; Robertson, C E; Frank, D N; Wilson, C C

    2014-07-01

    Human immunodeficiency virus-1 (HIV-1) infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation. We investigated the impact of HIV-1 infection on the intestinal microbiome and its association with mucosal T-cell and dendritic cell (DC) frequency and activation, as well as with levels of systemic T-cell activation, inflammation, and microbial translocation. Bacterial 16S ribosomal DNA sequencing was performed on colon biopsies and fecal samples from subjects with chronic, untreated HIV-1 infection and uninfected control subjects. Colon biopsies of HIV-1-infected subjects had increased abundances of Proteobacteria and decreased abundances of Firmicutes compared with uninfected donors. Furthermore at the genus level, a significant increase in Prevotella and decrease in Bacteroides was observed in HIV-1-infected subjects, indicating a disruption in the Bacteroidetes bacterial community structure. This HIV-1-associated increase in Prevotella abundance was associated with increased numbers of activated colonic T cells and myeloid DCs. Principal coordinates analysis demonstrated an HIV-1-related change in the microbiome that was associated with increased mucosal cellular immune activation, microbial translocation, and blood T-cell activation. These observations suggest that an important relationship exists between altered mucosal bacterial communities and intestinal inflammation during chronic HIV-1 infection. PMID:24399150

  9. An altered intestinal mucosal microbiome in HIV-1 infection is associated with mucosal and systemic immune activation and endotoxemia

    PubMed Central

    Dillon, SM; Lee, EJ; Kotter, CV; Austin, GL; Dong, Z; Hecht, DK; Gianella, S; Siewe, B; Smith, DM; Landay, AL; Robertson, CE; Frank, DN; Wilson, CC

    2014-01-01

    HIV-1 infection disrupts the intestinal immune system, leading to microbial translocation and systemic immune activation. We investigated the impact of HIV-1 infection on the intestinal microbiome and its association with mucosal T cell and dendritic cell (DC) frequency and activation, as well as with levels of systemic T cell activation, inflammation and microbial translocation. Bacterial 16S ribosomal DNA sequencing was performed on colon biopsies and fecal samples from subjects with chronic, untreated HIV-1 infection and uninfected control subjects. Colon biopsies of HIV-1 infected subjects had increased abundances of Proteobacteria and decreased abundances of Firmicutes compared to uninfected donors. Furthermore at the genus level, a significant increase in Prevotella and decrease in Bacteroides was observed in HIV-1 infected subjects, indicating a disruption in the Bacteroidetes bacterial community structure. This HIV-1-associated increase in Prevotella abundance was associated with increased numbers of activated colonic T cells and myeloid DCs. Principal coordinates analysis demonstrated an HIV-1-related change in the microbiome that was associated with increased mucosal cellular immune activation, microbial translocation and blood T cell activation. These observations suggest that an important relationship exists between altered mucosal bacterial communities and intestinal inflammation during chronic HIV-1 infection. PMID:24399150

  10. Inhibition of HIV-1 reactivation by a telomerase-derived peptide in a HSP90-dependent manner.

    PubMed

    Kim, Hong; Choi, Myung-Soo; Inn, Kyung-Soo; Kim, Bum-Joon

    2016-01-01

    A peptide vaccine designed to induce T-cell immunity to telomerase, GV1001, has been shown to modulate cellular signaling pathways and confer a direct anti-cancer effect through the interaction with heat shock protein (HSP) 90 and 70. Here, we have found that GV1001 can modulate transactivation protein-mediated human immunodeficiency virus (HIV)-1 transactivation in an HSP90-dependent manner. GV1001 treatment resulted in significant suppression of HIV-1 replication and rescue of infected cells from death by HIV-1. Transactivation of HIV-long terminal repeat (LTR) was inhibited by GV1001, indicating that GV1001 suppressed the transcription from proviral HIV DNA. The anti-HIV-1 activity of GV1001 was completely abrogated by an HSP90-neutralizing antibody, indicating that the antiviral activity depends on HSP90. Further mechanistic studies revealed that GV1001 suppresses basal NF-κB activation, which is required for HIV-1 LTR transactivation in an HSP90-dependent manner. Inhibition of LTR transactivation by GV1001 suggests its potential to suppress HIV-1 reactivation from latency. Indeed, PMA-mediated reactivation of HIV-1 from latent infected cells was suppressed by GV1001. The results suggest the potential therapeutic use of GV1001, a peptide proven to be safe for human use, as an anti-HIV-1 agent to suppress the reactivation from latently infected cells. PMID:27363520

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

    PubMed

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

    2014-08-01

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

  12. Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening.

    PubMed

    La, Jennifer; Latham, Catherine F; Tinetti, Ricky N; Johnson, Adam; Tyssen, David; Huber, Kelly D; Sluis-Cremer, Nicolas; Simpson, Jamie S; Headey, Stephen J; Chalmers, David K; Tachedjian, Gilda

    2015-06-01

    Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment. PMID:26038551

  13. Identification of mechanistically distinct inhibitors of HIV-1 reverse transcriptase through fragment screening

    PubMed Central

    La, Jennifer; Latham, Catherine F.; Tinetti, Ricky N.; Johnson, Adam; Tyssen, David; Huber, Kelly D.; Sluis-Cremer, Nicolas; Simpson, Jamie S.; Headey, Stephen J.; Chalmers, David K.; Tachedjian, Gilda

    2015-01-01

    Fragment-based screening methods can be used to discover novel active site or allosteric inhibitors for therapeutic intervention. Using saturation transfer difference (STD) NMR and in vitro activity assays, we have identified fragment-sized inhibitors of HIV-1 reverse transcriptase (RT) with distinct chemical scaffolds and mechanisms compared to nonnucleoside RT inhibitors (NNRTIs) and nucleoside/nucleotide RT inhibitors (NRTIs). Three compounds were found to inhibit RNA- and DNA-dependent DNA polymerase activity of HIV-1 RT in the micromolar range while retaining potency against RT variants carrying one of three major NNRTI resistance mutations: K103N, Y181C, or G190A. These compounds also inhibit Moloney murine leukemia virus RT but not the Klenow fragment of Escherichia coli DNA polymerase I. Steady-state kinetic analyses demonstrate that one of these fragments is a competitive inhibitor of HIV-1 RT with respect to deoxyribonucleoside triphosphate (dNTP) substrate, whereas a second compound is a competitive inhibitor of RT polymerase activity with respect to the DNA template/primer (T/P), and consequently also inhibits RNase H activity. The dNTP competing RT inhibitor retains activity against the NRTI-resistant mutants K65R and M184V, demonstrating a drug resistance profile distinct from the nucleotide competing RT inhibitors indolopyridone-1 (INDOPY-1) and 4-dimethylamino-6-vinylpyrimidine-1 (DAVP-1). In antiviral assays, the T/P competing compound inhibits HIV-1 replication at a step consistent with an RT inhibitor. Screening of additional structurally related compounds to the three fragments led to the discovery of molecules with improved potency against HIV-1 RT. These fragment inhibitors represent previously unidentified scaffolds for development of novel drugs for HIV-1 prevention or treatment. PMID:26038551

  14. SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4+ T-cells

    PubMed Central

    2012-01-01

    Background Quiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription. Results Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4+ T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment. Conclusion Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4+ T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4+ T-cells and could impact our understanding of HIV-1 mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks. PMID:23092122

  15. High throughput functional analysis of HIV-1 env genes without cloning

    PubMed Central

    Kirchherr, Jennifer L; Lu, Xiaozhi; Kasongo, Webster; Chalwe, Victor; Mwananyanda, Lawrence; Musonda, Rosemary M; Xia, Shi-Mao; Scearce, Richard M; Liao, Hua-Xin; Montefiori, David C; Haynes, Barton F; Gao, Feng

    2007-01-01

    Functional human immunodeficiency virus type -1 env clones have been widely used for vaccine design, neutralization assays, and pathogenesis studies. However, obtaining bona fide functional env clones is a time consuming and labor intensive process. A new high throughput method has been developed to characterize HIV-1 env genes. Multiple rev/env gene cassettes were obtained from each of seven HIV-1 strains using single genome amplification (SGA) PCR. The CMV promoter was amplified separately by PCR. A promoter PCR (pPCR) method was developed to link both PCR products using an overlapping PCR method. Pseudovirions were generated by cotransfection of pPCR products and pSG3Δenv backbone into 293T cells. After infecting TZM-bl cells, 75 out of 87 (86%) of the rev/env gene cassettes were functional. Pseudoviruses generated with pPCR products or corresponding plasmid DNA showed similar sensitivity to six HIV-1 positive sera and three monoclonal antibodies, suggesting neutralization properties are not altered in pPCR pseudovirions. Furthermore, sufficient amounts of pseudovirions can be obtained for a large number of neutralization assays. The new pPCR method eliminates cloning, transformation, and plasmid DNA preparation steps in the generation of HIV-1 pseudovirions, this allows for quick analysis of multiple env genes from HIV-1 infected individuals. PMID:17416428

  16. A point-of-care PCR test for HIV-1 detection in resource-limited settings.

    PubMed

    Jangam, Sujit R; Agarwal, Abhishek K; Sur, Kunal; Kelso, David M

    2013-04-15

    A low-cost, fully integrated sample-to-answer, quantitative PCR (qPCR) system that can be used for detection of HIV-1 proviral DNA in infants at the point-of-care in resource-limited settings has been developed and tested. The system is based on a novel DNA extraction method, which uses a glass fiber membrane, a disposable assay card that includes on-board reagent storage, provisions for thermal cycling and fluorescence detection, and a battery-operated portable analyzer. The system is capable of automated PCR mix assembly using a novel reagent delivery system and performing qPCR. HIV-1 and internal control targets are detected using two spectrally separated fluorophores, FAM and Quasar 670. In this report, a proof-of-concept of the platform is demonstrated. Initial results with whole blood demonstrate that the test is capable of detecting HIV-1 in blood samples containing greater than 5000 copies of HIV-1. In resource-limited settings, a point-of-care HIV-1 qPCR test would greatly increase the number of test results that reach the infants caregivers, allowing them to pursue anti-retroviral therapy. PMID:23202333

  17. Multifaceted counter-APOBEC3G mechanisms employed by HIV-1 Vif

    PubMed Central

    Britan-Rosich, Elena; Nowarski, Roni; Kotler, Moshe

    2011-01-01

    In the absence of HIV-1 Vif protein, the host antiviral deaminase APOBEC3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative ssDNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity, and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here we show that: (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δvif virus associated A3G; (ii) Encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) Purified HIV-1 Vif protein and the Vif-derived peptide Vif25-39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes (PICs) following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G anti-viral activity. PMID:21763507

  18. The Nucleoside Analog D-carba T Blocks HIV-1 Reverse Transcription

    PubMed Central

    Boyer, Paul L.; Vu, B. Christie; Ambrose, Zandrea; Julias, John G.; Warnecke, Svenja; Liao, Chenzhong; Meier, Chris; Marquez, Victor E.; Hughes, Stephen H.

    2009-01-01

    A major pathway for HIV-1 resistance to nucleoside reverse transcriptase inhibitors (NRTIs) involves reverse transcriptase (RT) mutations that enhance ATP-dependent pyrophosphorolysis, which excises NRTIs from the end of viral DNA. We analyzed novel NRTIs for their ability to inhibit DNA synthesis of excision-proficient HIV-1 RT mutants. D-carba T is a carbocyclic nucleoside that has a 3′ hydroxyl on the pseudosugar. The 3′ hydroxyl group allows RT to incorporate additional dNTPs, which should protect D-carba TMP from excision. D-carba T can be converted to the triphosphate form by host cell kinases with moderate efficiency. D-carba T-TP is efficiently incorporated by HIV-1 RT; however, the next dNTP is added slowly to a D-carba TMP at the primer terminus. D-carba T effectively inhibits viral vectors that replicate using NRTI-resistant HIV-1 RTs, and there is no obvious toxicity in cultured cells. NRTIs based on the carbocyclic pseudosugar may offer an effective approach for the treatment of HIV-1 infections. PMID:19678643

  19. 8-Modified-2′-Deoxyadenosine Analogues Induce Delayed Polymerization Arrest during HIV-1 Reverse Transcription

    PubMed Central

    Sleiman, Marwan; Smyth, Redmond; Ben Gaied, Nouha; Barhoum, Patrick; Laumond, Géraldine; Bec, Guillaume; Götte, Matthias; Mak, Johnson; Aubertin, Anne-Marie; Burger, Alain; Marquet, Roland

    2011-01-01

    The occurrence of resistant viruses to any of the anti-HIV-1 compounds used in the current therapies against AIDS underlies the urge for the development of new drug targets and/or new drugs acting through novel mechanisms. While all anti-HIV-1 nucleoside analogues in clinical use and in clinical trials rely on ribose modifications for activity, we designed nucleosides with a natural deoxyribose moiety and modifications of position 8 of the adenine base. Such modifications might induce a steric clash with helix αH in the thumb domain of the p66 subunit of HIV-1 RT at a distance from the catalytic site, causing delayed chain termination. Eleven new 2′-deoxyadenosine analogues modified on position 8 of the purine base were synthesized and tested in vitro and in cell-based assays. In this paper we demonstrate for the first time that chemical modifications on position 8 of 2′-deoxyadenosine induce delayed chain termination in vitro, and also inhibit DNA synthesis when incorporated in a DNA template strand. Furthermore, one of them had moderate anti-HIV-1 activity in cell-culture. Our results constitute a proof of concept indicating that modification on the base moiety of nucleosides can induce delayed polymerization arrest and inhibit HIV-1 replication. PMID:22087320

  20. Effect of rAd5-Vector HIV-1 Preventive Vaccines on HIV-1 Acquisition: A Participant-Level Meta-Analysis of Randomized Trials

    PubMed Central

    Huang, Yunda; Follmann, Dean; Nason, Martha; Zhang, Lily; Huang, Ying; Mehrotra, Devan V.; Moodie, Zoe; Metch, Barbara; Janes, Holly; Keefer, Michael C.; Churchyard, Gavin; Robb, Merlin L.; Fast, Patricia E.; Duerr, Ann; McElrath, M. Juliana; Corey, Lawrence; Mascola, John R.; Graham, Barney S.; Sobieszczyk, Magdalena E.; Kublin, James G.; Robertson, Michael; Hammer, Scott M.; Gray, Glenda E.; Buchbinder, Susan P.; Gilbert, Peter B.

    2015-01-01

    Background Three phase 2b, double-blind, placebo-controlled, randomized efficacy trials have tested recombinant Adenovirus serotype-5 (rAd5)-vector preventive HIV-1 vaccines: MRKAd5 HIV-1 gag/pol/nef in Step and Phambili, and DNA/rAd5 HIV-1 env/gag/pol in HVTN505. Due to efficacy futility observed at the first interim analysis in Step and HVTN505, participants of all three studies were unblinded to their vaccination assignments during the study but continued follow–up. Rigorous meta-analysis can provide crucial information to advise the future utility of rAd5-vector vaccines. Methods We included participant-level data from all three efficacy trials, and three Phase 1–2 trials evaluating the HVTN505 vaccine regimen. We predefined two co-primary analysis cohorts for assessing the vaccine effect on HIV-1 acquisition. The modified-intention-to-treat (MITT) cohort included all randomly assigned participants HIV-1 uninfected at study entry, who received at least the first vaccine/placebo, and the Ad5 cohort included MITT participants who received at least one dose of rAd5-HIV vaccine or rAd5-placebo. Multivariable Cox regression models were used to estimate hazard ratios (HRs) of HIV-1 infection (vaccine vs. placebo) and evaluate HR variation across vaccine regimens, time since vaccination, and subgroups using interaction tests. Findings Results are similar for the MITT and Ad5 cohorts; we summarize MITT cohort results. Pooled across the efficacy trials, over all follow-up time 403 (n = 224 vaccine; n = 179 placebo) of 6266 MITT participants acquired HIV-1, with a non-significantly higher incidence in vaccine recipients (HR 1.21, 95% CI 0.99–1.48, P = 0.06). The HRs significantly differed by vaccine regimen (interaction P = 0.03; MRKAd5 HR 1.41, 95% CI 1.11–1.78, P = 0.005 vs. DNA/rAd5 HR 0.88, 95% CI 0.61–1.26, P = 0.48). Results were similar when including the Phase 1–2 trials. Exploratory analyses based on the efficacy trials supported that the MRKAd5

  1. The HPA axis in HIV-1 infection.

    PubMed

    Kumar, Mahendra; Kumar, Adarsh M; Waldrop, Drenna; Antoni, Michael H; Schneiderman, Neil; Eisdorfer, Carl

    2002-10-01

    Several lines of evidence suggest that neuroendocrine abnormalities in general and HPA axis activity in particular occur in both HIV-1 infection and individuals engaging in chronic drug use. For instance, our studies showing attenuated norepinephrine as well as ACTH and cortisol responses to a cold pressor challenge in asymptomatic HIV-1 persons support such a concept. Furthermore, our data on investigations on mirror-star tracing and speech challenges also support the finding that neuroendocrine responses are compromised in HIV-1 infection. Although the mechanisms leading to adverse effects on HPA axis activity in HIV infection are not fully understood, several lines of evidence suggest that a number of mechanisms may be involved, including homologies in molecular structures of various mediators of neuroendocrine activity and HIV-related structures, HIV as a chronic stress model, and virus-induced toxic factors. This article reviews our recent findings in this area and also presents research hypotheses needed for testing and understanding the mechanisms involved in the development of neuroendocrine abnormalities in HIV-1-infected injection drug users. PMID:12394788

  2. HIV-1 remodels the nuclear pore complex

    PubMed Central

    Monette, Anne; Panté, Nelly

    2011-01-01

    Human immunodeficiency virus type 1 (HIV-1) commandeers host cell proteins and machineries for its replication. Our earlier work showed that HIV-1 induced the cytoplasmic retention of nucleocytoplasmic shuttling and ribonucleic acid (RNA)–binding proteins. This retention is dependent on nuclear export of the viral genomic RNA and on changes in the localization and expression level of the nucleoporin (Nup) p62 (Nup62). To further characterize the extent of perturbation induced by HIV-1, we performed proteomics analyses of nuclear envelopes (NEs) isolated from infected T cells. Infection induced extensive changes in the composition of the NE and its associated proteins, including a remarkable decrease in the abundance of Nups. Immunogold electron microscopy revealed the translocation of Nups into the cytoplasm. Nup62 was identified as a component of purified virus, and small interfering RNA depletion studies revealed an important role for this Nup in virus gene expression and infectivity. This detailed analysis highlights the profound effects on NE composition induced by HIV-1 infection, providing further evidence of the magnitude of viral control over the cell biology of its host. PMID:21576391

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

    PubMed

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

    2007-07-01

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

  4. HIV-1 transcription and latency: an update

    PubMed Central

    2013-01-01

    Combination antiretroviral therapy, despite being potent and life-prolonging, is not curative and does not eradicate HIV-1 infection since interruption of treatment inevitably results in a rapid rebound of viremia. Reactivation of latently infected cells harboring transcriptionally silent but replication-competent proviruses is a potential source of persistent residual viremia in cART-treated patients. Although multiple reservoirs may exist, the persistence of resting CD4+ T cells carrying a latent infection represents a major barrier to eradication. In this review, we will discuss the latest reports on the molecular mechanisms that may regulate HIV-1 latency at the transcriptional level, including transcriptional interference, the role of cellular factors, chromatin organization and epigenetic modifications, the viral Tat trans-activator and its cellular cofactors. Since latency mechanisms may also operate at the post-transcriptional level, we will consider inhibition of nuclear RNA export and inhibition of translation by microRNAs as potential barriers to HIV-1 gene expression. Finally, we will review the therapeutic approaches and clinical studies aimed at achieving either a sterilizing cure or a functional cure of HIV-1 infection, with a special emphasis on the most recent pharmacological strategies to reactivate the latent viruses and decrease the pool of viral reservoirs. PMID:23803414

  5. Activities, crystal structures and molecular dynamics of dihydro-1H-isoindole derivatives, inhibitors of HIV-1 integrase

    PubMed Central

    Métifiot, Mathieu; Maddali, Kasthuraiah; Johnson, Barry C.; Hare, Stephen; Smith, Steven J.; Zhao, XueZhi; Marchand, Christophe; Burke, Terrence R.; Hughes, Stephen H.; Cherepanov, Peter; Pommier, Yves

    2013-01-01

    Based on a series of lactam and phthalimide derivatives that inhibit HIV-1 integrase, we developed a new derivative, XZ-259, with biochemical and antiviral activities comparable to raltegravir. We determined the crystal structures of XZ-259 and four other derivatives in complex with the prototype foamy virus intasome. The compounds bind at the integrase-Mg2+-DNA interface of the integrase active site. In biochemical and antiviral assays, XZ-259 inhibits raltegravir-resistant HIV-1 integrases harboring the Y143R mutation. Molecular modeling is also presented suggesting that XZ-259 can bind in the HIV-1 intasome with its dimethyl sulfonamide group adopting two opposite orientations. Molecular dynamics analyses of the HIV-1 intasome highlight the importance of the viral DNA in drug potency. PMID:23075516

  6. The role of lysine 186 in HIV-1 integrase multimerization

    SciTech Connect

    Berthoux, Lionel; Sebastian, Sarah; Muesing, Mark A.; Luban, Jeremy . E-mail: luban@irb.unisi.ch

    2007-07-20

    HIV-1 integrase (IN) catalyzes biochemical reactions required for viral cDNA insertion into host cell chromosomal DNA, an essential step in the HIV-1 replication cycle. In one of these reactions, the two ends of the linear viral cDNA are believed to be simultaneously ligated to chromosomal DNA by a tetrameric form of IN. The structure of the full-length IN tetramer is not known but a model consisting of the N-terminal domain and the catalytic core revealed basic residues 186 to 188 at the interface between the two IN dimers. We found that alteration of these residues, in particular changing IN lysine residue 186 to glutamate (K186Q), impairs IN oligomerization in the yeast two-hybrid system and decreases oligomeric forms of IN within virions. When expressed independently of other viral proteins in human cells, IN-K186Q did not concentrate in the nucleus as did wild-type IN. Co-expression of wild-type IN restored the multimerization defects of IN-K186Q, in both the two-hybrid system and in virions, and also rescued the nuclear targeting defects. Virions bearing IN-K186Q were not infectious in a single cycle of replication but when mixed virions containing two different IN mutants were produced, IN-K186Q was capable of complementing the catalytically inactive mutant IN-D116A. Our biochemical and functional data support the crystallographic model in which IN residue K186 lies at the interface between IN dimers and suggest that tetramerization is important, not only for concerted integration, but also for IN nuclear targeting.

  7. HIV-1 subtype C is not associated with higher risk of heterosexual HIV-1 transmission: a multinational study among African HIV-1 serodiscordant couples

    PubMed Central

    Kahle, Erin; Campbell, Mary; Lingappa, Jairam; Donnell, Deborah; Celum, Connie; Ondondo, Raphael; Mujugira, Andrew; Fife, Kenneth; Mugo, Nelly; Kapiga, Saidi; Mullins, James I.; Baeten, Jared M.

    2014-01-01

    Background HIV-1 subtype C has emerged as the most prevalent strain of HIV-1 worldwide, leading to speculation that subtype C may be more transmissible than other subtypes. We compared the risk of HIV-1 transmission for subtype C versus non-C subtypes (A, D, G and recombinant forms) among heterosexual African HIV-1 serodiscordant couples. Methods We conducted a nested case-control analysis using data from two prospective cohort studies of heterosexual HIV-1 serodiscordant couples from 6 countries in eastern and southern Africa. Cases (N=121) included incident HIV-1 transmissions that were established as linked within the serodiscordant partnership by viral sequencing; controls (N=501) were non-transmitting HIV-1 infected partners. Subtype was determined for partial env and gag genes. Multiple logistic regression controlled for age and gender of the HIV-1 infected partner and self-reported unprotected sex. Plasma and genital HIV-1 RNA concentrations were compared between subtype C and non-C subtypes using generalized estimating equations. Results HIV-1 subtype C was not associated with increased risk of HIV-1 transmission compared to non-C subtypes: env adjusted odds ratio (adjOR) 1.14 (95% confidence interval [CI] 0.74–1.75, p=0.6) and gag adjOR 0.98 (95% CI 0.63–1.52, p=0.9). Plasma and genital HIV-1 RNA levels did not differ significantly for subtype C versus non-C. Conclusion In a geographically diverse population of heterosexual African HIV-1 serodiscordant couples, subtype C was not associated with greater risk of HIV-1 transmission compared to non-C subtypes, arguing against the hypothesis that subtype C is more transmissible compared to other common subtypes. PMID:24413311

  8. HIV-1 therapy with monoclonal antibody 3BNC117 elicits host immune responses against HIV-1.

    PubMed

    Schoofs, Till; Klein, Florian; Braunschweig, Malte; Kreider, Edward F; Feldmann, Anna; Nogueira, Lilian; Oliveira, Thiago; Lorenzi, Julio C C; Parrish, Erica H; Learn, Gerald H; West, Anthony P; Bjorkman, Pamela J; Schlesinger, Sarah J; Seaman, Michael S; Czartoski, Julie; McElrath, M Juliana; Pfeifer, Nico; Hahn, Beatrice H; Caskey, Marina; Nussenzweig, Michel C

    2016-05-20

    3BNC117 is a broad and potent neutralizing antibody to HIV-1 that targets the CD4 binding site on the viral envelope spike. When administered passively, this antibody can prevent infection in animal models and suppress viremia in HIV-1-infected individuals. Here we report that HIV-1 immunotherapy with a single injection of 3BNC117 affects host antibody responses in viremic individuals. In comparison to untreated controls that showed little change in their neutralizing activity over a 6-month period, 3BNC117 infusion significantly improved neutralizing responses to heterologous tier 2 viruses in nearly all study participants. We conclude that 3BNC117-mediated immunotherapy enhances host humoral immunity to HIV-1. PMID:27199429

  9. Enhanced clearance of HIV-1-infected cells by broadly neutralizing antibodies against HIV-1 in vivo.

    PubMed

    Lu, Ching-Lan; Murakowski, Dariusz K; Bournazos, Stylianos; Schoofs, Till; Sarkar, Debolina; Halper-Stromberg, Ariel; Horwitz, Joshua A; Nogueira, Lilian; Golijanin, Jovana; Gazumyan, Anna; Ravetch, Jeffrey V; Caskey, Marina; Chakraborty, Arup K; Nussenzweig, Michel C

    2016-05-20

    Antiretroviral drugs and antibodies limit HIV-1 infection by interfering with the viral life cycle. In addition, antibodies also have the potential to guide host immune effector cells to kill HIV-1-infected cells. Examination of the kinetics of HIV-1 suppression in infected individuals by passively administered 3BNC117, a broadly neutralizing antibody, suggested that the effects of the antibody are not limited to free viral clearance and blocking new infection but also include acceleration of infected cell clearance. Consistent with these observations, we find that broadly neutralizing antibodies can target CD4(+) T cells infected with patient viruses and can decrease their in vivo half-lives by a mechanism that requires Fcγ receptor engagement in a humanized mouse model. The results indicate that passive immunotherapy can accelerate elimination of HIV-1-infected cells. PMID:27199430

  10. Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication Studies

    PubMed Central

    Lysenko, Elena S.; Bosch, Ronald J.; Lai, Jun; Chioma, Stanley; Emad, Fatemeh; Abdel-Mohsen, Mohamed; Hoh, Rebecca; Hecht, Frederick; Hunt, Peter; Somsouk, Ma; Wong, Joseph; Johnston, Rowena; Siliciano, Robert F.; Richman, Douglas D.; O'Doherty, Una; Palmer, Sarah; Deeks, Steven G.; Siliciano, Janet D.

    2013-01-01

    HIV-1 reservoirs preclude virus eradication in patients receiving highly active antiretroviral therapy (HAART). The best characterized reservoir is a small, difficult-to-quantify pool of resting memory CD4+ T cells carrying latent but replication-competent viral genomes. Because strategies targeting this latent reservoir are now being tested in clinical trials, well-validated high-throughput assays that quantify this reservoir are urgently needed. Here we compare eleven different approaches for quantitating persistent HIV-1 in 30 patients on HAART, using the original viral outgrowth assay for resting CD4+ T cells carrying inducible, replication-competent viral genomes as a standard for comparison. PCR-based assays for cells containing HIV-1 DNA gave infected cell frequencies at least 2 logs higher than the viral outgrowth assay, even in subjects who started HAART during acute/early infection. This difference may reflect defective viral genomes. The ratio of infected cell frequencies determined by viral outgrowth and PCR-based assays varied dramatically between patients. Although strong correlations with the viral outgrowth assay could not be formally excluded for most assays, correlations achieved statistical significance only for integrated HIV-1 DNA in peripheral blood mononuclear cells and HIV-1 RNA/DNA ratio in rectal CD4+ T cells. Residual viremia was below the limit of detection in many subjects and did not correlate with the viral outgrowth assays. The dramatic differences in infected cell frequencies and the lack of a precise correlation between culture and PCR-based assays raise the possibility that the successful clearance of latently infected cells may be masked by a larger and variable pool of cells with defective proviruses. These defective proviruses are detected by PCR but may not be affected by reactivation strategies and may not require eradication to accomplish an effective cure. A molecular understanding of the discrepancy between infected cell

  11. Comparison of Newly Assembled Full Length HIV-1 Integrase With Prototype Foamy Virus Integrase: Structure-Function Prospective

    PubMed Central

    Dayer, Mohammad Reza

    2016-01-01

    Background Drug design against human immunodeficiency virus type 1 (HIV-1) integrase through its mechanistic study is of great interest in the area in biological research. The main obstacle in this area is the absence of the full-length crystal structure for HIV-1 integrase to be used as a model. A complete structure, similar to HIV-1 of a prototype foamy virus integrase in complex with DNA, including all conservative residues, is available and has been extensively used in recent investigations. Objectives The aim of this study was to determine whether the above model is precisely representative of HIV-1 integrase. This would critically determine the success of any designed drug using the model in deactivation of integrase and AIDS treatment. Materials and Methods Primarily, a new structure for HIV-1 was constructed, using a crystal structure of prototype foamy virus as the starting structure. The constructed structure of HIV-1 integrase was simultaneously simulated with a prototype foamy virus integrase on a separate occasion. Results Our results indicate that the HIV-1 system behaves differently from the prototype foamy virus in terms of folding, hydration, hydrophobicity of binding site and stability. Conclusions Based on our findings, we can conclude that HIV-1 integrase is vastly different from the prototype foamy virus integrase and does not resemble it, and the modeling output of the prototype foamy virus simulations could not be simply generalized to HIV-1 integrase. Therefore, our HIV-1 model seems to be more representative and more useful for future research. PMID:27540450

  12. Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr.

    PubMed

    Datta, Prasun K; Deshmane, Satish; Khalili, Kamel; Merali, Salim; Gordon, John C; Fecchio, Chiara; Barrero, Carlos A

    2016-09-01

    HIV-1 infected macrophages play a significant role in the neuropathogenesis of AIDS. HIV-1 viral protein R (Vpr) not only facilitates HIV-1 infection but also contribute to long-lived persistence in macrophages. Our previous studies using SILAC-based proteomic analysis showed that the expression of critical metabolic enzymes in the glycolytic pathway and tricarboxylic acid (TCA) cycle were altered in response to Vpr expression in macrophages. We hypothesized that Vpr-induced modulation of glycolysis and TCA cycle regulates glutamate metabolism and release in HIV-1 infected macrophages. We assessed the amount of specific metabolites induced by Vpr and HIV-1 in macrophages at the intracellular and extracellular level in a time-dependent manner utilizing multiple reaction monitoring (MRM) targeted metabolomics. In addition, stable isotope-labeled glucose and an MRM targeted metabolomics assay were used to evaluate the de novo synthesis and release of glutamate in Vpr overexpressing macrophages and HIV-1 infected macrophages, throughout the metabolic flux of glycolytic pathway and TCA cycle activation. The metabolic flux studies demonstrated an increase in glucose uptake, glutamate release and accumulation of α-ketoglutarate (α-KG) and glutamine in the extracellular milieu in Vpr expressing and HIV-1 infected macrophages. Interestingly, glutamate pools and other intracellular intermediates (glucose-6-phosphate (G6P), fructose-6-phosphate (F6P), citrate, malate, α-KG, and glutamine) showed a decreased trend except for fumarate, in contrast to the glutamine accumulation observed in the extracellular space in Vpr overexpressing macrophages. Our studies demonstrate that dysregulation of mitochondrial glutamate metabolism induced by Vpr in HIV-1 infected macrophages commonly seen, may contribute to neurodegeneration via excitotoxic mechanisms in the context of NeuroAIDS. PMID:27245560

  13. Restricting HIV-1 pathways for escape using rationally designed anti–HIV-1 antibodies

    PubMed Central

    Klein, Florian; Horwitz, Joshua A.; Halper-Stromberg, Ariel; Sather, D. Noah; Marcovecchio, Paola M.; Lee, Terri; West, Anthony P.; Gao, Han; Seaman, Michael S.; Stamatatos, Leonidas; Nussenzweig, Michel C.; Bjorkman, Pamela J.

    2013-01-01

    Recently identified broadly neutralizing antibodies (bNAbs) that potently neutralize most HIV-1 strains are key to potential antibody-based therapeutic approaches to combat HIV/AIDS in the absence of an effective vaccine. Increasing bNAb potencies and resistance to common routes of HIV-1 escape through mutation would facilitate their use as therapeutics. We previously used structure-based design to create the bNAb NIH45-46G54W, which exhibits superior potency and/or breadth compared with other bNAbs. We report new, more effective NIH45-46G54W variants designed using analyses of the NIH45-46–gp120 complex structure and sequences of NIH45-46G54W–resistant HIV-1 strains. One variant, 45-46m2, neutralizes 96% of HIV-1 strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti–HIV-1 antibody to date. A description of its mechanism is presented based on a 45-46m2–gp120 crystal structure. A second variant, 45-46m7, designed to thwart HIV-1 resistance to NIH45-46G54W arising from mutations in a gp120 consensus sequence, targets a common route of HIV-1 escape. In combination, 45-46m2 and 45-46m7 reduce the possible routes for the evolution of fit viral escape mutants in HIV-1YU-2–infected humanized mice, with viremic control exhibited when a third antibody, 10–1074, was added to the combination. PMID:23712429

  14. Elevated Cytokine and Chemokine Levels in the Placenta Are Associated With in utero HIV-1 Mother-To-Child Transmission

    PubMed Central

    Kumar, Surender B.; Rice, Cara E.; Milner, Danny A.; Ramirez, Nilsa C.; Ackerman, William E.; Mwapasa, Victor; Turner, Abigail Norris; Kwiek, Jesse J.

    2012-01-01

    Objective To determine if there is an association between cytokine and chemokine levels in plasma isolated from the placenta and HIV-1 mother-to-child transmission (MTCT). Design We designed a case-control study of HIV-infected, pregnant women enrolled in the Malaria and HIV in Pregnancy cohort. Participants were recruited in Blantyre, Malawi from 2000-04. Cases were women whose children were HIV-1 DNA-positive at birth (in utero MTCT) or HIV-1 DNA-negative at birth and HIV-1 DNA-positive at 6-weeks post-partum (intrapartum MTCT); controls were women whose children were HIV-1 DNA-negative both at birth and 6-weeks post-partum. Methods After delivery, blood was isolated from an incision on the basal plate of the placenta. We used a Luminex assay to simultaneously quantify 27 cytokines, chemokines, and growth factors in placental plasma. HIV-1 RNA copies were quantified with the Roche Amplicor kit. Results Levels of IL-4, IL-5, IL-6, IL-7, IL-9, eotaxin, IL1Ra and IP-10 were significantly elevated in placental plasma isolated from cases of in utero HIV-1 MTCT. In contrast, only GCSF was elevated in placental plasma isolated from cases of intrapartum MTCT. After adjusting for maternal age, gestational age, and peripheral CD4+ T cell count, every log10 increase in placental IP-10 was associated with a three-fold increase in the prevalence of in utero HIV-1 MTCT. Conclusions Elevated cytokine and chemokine levels in placental plasma were associated with in utero and not intrapartum MTCT. IP-10, which is both a T-cell chemokine and potentiator of HIV-replication, was robustly and independently associated with prevalent, in utero MTCT. PMID:22301415

  15. Interaction between Reverse Transcriptase and Integrase Is Required for Reverse Transcription during HIV-1 Replication

    PubMed Central

    Tekeste, Shewit S.; Wilkinson, Thomas A.; Weiner, Ethan M.; Xu, Xiaowen; Miller, Jennifer T.; Le Grice, Stuart F. J.; Clubb, Robert T.

    2015-01-01

    ABSTRACT Human immunodeficiency virus type 1 (HIV-1) replication requires reverse transcription of its RNA genome into a double-stranded cDNA copy, which is then integrated into the host cell chromosome. The essential steps of reverse transcription and integration are catalyzed by the viral enzymes reverse transcriptase (RT) and integrase (IN), respectively. In vitro, HIV-1 RT can bind with IN, and the C-terminal domain (CTD) of IN is necessary and sufficient for this binding. To better define the RT-IN interaction, we performed nuclear magnetic resonance (NMR) spectroscopy experiments to map a binding surface on the IN CTD in the presence of RT prebound to a duplex DNA construct that mimics the primer-binding site in the HIV-1 genome. To determine the biological significance of the RT-IN interaction during viral replication, we used the NMR chemical shift mapping information as a guide to introduce single amino acid substitutions of nine different residues on the putative RT-binding surface in the IN CTD. We found that six viral clones bearing such IN substitutions (R231E, W243E, G247E, A248E, V250E, and I251E) were noninfectious. Further analyses of the replication-defective IN mutants indicated that the block in replication took place specifically during early reverse transcription. The recombinant INs purified from these mutants, though retaining enzymatic activities, had diminished ability to bind RT in a cosedimentation assay. The results indicate that the RT-IN interaction is functionally relevant during the reverse transcription step of the HIV-1 life cycle. IMPORTANCE To establish a productive infection, human immunodeficiency virus type 1 (HIV-1) needs to reverse transcribe its RNA genome to create a double-stranded DNA copy and then integrate this viral DNA genome into the chromosome of the host cell. These two essential steps are catalyzed by the HIV-1 enzymes reverse transcriptase (RT) and integrase (IN), respectively. We have shown previously that IN

  16. Transplanting Supersites of HIV-1 Vulnerability

    PubMed Central

    Yang, Yongping; Gorman, Jason; Ofek, Gilad; Srivatsan, Sanjay; Druz, Aliaksandr; Lees, Christopher R.; Lu, Gabriel; Soto, Cinque; Stuckey, Jonathan; Burton, Dennis R.; Koff, Wayne C.; Connors, Mark; Kwon, Peter D.

    2014-01-01

    One strategy for isolating or eliciting antibodies against a specific target region on the envelope glycoprotein trimer (Env) of the human immunodeficiency virus type 1 (HIV-1) involves the creation of site transplants, which present the target region on a heterologous protein scaffold with preserved antibody-binding properties. If the target region is a supersite of HIV-1 vulnerability, recognized by a collection of broadly neutralizing antibodies, this strategy affords the creation of “supersite transplants”, capable of binding (and potentially eliciting) antibodies similar to the template collection of effective antibodies. Here we transplant three supersites of HIV-1 vulnerability, each targeted by effective neutralizing antibodies from multiple donors. To implement our strategy, we chose a single representative antibody against each of the target supersites: antibody 10E8, which recognizes the membrane-proximal external region (MPER) on the HIV-1 gp41 glycoprotein; antibody PG9, which recognizes variable regions one and two (V1V2) on the HIV-1 gp120 glycoprotein; and antibody PGT128 which recognizes a glycopeptide supersite in variable region 3 (glycan V3) on gp120. We used a structural alignment algorithm to identify suitable acceptor proteins, and then designed, expressed, and tested antigenically over 100-supersite transplants in a 96-well microtiter-plate format. The majority of the supersite transplants failed to maintain the antigenic properties of their respective template supersite. However, seven of the glycan V3-supersite transplants exhibited nanomolar affinity to effective neutralizing antibodies from at least three donors and recapitulated the mannose9-N-linked glycan requirement of the template supersite. The binding of these transplants could be further enhanced by placement into self-assembling nanoparticles. Essential elements of the glycan V3 supersite, embodied by as few as 3 N-linked glycans and ∼25 Env residues, can be segregated

  17. Strand Transfer and Elongation of HIV-1 Reverse Transcription Is Facilitated by Cell Factors In Vitro

    PubMed Central

    Warrilow, David; Warren, Kylie; Harrich, David

    2010-01-01

    Recent work suggests a role for multiple host factors in facilitating HIV-1 reverse transcription. Previously, we identified a cellular activity which increases the efficiency of HIV-1 reverse transcription in vitro. Here, we describe aspects of the activity which shed light on its function. The cellular factor did not affect synthesis of strong-stop DNA but did improve downstream DNA synthesis. The stimulatory activity was isolated by gel filtration in a single fraction of the exclusion volume. Velocity-gradient purified HIV-1, which was free of detectable RNase activity, showed poor reverse transcription efficiency but was strongly stimulated by partially purified cell proteins. Hence, the cell factor(s) did not inactivate an RNase activity that might degrade the viral genomic RNA and block completion of reverse transcription. Instead, the cell factor(s) enhanced first strand transfer and synthesis of late reverse transcription suggesting it stabilized the reverse transcription complex. The factor did not affect lysis of HIV-1 by Triton X-100 in the endogenous reverse transcription (ERT) system, and ERT reactions with HIV-1 containing capsid mutations, which varied the biochemical stability of viral core structures and impeded reverse transcription in cells, showed no difference in the ability to be stimulated by the cell factor(s) suggesting a lack of involvement of the capsid in the in vitro assay. In addition, reverse transcription products were found to be resistant to exogenous DNase I activity when the active fraction was present in the ERT assay. These results indicate that the cell factor(s) may improve reverse transcription by facilitating DNA strand transfer and DNA synthesis. It also had a protective function for the reverse transcription products, but it is unclear if this is related to improved DNA synthesis. PMID:20949087

  18. Use of ATP analogs to inhibit HIV-1 transcription.

    PubMed

    Narayanan, Aarthi; Sampey, Gavin; Van Duyne, Rachel; Guendel, Irene; Kehn-Hall, Kylene; Roman, Jessica; Currer, Robert; Galons, Hervé; Oumata, Nassima; Joseph, Benoît; Meijer, Laurent; Caputi, Massimo; Nekhai, Sergei; Kashanchi, Fatah

    2012-10-10

    Human immunodeficiency virus type 1 (HIV-1) is the etiological agent of AIDS. Chronic persistent infection is an important reason for the presence of "latent cell populations" even after Anti-Retroviral Therapy (ART). We have analyzed the effect of ATP analogs in inhibiting cdk9/T1 complex in infected cells. A third generation drug named CR8#13 is an effective inhibitor of Tat activated transcription. Following drug treatment, we observed a decreased loading of cdk9 onto the HIV-1 DNA. We found multiple novel cdk9/T1 complexes present in infected and uninfected cells with one complex being unique to infected cells. This complex is sensitive to CR8#13 in kinase assays. Treatment of PBMC with CR8#13 does not kill infected cells as compared to Flavopiridol. Interestingly, there is a difference in sensitivity of various clades to these analogs. Collectively, these results point to targeting novel complexes for inhibition of cellular proteins that are unique to infected cells. PMID:22771113

  19. Recombinant Viruses and Early Global HIV-1 Epidemic

    PubMed Central

    Robbins, Kenneth E.; Pieniazek, Danuta; Schaefer, Amanda; Nzilambi, Nzila; Quinn, Thomas C.; St. Louis, Michael E.; Youngpairoj, Ae S.; Phillips, Jonathan; Jaffe, Harold W.; Folks, Thomas M.

    2004-01-01

    Central Africa was the epicenter of the HIV type 1 (HIV-1) pandemic. Understanding the early epidemic in the Democratic Republic of the Congo, formerly Zaire, could provide insight into how HIV evolved and assist vaccine design and intervention efforts. Using enzyme immunosorbent assays, we tested 3,988 serum samples collected in Kinshasa in the mid-1980s and confirmed seroreactivity by Western blot. Polymerase chain reaction of gag p17, env C2V3C3, and/or gp41; DNA sequencing; and genetic analyses were performed. Gene regions representing all the HIV-1 group M clades and unclassifiable sequences were found. From two or three short gene regions, 37% of the strains represented recombinant viruses, multiple infections, or both, which suggests that if whole genome sequences were available, most of these strains would have mosaic genomes. We propose that the HIV epidemic was well established in central Africa by the early 1980s and that some recombinant viruses most likely seeded the early global epidemic. PMID:15324542

  20. Intra-spike crosslinking overcomes antibody evasion by HIV-1.

    PubMed

    Galimidi, Rachel P; Klein, Joshua S; Politzer, Maria S; Bai, Shiyu; Seaman, Michael S; Nussenzweig, Michel C; West, Anthony P; Bjorkman, Pamela J

    2015-01-29

    Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. We postulated that anti-HIV-1 antibodies primarily bind monovalently because HIV's low spike density impedes bivalent binding through inter-spike crosslinking, and the spike structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a "molecular ruler" to measure intra-epitope distances on virion-bound spikes and construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders of magnitude increased potency (>100-fold average increases across virus panels) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection. PMID:25635457

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

    SciTech Connect

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

    2011-11-23

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

  2. Vaccine-elicited Human T Cells Recognizing Conserved Protein Regions Inhibit HIV-1

    PubMed Central

    Borthwick, Nicola; Ahmed, Tina; Ondondo, Beatrice; Hayes, Peter; Rose, Annie; Ebrahimsa, Umar; Hayton, Emma-Jo; Black, Antony; Bridgeman, Anne; Rosario, Maximillian; Hill, Adrian VS; Berrie, Eleanor; Moyle, Sarah; Frahm, Nicole; Cox, Josephine; Colloca, Stefano; Nicosia, Alfredo; Gilmour, Jill; McMichael, Andrew J; Dorrell, Lucy; Hanke, Tomáš

    2014-01-01

    Virus diversity and escape from immune responses are the biggest challenges to the development of an effective vaccine against HIV-1. We hypothesized that T-cell vaccines targeting the most conserved regions of the HIV-1 proteome, which are common to most variants and bear fitness costs when mutated, will generate effectors that efficiently recognize and kill virus-infected cells early enough after transmission to potentially impact on HIV-1 replication and will do so more efficiently than whole protein-based T-cell vaccines. Here, we describe the first-ever administration of conserved immunogen vaccines vectored using prime-boost regimens of DNA, simian adenovirus and modified vaccinia virus Ankara to uninfected UK volunteers. The vaccine induced high levels of effector T cells that recognized virus-infected autologous CD4+ cells and inhibited HIV-1 replication by up to 5.79 log10. The virus inhibition was mediated by both Gag- and Pol- specific effector CD8+ T cells targeting epitopes that are typically subdominant in natural infection. These results provide proof of concept for using a vaccine to target T cells at conserved epitopes, showing that these T cells can control HIV-1 replication in vitro. PMID:24166483

  3. APOBEC3G ubiquitination by Nedd4-1 favors its packaging into HIV-1 particles.

    PubMed

    Dussart, Sylvie; Douaisi, Marc; Courcoul, Marianne; Bessou, Gilles; Vigne, Robert; Decroly, Etienne

    2005-01-21

    APOBEC3G is a cytidine deaminase that limits the replication of many retroviruses. This antiviral host factor is packaged into retrovirus particles, where it targets single-stranded DNA generated during reverse transcription and induces up to 2% of G-to-A mutations, which are lethal for the HIV-1 provirus. Vif protein counteracts this antiviral factor by decreasing its packaging into lentivirus particles. Here, we demonstrate that Nedd4-1, an HECT E3 ubiquitin ligase, interacts with APOBEC3G, through its WW2 and WW3 domains. As a result of this interaction, APOBEC3G undergoes post-translational modification by addition of ubiquitin moieties. Accordingly, we demonstrate that the dominant negative Nedd4-1 C/S form prevents APOBEC3G ubiquitination. Moreover, the packaging of APOBEC3G into Pr55 Gag virus-like particles and into HIV-1 virions is reduced when Nedd4-1 C/S is expressed. During HIV-1 viral production in the presence of APOBEC3G, Nedd4-1 C/S restores partially the infectivity of Deltavif HIV-1. We conclude that the ubiquitination of APOBEC3G by Nedd4-1 favors its targeting to the virus assembly site where APOBEC3G interacts with Gag and is packaged into HIV-1 particles in the absence of Vif. PMID:15581898

  4. 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

  5. HIV-1 cellular and tissue replication patterns in infected humanized mice

    PubMed Central

    Araínga, Mariluz; Su, Hang; Poluektova, Larisa Y.; Gorantla, Santhi; Gendelman, Howard E.

    2016-01-01

    Humanized mice have emerged as a testing platform for HIV-1 pathobiology by reflecting natural human disease processes. Their use to study HIV-1 biology, virology, immunology, pathogenesis and therapeutic development has served as a robust alternative to more-well developed animal models for HIV/AIDS. A critical component in reflecting such human pathobiology rests in defining the tissue and cellular sites for HIV-1 infection. To this end, we examined the tissue sites for viral infection in bone marrow, blood, spleens, liver, gut, brain, kidney and lungs of human CD34+ hematopoietic stem cell engrafted virus-infected NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ mice. Cells were analyzed by flow cytometry and sorted from species mixtures defined as CD34+ lineage negative progenitor cells, CD14+CD16+ monocyte-macrophages and central, stem cell and effector memory T cells. The cell distribution and viral life cycle were found dependent on the tissue compartment and time of infection. Cell subsets contained HIV-1 total and integrated DNA as well as multi-spliced and unspliced RNA in divergent proportions. The data support the idea that humanized mice can provide a means to examine the multifaceted sites of HIV-1 replication including, but not limited to progenitor cells and monocyte-macrophages previously possible only in macaques and human. PMID:26996968

  6. Nup153 and Nup98 bind the HIV-1 core and contribute to the early steps of HIV-1 replication

    SciTech Connect

    Di Nunzio, Francesca; Fricke, Thomas; Miccio, Annarita; Valle-Casuso, Jose Carlos; Perez, Patricio; Souque, Philippe; Rizzi, Ermanno; Severgnini, Marco; Mavilio, Fulvio; Charneau, Pierre; Diaz-Griffero, Felipe

    2013-05-25

    The early steps of HIV-1 replication involve the entry of HIV-1 into the nucleus, which is characterized by viral interactions with nuclear pore components. HIV-1 developed an evolutionary strategy to usurp the nuclear pore machinery and chromatin in order to integrate and efficiently express viral genes. In the current work, we studied the role of nucleoporins 153 and 98 (Nup153 and Nup98) in infection of human Jurkat lymphocytes by HIV-1. We showed that Nup153-depleted cells exhibited a defect in nuclear import, while depletion of Nup 98 caused a slight defect in HIV integration. To explore the biochemical viral determinants for the requirement of Nup153 and Nup98 during HIV-1 infection, we tested the ability of these nucleoporins to interact with HIV-1 cores. Our findings showed that both nucleoporins bind HIV-1 cores suggesting that this interaction is important for HIV-1 nuclear import and/or integration. Distribution analysis of integration sites in Nup153-depleted cells revealed a reduced tendency of HIV-1 to integrate in intragenic sites, which in part could account for the large infectivity defect observed in Nup153-depleted cells. Our work strongly supports a role for Nup153 in HIV-1 nuclear import and integration. - Highlights: ► We studied the role of Nup98 and Nup153 in HIV-1 infection. ► Nup98 binds the HIV-1 core and is involved in HIV-1 integration. ► Nup153 binds the HIV-1 core and is involved in HIV-1 nuclear import. ► Depletion of Nup153 decreased the integration of HIV-1 in transcriptionally active sites.

  7. Enhanced HIV-1 neutralization by antibody heteroligation

    PubMed Central

    Mouquet, Hugo; Warncke, Malte; Scheid, Johannes F.; Seaman, Michael S.; Nussenzweig, Michel C.

    2012-01-01

    Passive transfer of broadly neutralizing human antibodies against HIV-1 protects macaques against infection. However, HIV-1 uses several strategies to escape antibody neutralization, including mutation of the gp160 viral surface spike, a glycan shield to block antibody access to the spike, and expression of a limited number of viral surface spikes, which interferes with bivalent antibody binding. The latter is thought to decrease antibody apparent affinity or avidity, thereby interfering with neutralizing activity. To test the idea that increasing apparent affinity might enhance neutralizing activity, we engineered bispecific anti–HIV-1 antibodies (BiAbs) that can bind bivalently by virtue of one scFv arm that binds to gp120 and a second arm to the gp41 subunit of gp160. The individual arms of the BiAbs preserved the binding specificities of the original anti-HIV IgG antibodies and together bound simultaneously to gp120 and gp41. Heterotypic bivalent binding enhanced neutralization compared with the parental antibodies. We conclude that antibody recognition and viral neutralization of HIV can be improved by heteroligation. PMID:22219363

  8. A radiometric assay for HIV-1 protease

    SciTech Connect

    Hyland, L.J.; Dayton, B.D.; Moore, M.L.; Shu, A.Y.; Heys, J.R.; Meek, T.D. )

    1990-08-01

    A rapid, high-throughput radiometric assay for HIV-1 protease has been developed using ion-exchange chromatography performed in 96-well filtration plates. The assay monitors the activity of the HIV-1 protease on the radiolabeled form of a heptapeptide substrate, (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2, which is based on the p17-p24 cleavage site found in the viral polyprotein substrate Pr55gag. Specific cleavage of this uncharged heptapeptide substrate by HIV-1 protease releases the anionic product (tyrosyl-3,5-3H)Ac-Ser-Gln-Asn-Tyr, which is retained upon minicolumns of the anion-exchange resin AG1-X8. Protease activity is determined from the recovery of this radiolabeled product following elution with formic acid. This facile and highly sensitive assay may be utilized for steady-state kinetic analysis of the protease, for measurements of enzyme activity during its purification, and as a routine assay for the evaluation of protease inhibitors from natural product or synthetic sources.

  9. Polyclonal B-cell activation reveals antibodies against human immunodeficiency virus type 1 (HIV-1) in HIV-1-seronegative individuals.

    PubMed Central

    Jehuda-Cohen, T; Slade, B A; Powell, J D; Villinger, F; De, B; Folks, T M; McClure, H M; Sell, K W; Ahmed-Ansari, A

    1990-01-01

    Identification of human immunodeficiency virus type 1 (HIV-1)-infected individuals is of paramount importance for the control of the spread of AIDS worldwide. Currently, the vast majority of screening centers throughout the world rely on serological techniques. As such, clinically asymptomatic but HIV-infected, seronegative individuals are rarely identified. In this report we show that 18% (30/165) of seronegative individuals who were considered to be a unique cohort of patients at high risk for HIV infection had circulating B cells that, upon in vitro polyclonal activation with pokeweed mitogen, produced antibodies reactive with HIV. Furthermore, polymerase chain reaction analysis of DNA obtained from aliquots of the peripheral blood mononuclear cells from these seronegative but pokeweed mitogen assay-positive individuals tested revealed the presence of HIV-specific sequences in a significant number of samples. In addition, depletion of CD8+ T cells from peripheral blood mononuclear cells of HIV-1-seronegative individuals prior to in vitro culture with pokeweed mitogen resulted in increased sensitivity for detecting HIV-reactive antibodies. This assay has obvious epidemiological implications, especially in the case of high-risk groups, and also provides a simple technique to enhance detection of HIV-infected individuals. Of further interest is the determination of the mechanisms related to the lack of HIV-specific antibodies in the serum of these infected individuals. Images PMID:2111024

  10. Suppression of HIV-1 Infectivity by Human Glioma Cells.

    PubMed

    Hoque, Sheikh Ariful; Tanaka, Atsushi; Islam, Salequl; Ahsan, Gias Uddin; Jinno-Oue, Atsushi; Hoshino, Hiroo

    2016-05-01

    HIV-1 infection to the central nervous system (CNS) is very common in AIDS patients. The predominant cell types infected in the brain are monocytes and macrophages, which are surrounded by several HIV-1-resistant cell types, such as astrocytes, oligodendrocytes, neurons, and microvascular cells. The effect of these HIV-1-resistant cells on HIV-1 infection is largely unknown. In this study, we examined the stability of HIV-1 cultured with several human glioblastoma cell lines, for example, NP-2, U87MG, T98G, and A172, to determine whether these HIV-1-resistant brain cells could enhance or suppress HIV-1 infection and thus modulate HIV-1 infection in the CNS. The HIV-1 titer was determined using the MAGIC-5A indicator cell line as well as naturally occurring CD4(+) T cells. We found that the stability of HIV-1 incubated with NP-2 or U87MG cells at 37°C was significantly shorter (half-life, 2.5-4 h) compared to that of HIV-1 incubated with T98G or A172 cells or in culture medium without cells (half-life, 8-18 h). The spent culture media (SCM) of NP-2 and U87MG cells had the ability to suppress both R5- and X4-HIV-1 infection by inhibiting HIV-1 attachment to target cells. This inhibitory effect was eliminated by the treatment of the SCM with chondroitinase ABC but not heparinase, suggesting that the inhibitory factor(s) secreted by NP-2 and U87MG cells was chiefly mediated by chondroitin sulfate (CS) or CS-like moiety. Thus, this study reveals that some but not all glioma cells secrete inhibitory molecules to HIV-1 infection that may contribute in lowering HIV-1 infection in the CNS in vivo. PMID:26650729

  11. Quantification of the Epitope Diversity of HIV-1-Specific Binding Antibodies by Peptide Microarrays for Global HIV-1 Vaccine Development

    PubMed Central

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-01-01

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6,564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research. PMID:25445329

  12. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    SciTech Connect

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth of IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.

  13. Quantification of the epitope diversity of HIV-1-specific binding antibodies by peptide microarrays for global HIV-1 vaccine development

    DOE PAGESBeta

    Stephenson, Kathryn E.; Neubauer, George H.; Reimer, Ulf; Pawlowski, Nikolaus; Knaute, Tobias; Zerweck, Johannes; Korber, Bette T.; Barouch, Dan H.

    2014-11-14

    An effective vaccine against human immunodeficiency virus type 1 (HIV-1) will have to provide protection against a vast array of different HIV-1 strains. Current methods to measure HIV-1-specific binding antibodies following immunization typically focus on determining the magnitude of antibody responses, but the epitope diversity of antibody responses has remained largely unexplored. Here we describe the development of a global HIV-1 peptide microarray that contains 6564 peptides from across the HIV-1 proteome and covers the majority of HIV-1 sequences in the Los Alamos National Laboratory global HIV-1 sequence database. Using this microarray, we quantified the magnitude, breadth, and depth ofmore » IgG binding to linear HIV-1 sequences in HIV-1-infected humans and HIV-1-vaccinated humans, rhesus monkeys and guinea pigs. The microarray measured potentially important differences in antibody epitope diversity, particularly regarding the depth of epitope variants recognized at each binding site. Our data suggest that the global HIV-1 peptide microarray may be a useful tool for both preclinical and clinical HIV-1 research.« less

  14. Kinase Control of Latent HIV-1 Infection: PIM-1 Kinase as a Major Contributor to HIV-1 Reactivation

    PubMed Central

    Duverger, Alexandra; Wolschendorf, Frank; Anderson, Joshua C.; Wagner, Frederic; Bosque, Alberto; Shishido, Takao; Jones, Jennifer; Planelles, Vicente; Willey, Christopher; Cron, Randall Q.

    2014-01-01

    Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation. PMID:24155393

  15. Kinase control of latent HIV-1 infection: PIM-1 kinase as a major contributor to HIV-1 reactivation.

    PubMed

    Duverger, Alexandra; Wolschendorf, Frank; Anderson, Joshua C; Wagner, Frederic; Bosque, Alberto; Shishido, Takao; Jones, Jennifer; Planelles, Vicente; Willey, Christopher; Cron, Randall Q; Kutsch, Olaf

    2014-01-01

    Despite the clinical relevance of latent HIV-1 infection as a block to HIV-1 eradication, the molecular biology of HIV-1 latency remains incompletely understood. We recently demonstrated the presence of a gatekeeper kinase function that controls latent HIV-1 infection. Using kinase array analysis, we here expand on this finding and demonstrate that the kinase activity profile of latently HIV-1-infected T cells is altered relative to that of uninfected T cells. A ranking of altered kinases generated from these kinome profile data predicted PIM-1 kinase as a key switch involved in HIV-1 latency control. Using genetic and pharmacologic perturbation strategies, we demonstrate that PIM-1 activity is indeed required for HIV-1 reactivation in T cell lines and primary CD4 T cells. The presented results thus confirm that kinases are key contributors to HIV-1 latency control. In addition, through mutational studies we link the inhibitory effect of PIM-1 inhibitor IV (PIMi IV) on HIV-1 reactivation to an AP-1 motif in the CD28-responsive element of the HIV-1 long terminal repeat (LTR). The results expand our conceptual understanding of the dynamic interactions of the host cell and the latent HIV-1 integration event and position kinome profiling as a research tool to reveal novel molecular mechanisms that can eventually be targeted to therapeutically trigger HIV-1 reactivation. PMID:24155393

  16. Recruitment of a SAP18-HDAC1 complex into HIV-1 virions and its requirement for viral replication.

    PubMed

    Sorin, Masha; Cano, Jennifer; Das, Supratik; Mathew, Sheeba; Wu, Xuhong; Davies, Kelvin P; Shi, Xuanling; Cheng, S-W Grace; Ott, David; Kalpana, Ganjam V

    2009-06-01

    HIV-1 integrase (IN) is a virally encoded protein required for integration of viral cDNA into host chromosomes. INI1/hSNF5 is a component of the SWI/SNF complex that interacts with HIV-1 IN, is selectively incorporated into HIV-1 (but not other retroviral) virions, and modulates multiple steps, including particle production and infectivity. To gain further insight into the role of INI1 in HIV-1 replication, we screened for INI1-interacting proteins using the yeast two-hybrid system. We found that SAP18 (Sin3a associated protein 18 kD), a component of the Sin3a-HDAC1 complex, directly binds to INI1 in yeast, in vitro and in vivo. Interestingly, we found that IN also binds to SAP18 in vitro and in vivo. SAP18 and components of a Sin3A-HDAC1 complex were specifically incorporated into HIV-1 (but not SIV and HTLV-1) virions in an HIV-1 IN-dependent manner. Using a fluorescence-based assay, we found that HIV-1 (but not SIV) virion preparations harbour significant deacetylase activity, indicating the specific recruitment of catalytically active HDAC into the virions. To determine the requirement of virion-associated HDAC1 to HIV-1 replication, an inactive, transdominant negative mutant of HDAC1 (HDAC1(H141A)) was utilized. Incorporation of HDAC1(H141A) decreased the virion-associated histone deacetylase activity. Furthermore, incorporation of HDAC1(H141A) decreased the infectivity of HIV-1 (but not SIV) virions. The block in infectivity due to virion-associated HDAC1(H141A) occurred specifically at the early reverse transcription stage, while entry of the virions was unaffected. RNA-interference mediated knock-down of HDAC1 in producer cells resulted in decreased virion-associated HDAC1 activity and a reduction in infectivity of these virions. These studies indicate that HIV-1 IN and INI1/hSNF5 bind SAP18 and selectively recruit components of Sin3a-HDAC1 complex into HIV-1 virions. Furthermore, HIV-1 virion-associated HDAC1 is required for efficient early post

  17. 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

  18. Phenotypic Correlates of HIV-1 Macrophage Tropism

    PubMed Central

    Arrildt, Kathryn T.; LaBranche, Celia C.; Joseph, Sarah B.; Dukhovlinova, Elena N.; Graham, William D.; Ping, Li-Hua; Schnell, Gretja; Sturdevant, Christa B.; Kincer, Laura P.; Mallewa, Macpherson; Heyderman, Robert S.; Van Rie, Annelies; Cohen, Myron S.; Spudich, Serena; Price, Richard W.; Montefiori, David C.

    2015-01-01

    ABSTRACT HIV-1 is typically CCR5 using (R5) and T cell tropic (T-tropic), targeting memory CD4+ T cells throughout acute and chronic infections. However, viruses can expand into alternative cells types. Macrophage-tropic (M-tropic) HIV-1 variants have evolved to infect macrophages, which have only low levels of surface CD4. Most M-tropic variants have been isolated from the central nervous system during late-stage chronic infection. We used the HIV-1 env genes of well-defined, subject-matched M-tropic and T-tropic viruses to characterize the phenotypic features of the M-tropic Env protein. We found that, compared to T-tropic viruses, M-tropic viruses infect monocyte-derived macrophages (MDMs) on average 28-fold more efficiently, use low-density CD4 more efficiently, have increased sensitivity to soluble CD4 (sCD4), and show trends toward sensitivity to some CD4 binding site antibodies but no difference in sensitivity to antibodies targeting the CD4-bound conformation. M-tropic viruses also displayed a trend toward resistance to neutralization by monoclonal antibodies targeting the V1/V2 region of Env, suggesting subtle changes in Env protein conformation. The paired M- and T-tropic viruses did not differ in autologous serum neutralization, temperature sensitivity, entry kinetics, intrinsic infectivity, or Env protein incorporation. We also examined viruses with modestly increased CD4 usage. These variants have significant sensitivity to sCD4 and may represent evolutionary intermediates. CD4 usage is strongly correlated with infectivity of MDMs over a wide range of CD4 entry phenotypes. These data suggest that emergence of M-tropic HIV-1 includes multiple steps in which a phenotype of increased sensitivity to sCD4 and enhanced CD4 usage accompany subtle changes in Env conformation. IMPORTANCE HIV-1 typically replicates in CD4+ T cells. However, HIV-1 can evolve to infect macrophages, especially within the brain. Understanding how CCR5-using macrophage-tropic viruses

  19. Novel mechanism and factor for regulation by HIV-1 Tat.

    PubMed Central

    Zhou, Q; Sharp, P A

    1995-01-01

    Tat regulation of human immunodeficiency virus (HIV) transcription is unique because of its specificity for an RNA target, TAR, and its ability to increase the efficiency of elongation by polymerase. A reconstituted reaction that is Tat-specific and TAR-dependent for activation of HIV transcription has been used to identify and partially purify a cellular activity that is required for trans-activation by Tat, but not by other activators. In the reaction, Tat stimulates the efficiency of elongation by polymerase, whereas Sp1 and other DNA sequence-specific transcription factors activate the rate of initiation. Furthermore, while TATA binding protein (TBP)-associated factors (TAFs) in the TFIID complex are required for activation by transcription factors, they are dispensable for Tat function. Thus, Tat acts through a novel mechanism, which is mediated by a specific host cellular factor, to stimulate HIV-1 gene expression. Images PMID:7835343

  20. HIV-1 Quasispecies Delineation by Tag Linkage Deep Sequencing

    PubMed Central

    Wu, Nicholas C.; De La Cruz, Justin; Al-Mawsawi, Laith Q.; Olson, C. Anders; Qi, Hangfei; Luan, Harding H.; Nguyen, Nguyen; Du, Yushen; Le, Shuai; Wu, Ting-Ting; Li, Xinmin; Lewis, Martha J.; Yang, Otto O.; Sun, Ren

    2014-01-01

    Trade-offs between throughput, read length, and error rates in high-throughput sequencing limit certain applications such as monitoring viral quasispecies. Here, we describe a molecular-based tag linkage method that allows assemblage of short sequence reads into long DNA fragments. It enables haplotype phasing with high accuracy and sensitivity to interrogate individual viral sequences in a quasispecies. This approach is demonstrated to deduce ∼2000 unique 1.3 kb viral sequences from HIV-1 quasispecies in vivo and after passaging ex vivo with a detection limit of ∼0.005% to ∼0.001%. Reproducibility of the method is validated quantitatively and qualitatively by a technical replicate. This approach can improve monitoring of the genetic architecture and evolution dynamics in any quasispecies population. PMID:24842159

  1. Increased Risk of HIV-1 Transmission in Pregnancy: A Prospective Study among African HIV-1 Serodiscordant Couples

    PubMed Central

    MUGO, Nelly R.; HEFFRON, Renee; DONNELL, Deborah; WALD, Anna; WERE, Edwin O.; REES, Helen; CELUM, Connie; KIARIE, James N.; COHEN, Craig R.; KAYINTEKORE, Kayitesi; BAETEN, Jared M.

    2011-01-01

    Background Physiologic and behavioral changes during pregnancy may alter HIV-1 susceptibility and infectiousness. Prospective studies exploring pregnancy and HIV-1 acquisition risk in women have found inconsistent results. No study has explored the effect of pregnancy on HIV-1 transmission risk from HIV-1 infected women to male partners. Methods In a prospective study of African HIV-1 serodiscordant couples, we evaluated the relationship between pregnancy and the risk of 1) HIV-1 acquisition among women and 2) HIV-1 transmission from women to men. Results 3321 HIV-1 serodiscordant couples were enrolled, 1085 (32.7%) with HIV-1 susceptible female partners and 2236 (67.3%) with susceptible male partners. HIV-1 incidence in women was 7.35 versus 3.01 per 100 person-years during pregnant and non-pregnant periods (hazard ratio [HR] 2.34, 95% confidence interval [CI] 1.33–4.09). This effect was attenuated and not statistically significant after adjusting for sexual behavior and other confounding factors (adjusted HR 1.71, 95% CI 0.93–3.12). HIV-1 incidence in male partners of infected women was 3.46 versus 1.58 per 100 person-years when their partners were pregnant versus not pregnant (HR 2.31, 95% CI 1.22–4.39). This effect was not attenuated in adjusted analysis (adjusted HR 2.47, 95% CI 1.26–4.85). Conclusions HIV-1 risk increased two-fold during pregnancy. Elevated risk of HIV-1 acquisition in pregnant women appeared in part to be explained by behavioral and other factors. This is the first study to show pregnancy increased the risk of female-to-male HIV-1 transmission, which may reflect biological changes of pregnancy that could increase HIV-1 infectiousness. PMID:21785321

  2. HIV-1 Virus-Like Particles Produced by Stably Transfected Drosophila S2 Cells: a Desirable Vaccine Component

    PubMed Central

    Yang, Lifei; Song, Yufeng; Li, Xiaomin; Huang, Xiaoxing; Liu, Jingjing; Ding, Heng; Zhu, Ping

    2012-01-01

    The development of a successful vaccine against human immunodeficiency virus type 1 (HIV-1) likely requires immunogens that elicit both broadly neutralizing antibodies against envelope spikes and T cell responses that recognize multiple viral proteins. HIV-1 virus-like particles (VLP), because they display authentic envelope spikes on the particle surface, may be developed into such immunogens. However, in one way or the other current systems for HIV-1 VLP production have many limitations. To overcome these, in the present study we developed a novel strategy to produce HIV-1 VLP using stably transfected Drosophila S2 cells. We cotransfected S2 cells with plasmids encoding HIV-1 envelope, Gag, and Rev proteins and a selection marker. After stably transfected S2 clones were established, HIV-1 VLP and their immunogenicity in mice were carefully evaluated. Here, we report that HIV-1 envelope proteins are properly cleaved, glycosylated, and incorporated into VLP with Gag. The amount of VLP released into culture supernatants is comparable to those produced by insect cells infected with recombinant baculoviruses. Moreover, cryo-electron microscopy tomography revealed average 17 spikes per purified VLP, and antigenic epitopes on the spikes were recognized by the broadly neutralizing antibodies 2G12, b12, VRC01, and 4E10 but not by PG16. Finally, mice primed with DNA and boosted with VLP in the presence of CpG exhibited anti-envelope antibody responses, including ELISA-binding, neutralizing, antibody-dependent cell-mediated cytotoxicity and antibody-dependent cell-mediated viral inhibition, as well as envelope and Gag-specific CD8 T cell responses. Thus, we conclude that HIV-1 VLP produced by the S2 expression system has many desirable features to be developed into a vaccine component against HIV-1. PMID:22553333

  3. Development of a novel anti-HIV-1 agent from within: Effect of chimeric Vpr-containing protease cleavage site residues on virus replication

    PubMed Central

    Serio, D.; Rizvi, T. A.; Cartas, M.; Kalyanaraman, V. S.; Weber, I. T.; Koprowski, H.; Srinivasan, A.

    1997-01-01

    Effective antiviral agents will be of great value in controlling virus replication and delaying the onset of HIV-1-related disease symptoms. Current therapy involves the use of antiviral agents that target the enzymatic functions of the virus, resulting in the emergence of resistant viruses to these agents, thus lowering their effectiveness. To overcome this problem, we have considered the idea of developing novel agents from within HIV-1 as inhibitors of virus replication. The specificity of the Vpr protein for the HIV-1 virus particle makes it an attractive molecule for the development of antiviral agents targeting the events associated with virus maturation. We have generated chimeric Vpr proteins containing HIV-1-specific sequences added to the C terminus of Vpr. These sequences correspond to nine cleavage sites of the Gag and Gag–Pol precursors of HIV-1. The chimeric Vpr constructs were introduced into HIV-1 proviral DNA to assess their effect on virus infectivity using single- and multiple-round replication assays. The virus particles generated exhibited a variable replication pattern depending on the protease cleavage site used as a fusion partner. Interestingly, the chimeric Vpr containing the cleavage sequences from the junction of p24 and p2, 24/2, completely abolished virus infectivity. These results show that chimeric proteins generated from within HIV-1 have the ability to suppress HIV-1 replication and make ideal agents for gene therapy or intracellular immunization to treat HIV-1 infection. PMID:9096396

  4. HIV-1 and herpes simplex virus type-2 genital shedding among co-infected women using self-collected swabs in Chiang Rai, Thailand.

    PubMed

    Forhan, S E; Dunne, E F; Sternberg, M R; Whitehead, S J; Leelawiwat, W; Thepamnuay, S; Chen, C; Evans-Strickfaden, Tt; McNicholl, J M; Markowitz, L E

    2012-08-01

    We analysed 528 genital self-collected swabs (SCS) from 67 HIV-1 and herpes simplex virus type-2 (HSV-2) co-infected women collected during the placebo month of a randomized crossover clinical trial of suppressive acyclovir in Chiang Rai, Thailand. In this first longitudinal study of HIV-1 and HSV-2 co-infected women using genital SCS specimens, we found frequent mucosal HIV-1 shedding. Overall, 372 (70%) swabs had detectable HIV-1 RNA with median HIV-1 viral load of 2.61 log(10) copies/swab. We found no statistically significant association between detectable HIV-1 RNA and HSV-2 DNA in the same SCS specimen (adjusted odds ratio [aOR] 1.40; 95% confidence intervals [CI], 0.78-2.60, P = 0.25). Only baseline HIV-1 plasma viral load was independently associated with genital HIV-1 RNA shedding (aOR, 7.6; 95% CI, 3.3-17.2, P < 0.0001). SCS may be useful for future HIV-1 and HSV-2 studies because this method allows for frequent genital sampling, and inclusion of genital sites other than the cervix. PMID:22930292

  5. Productive Replication and Evolution of HIV-1 in Ferret Cells

    PubMed Central

    Fadel, Hind J.; Saenz, Dyana T.; Guevara, Rebekah; von Messling, Veronika; Peretz, Mary

    2012-01-01

    A rodent or other small animal model for HIV-1 has not been forthcoming, with the principal obstacles being species-specific restriction mechanisms and deficits in HIV-1 dependency factors. Some Carnivorans may harbor comparatively fewer impediments. For example, in contrast to mice, the domestic cat genome encodes essential nonreceptor HIV-1 dependency factors. All Feliformia species and at least one Caniformia species also lack a major lentiviral restriction mechanism (TRIM5α/TRIMCyp proteins). Here we investigated cells from two species in another carnivore family, the Mustelidae, for permissiveness to the HIV-1 life cycle. Mustela putorius furo (domesticated ferret) primary cells and cell lines did not restrict HIV-1, feline immunodeficiency virus (FIV), equine infectious anemia virus (EIAV), or N-tropic murine leukemia virus (MLV) postentry and supported late HIV-1 life cycle steps comparably to human cells. The ferret TRIM5α gene exon 8, which encodes the B30.2 domain, was found to be pseudogenized. Strikingly, ferret (but not mink) cells engineered to express human HIV-1 entry receptors supported productive spreading replication, amplification, and serial passage of wild-type HIV-1. Nevertheless, produced virions had relatively reduced infectivity and the virus accrued G→A hypermutations, consistent with APOBEC3 protein pressure. Ferret cell-passaged HIV-1 also evolved amino acid changes in the capsid cyclophilin A binding loop. We conclude that the genome of this carnivore can provide essential nonreceptor HIV-1 dependency factors and that ferret APOBEC3 proteins with activity against HIV-1 are likely. Even so, unlike in cat cells, HIV-1 can replicate in ferret cells without vif substitution. The virus evolves in this novel nonprimate cell adaptive landscape. We suggest that further characterization of HIV-1 adaptation in ferret cells and delineation of Mustelidae restriction factor repertoires are warranted, with a view to the potential for an HIV-1

  6. Long-Term Antiretroviral Treatment Initiated at Primary HIV-1 Infection Affects the Size, Composition, and Decay Kinetics of the Reservoir of HIV-1-Infected CD4 T Cells

    PubMed Central

    Buzon, Maria J.; Martin-Gayo, Enrique; Pereyra, Florencia; Ouyang, Zhengyu; Sun, Hong; Li, Jonathan Z.; Piovoso, Michael; Shaw, Amy; Dalmau, Judith; Zangger, Nadine; Martinez-Picado, Javier; Zurakowski, Ryan; Yu, Xu G.; Telenti, Amalio; Walker, Bruce D.; Rosenberg, Eric S.

    2014-01-01

    ABSTRACT Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1

  7. HIV-1 VACCINES. HIV-1 neutralizing antibodies induced by native-like envelope trimers.

    PubMed

    Sanders, Rogier W; van Gils, Marit J; Derking, Ronald; Sok, Devin; Ketas, Thomas J; Burger, Judith A; Ozorowski, Gabriel; Cupo, Albert; Simonich, Cassandra; Goo, Leslie; Arendt, Heather; Kim, Helen J; Lee, Jeong Hyun; Pugach, Pavel; Williams, Melissa; Debnath, Gargi; Moldt, Brian; van Breemen, Mariëlle J; Isik, Gözde; Medina-Ramírez, Max; Back, Jaap Willem; Koff, Wayne C; Julien, Jean-Philippe; Rakasz, Eva G; Seaman, Michael S; Guttman, Miklos; Lee, Kelly K; Klasse, Per Johan; LaBranche, Celia; Schief, William R; Wilson, Ian A; Overbaugh, Julie; Burton, Dennis R; Ward, Andrew B; Montefiori, David C; Dean, Hansi; Moore, John P

    2015-07-10

    A challenge for HIV-1 immunogen design is the difficulty of inducing neutralizing antibodies (NAbs) against neutralization-resistant (tier 2) viruses that dominate human transmissions. We show that a soluble recombinant HIV-1 envelope glycoprotein trimer that adopts a native conformation, BG505 SOSIP.664, induced NAbs potently against the sequence-matched tier 2 virus in rabbits and similar but weaker responses in macaques. The trimer also consistently induced cross-reactive NAbs against more sensitive (tier 1) viruses. Tier 2 NAbs recognized conformational epitopes that differed between animals and in some cases overlapped with those recognized by broadly neutralizing antibodies (bNAbs), whereas tier 1 responses targeted linear V3 epitopes. A second trimer, B41 SOSIP.664, also induced a strong autologous tier 2 NAb response in rabbits. Thus, native-like trimers represent a promising starting point for the development of HIV-1 vaccines aimed at inducing bNAbs. PMID:26089353

  8. Immunodominant HIV-1 Cd4+ T Cell Epitopes in Chronic Untreated Clade C HIV-1 Infection

    PubMed Central

    Ramduth, Danni; Day, Cheryl L.; Thobakgale, Christina F.; Mkhwanazi, Nompumelelo P.; de Pierres, Chantal; Reddy, Sharon; van der Stok, Mary; Mncube, Zenele; Nair, Kriebashne; Moodley, Eshia S.; Kaufmann, Daniel E.; Streeck, Hendrik; Coovadia, Hoosen M.; Kiepiela, Photini; Goulder, Philip J. R.; Walker, Bruce D.

    2009-01-01

    Background A dominance of Gag-specific CD8+ T cell responses is significantly associated with a lower viral load in individuals with chronic, untreated clade C human immunodeficiency virus type 1 (HIV-1) infection. This association has not been investigated in terms of Gag-specific CD4+ T cell responses, nor have clade C HIV-1–specific CD4+ T cell epitopes, likely a vital component of an effective global HIV-1 vaccine, been identified. Methodology/Principal Findings Intracellular cytokine staining was conducted on 373 subjects with chronic, untreated clade C infection to assess interferon-gamma (IFN-γ) responses by CD4+ T cells to pooled Gag peptides and to determine their association with viral load and CD4 count. Gag-specific IFN-γ–producing CD4+ T cell responses were detected in 261/373 (70%) subjects, with the Gag responders having a significantly lower viral load and higher CD4 count than those with no detectable Gag response (p<0.0001 for both parameters). To identify individual peptides targeted by HIV-1–specific CD4+ T cells, separate ELISPOT screening was conducted on CD8-depleted PBMCs from 32 chronically infected untreated subjects, using pools of overlapping peptides that spanned the entire HIV-1 clade C consensus sequence, and reconfirmed by flow cytometry to be CD4+ mediated. The ELISPOT screening identified 33 CD4+ peptides targeted by 18/32 patients (56%), with 27 of the 33 peptides located in the Gag region. Although the breadth of the CD4+ responses correlated inversely with viral load (p = 0.015), the magnitude of the response was not significantly associated with viral load. Conclusions/Significance These data indicate that in chronic untreated clade C HIV-1 infection, IFN-γ–secreting Gag-specific CD4+ T cell responses are immunodominant, directed at multiple distinct epitopes, and associated with viral control. PMID:19352428

  9. A Directed Molecular Evolution Approach to Improved Immunogenicity of the HIV-1 Envelope Glycoprotein

    PubMed Central

    Du, Sean X.; Xu, Li; Zhang, Wenge; Tang, Susan; Boenig, Rebecca I.; Chen, Helen; Mariano, Ellaine B.; Zwick, Michael B.; Parren, Paul W. H. I.; Burton, Dennis R.; Wrin, Terri; Petropoulos, Christos J.; Ballantyne, John A.; Chambers, Michael; Whalen, Robert G.

    2011-01-01

    A prophylactic vaccine is needed to slow the spread of HIV-1 infection. Optimization of the wild-type envelope glycoproteins to create immunogens that can elicit effective neutralizing antibodies is a high priority. Starting with ten genes encoding subtype B HIV-1 gp120 envelope glycoproteins and using in vitro homologous DNA recombination, we created chimeric gp120 variants that were screened for their ability to bind neutralizing monoclonal antibodies. Hundreds of variants were identified with novel antigenic phenotypes that exhibit considerable sequence diversity. Immunization of rabbits with these gp120 variants demonstrated that the majority can induce neutralizing antibodies to HIV-1. One novel variant, called ST-008, induced significantly improved neutralizing antibody responses when assayed against a large panel of primary HIV-1 isolates. Further study of various deletion constructs of ST-008 showed that the enhanced immunogenicity results from a combination of effective DNA priming, an enhanced V3-based response, and an improved response to the constant backbone sequences. PMID:21738594

  10. Similarities in the HIV-1 and ASV Integrease Active Site Upon Metal Binding

    SciTech Connect

    Lins, Roberto D.; Straatsma, TP; Briggs, J. M.

    2000-04-05

    The HIV-1 integrase, which is essential for viral replication, catalyzes the insertion of viral DNA into the host chromosome thereby recruiting host cell machinery into making viral proteins. It represents the third main HIV enzyme target for inhibitor design, the first two being the reverse transcriptase and the protease. We report here a fully hydrated 2 ns molecular dynamics simulation performed using parallel NWChem3.2.1 with the AMBER95 force field. The HIV-1 integrase catalytic domain previously determined by crystallography (1B9D) and modeling including two Mg2+ ions placed into the active site based on an alignment against an ASV integrase structure containing two divalent metals (1VSH), was used as the starting structure. The simulation reveals a high degree of flexibility in the region of residues 140-149 even in the presence of a second divalent metal ion and a dramatic conformational change of the side chain of E152 when the second metal ion is present. This study shows similarities in the behavior of the catalytic residues in the HIV-1 and ASV integrases upon metal binding. The present simulation also provides support to the hypothesis that the second metal ion is likely to be carried into the HIV-1 integrase active site by the substrate, a strand of DNA.

  11. Acceleration of age-associated methylation patterns in HIV-1-infected adults.

    PubMed

    Rickabaugh, Tammy M; Baxter, Ruth M; Sehl, Mary; Sinsheimer, Janet S; Hultin, Patricia M; Hultin, Lance E; Quach, Austin; Martínez-Maza, Otoniel; Horvath, Steve; Vilain, Eric; Jamieson, Beth D

    2015-01-01

    Patients with treated HIV-1-infection experience earlier occurrence of aging-associated diseases, raising speculation that HIV-1-infection, or antiretroviral treatment, may accelerate aging. We recently described an age-related co-methylation module comprised of hundreds of CpGs; however, it is unknown whether aging and HIV-1-infection exert negative health effects through similar, or disparate, mechanisms. We investigated whether HIV-1-infection would induce age-associated methylation changes. We evaluated DNA methylation levels at >450,000 CpG sites in peripheral blood mononuclear cells (PBMC) of young (20-35) and older (36-56) adults in two separate groups of participants. Each age group for each data set consisted of 12 HIV-1-infected and 12 age-matched HIV-1-uninfected samples for a total of 96 samples. The effects of age and HIV-1 infection on methylation at each CpG revealed a strong correlation of 0.49, p<1 x 10(-200) and 0.47, p<1 x 10(-200). Weighted gene correlation network analysis (WGCNA) identified 17 co-methylation modules; module 3 (ME3) was significantly correlated with age (cor=0.70) and HIV-1 status (cor=0.31). Older HIV-1+ individuals had a greater number of hypermethylated CpGs across ME3 (p=0.015). In a multivariate model, ME3 was significantly associated with age and HIV status (Data set 1: βage=0.007088, p=2.08 x 10(-9); βHIV=0.099574, p=0.0011; Data set 2: βage=0.008762, p=1.27 x 10(-5); βHIV=0.128649, p=0.0001). Using this model, we estimate that HIV-1 infection accelerates age-related methylation by approximately 13.7 years in data set 1 and 14.7 years in data set 2. The genes related to CpGs in ME3 are enriched for polycomb group target genes known to be involved in cell renewal and aging. The overlap between ME3 and an aging methylation module found in solid tissues is also highly significant (Fisher-exact p=5.6 x 10(-6), odds ratio=1.91). These data demonstrate that HIV-1 infection is associated with methylation patterns that are

  12. Selection of 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamers that bind HIV-1 reverse transcriptase with picomolar affinity

    PubMed Central

    Alves Ferreira-Bravo, Irani; Cozens, Christopher; Holliger, Philipp; DeStefano, Jeffrey J.

    2015-01-01

    Using a Systematic Evolution of Ligands by Exponential Enrichment (SELEX) protocol capable of selecting xeno-nucleic acid (XNA) aptamers, a 2′-deoxy-2′-fluoroarabinonucleotide (FANA) aptamer (referred to as FA1) to HIV-1 reverse transcriptase (HIV-1 RT) was selected. FA1 bound HIV-1 RT with KD,app values in the low pM range under different ionic conditions. Comparisons to published HIV-1 RT RNA and DNA aptamers indicated that FA1 bound at least as well as these aptamers. FA1 contained a 20 nucleotide 5′ DNA sequence followed by a 57 nucleotide region of FANA nucleotides. Removal of the fourteen 5′ DNA nucleotides did not affect binding. FA1's predicted structure was composed of four stems and four loops. All stem nucleotides could be modified to G-C base pairs (14 total changes) with a small effect on binding. Eliminating or altering most loop sequences reduced or abolished tight binding. Overall, results suggested that the structure and the sequence of FA1 were important for binding. FA1 showed strong inhibition of HIV-1 RT in extension assays while no specific binding to avian myeloblastosis or Moloney murine leukemia RTs was detected. A complete DNA version of FA1 showed low binding to HIV-1 RT, emphasizing the unique properties of FANA in HIV-1 RT binding. PMID:26476448

  13. HIV-1 Protease, Reverse Transcriptase, and Integrase Variation

    PubMed Central

    Sankaran, Kris; Varghese, Vici; Winters, Mark A.; Hurt, Christopher B.; Eron, Joseph J.; Parkin, Neil; Holmes, Susan P.; Holodniy, Mark; Shafer, Robert W.

    2016-01-01

    ABSTRACT HIV-1 protease (PR), reverse transcriptase (RT), and integrase (IN) variability presents a challenge to laboratories performing genotypic resistance testing. This challenge will grow with increased sequencing of samples enriched for proviral DNA such as dried blood spots and increased use of next-generation sequencing (NGS) to detect low-abundance HIV-1 variants. We analyzed PR and RT sequences from >100,000 individuals and IN sequences from >10,000 individuals to characterize variation at each amino acid position, identify mutations indicating APOBEC-mediated G-to-A editing, and identify mutations resulting from selective drug pressure. Forty-seven percent of PR, 37% of RT, and 34% of IN positions had one or more amino acid variants with a prevalence of ≥1%. Seventy percent of PR, 60% of RT, and 60% of IN positions had one or more variants with a prevalence of ≥0.1%. Overall 201 PR, 636 RT, and 346 IN variants had a prevalence of ≥0.1%. The median intersubtype prevalence ratios were 2.9-, 2.1-, and 1.9-fold for these PR, RT, and IN variants, respectively. Only 5.0% of PR, 3.7% of RT, and 2.0% of IN variants had a median intersubtype prevalence ratio of ≥10-fold. Variants at lower prevalences were more likely to differ biochemically and to be part of an electrophoretic mixture compared to high-prevalence variants. There were 209 mutations indicative of APOBEC-mediated G-to-A editing and 326 mutations nonpolymorphic treatment selected. Identification of viruses with a high number of APOBEC-associated mutations will facilitate the quality control of dried blood spot sequencing. Identifying sequences with a high proportion of rare mutations will facilitate the quality control of NGS. IMPORTANCE Most antiretroviral drugs target three HIV-1 proteins: PR, RT, and IN. These proteins are highly variable: many different amino acids can be present at the same position in viruses from different individuals. Some of the amino acid variants cause drug

  14. An empiric risk scoring tool for identifying high-risk heterosexual HIV-1 serodiscordant couples for targeted HIV-1 prevention

    PubMed Central

    KAHLE, Erin M.; HUGHES, James P.; LINGAPPA, Jairam R.; JOHN-STEWART, Grace; CELUM, Connie; NAKKU-JOLOBA, Edith; NJUGUNA, Stella; MUGO, Nelly; BUKUSI, Elizabeth; MANONGI, Rachel; BAETEN, Jared M.

    2012-01-01

    Background and objectives Heterosexual HIV-1 serodiscordant couples are increasingly recognized as an important source of new HIV-1 infections in sub-Saharan Africa. A simple risk assessment tool could be useful for identifying couples at highest risk for HIV-1 transmission. Methods Using data from three prospective studies of HIV-1 serodiscordant couples from seven African countries and standard methods for development of clinical prediction rules, we derived and validated a risk scoring tool developed from multivariate modeling and composed of key predictors for HIV-1 risk that could be measured in standard research and clinical settings. Results The final risk score included age of the HIV-1 uninfected partner, married and/or cohabiting partnership, number of children, unprotected sex, uncircumcised male HIV-1 uninfected partner, and plasma HIV-1 RNA in the HIV-1 infected partner. The maximum risk score was 12, scores ≥5 were associated with an annual HIV-1 incidence of >3%, and couples with a score ≥6 accounted for only 28% of the population but 67% of HIV-1 transmissions. The area under the curve for predictive ability of the score was 0.74 (95% CI 0.70–0.78). Internal and external validation showed similar predictive ability of the risk score, even when plasma viral load was excluded from the risk score. Conclusions A discrete combination of clinical and behavioral characteristics defines highest-risk HIV-1 serodiscordant couples. Discriminating highest-risk couples for HIV-1 prevention programs and clinical trials using a validated risk score could improve research efficiency and maximize the impact of prevention strategies for reducing HIV-1 transmission. PMID:23187945

  15. Synaptodendritic recovery following HIV-1 Tat exposure: Neurorestoration by phytoestrogens

    PubMed Central

    Bertrand, SJ; Mactutus, CF; Aksenova, MV; Espensen-Sturges, TD; Booze, RM

    2013-01-01

    HIV-1 infects the brain and, despite antiretroviral therapy, many infected individuals suffer from HIV-1-associated neurocognitive disorders (HAND). HAND is associated with dendritic simplification and synaptic loss. Prevention of synaptodendritic damage may ameliorate or forestall neurocognitive decline in latent HIV-1 infections. The HIV-1 transactivating protein (Tat) is produced during viral latency in the brain and may cause synaptodendritic damage. The present study examined the integrity of the dendritic network after exposure to HIV-1 Tat by labeling filamentous actin (F-actin)-rich structures (puncta) in primary neuronal cultures. After 24 hours of treatment, HIV-1 Tat was associated with the dendritic arbor and produced a significant reduction of F-actin-labeled dendritic puncta as well as loss of dendrites. Pretreatment with either of two plant-derived phytoestrogen compounds (daidzein and liquiritigenin), significantly reduced synaptodendritic damage following HIV-1 Tat treatment. Additionally, 6 days after HIV-1 Tat treatment, treatment with either daidzein or liquiritigenin enhanced recovery, via the estrogen receptor, from HIV-1 Tat-induced synaptodendritic damage. These results suggest that either liquiritigenin or daidzein may not only attenuate acute synaptodendritic injury in HIV-1, but also promote recovery from synaptodendritic damage. PMID:23875777

  16. The in vitro Biochemical Characterization of an HIV-1 Restriction Factor APOBEC3F: Importance of Loop 7 on Both CD1 and CD2 for DNA Binding and Deamination.

    PubMed

    Chen, Qihan; Xiao, Xiao; Wolfe, Aaron; Chen, Xiaojiang S

    2016-07-01

    APOBEC3F (A3F) is a member of the apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like (APOBEC) family of proteins that can deaminate cytosine (C) to uracil (U) on nucleic acids. A3F is one of the four APOBEC members with two Zn-coordinated homologous cytosine deaminase (CD) domains, with the others being A3G, A3D, and A3B. Here we report the in vitro characterization of DNA binding and deaminase activities using purified wild-type and various mutant proteins of A3F from an Escherichia coli expression system. We show that even though CD1 is catalytically inactive and CD2 is the active deaminase domain, presence of CD1 on the N-terminus of CD2 enhances the deaminase activity by over an order of magnitude. This enhancement of CD2 catalytic activity is mainly through the increase of substrate single-stranded (ss) DNA binding by the N-terminal CD1 domain. We further show that the loop 7 of both CD1 and CD2 of A3F plays an important role for ssDNA binding for each individual domain, as well as for the deaminase activity of CD2 domain in the full-length A3F. PMID:27063502

  17. Nicotine mediates expression of genes related to antioxidant capacity and oxidative stress response in HIV-1 transgenic rat brain.

    PubMed

    Song, Guohua; Nesil, Tanseli; Cao, Junran; Yang, Zhongli; Chang, Sulie L; Li, Ming D

    2016-02-01

    Oxidative stress plays an important role in the progression of HIV-1 infection. Nicotine can either protect neurons from neurodegeneration or induce oxidative stress, depending on its dose and degree of oxidative stress impairment. However, the relationship between nicotine and oxidative stress in the HIV-1-infected individuals remains largely unknown. The purpose of this study was to determine the effect of nicotine on expression of genes related to the glutathione (GSH)-centered antioxidant system and oxidative stress in the nucleus accumbens (NAc) and ventral tegmental area (VTA) of HIV-1 transgenic (HIV-1Tg) and F344 control rats. Adult HIV-1Tg and F344 rats received nicotine (0.4 mg/kg, base, s.c.) or saline injections once per day for 27 days. At the end of treatment, various brain regions including the NAc and VTA were collected from each rat. Following total RNA extraction and complementary DNA (cDNA) synthesis of each sample, quantitative reverse transcription PCR (RT-PCR) analysis was performed for 43 oxidative-stress-related genes. Compared with F344 control rats, HIV-1Tg rats showed a significant downregulation of genes involved in ATPase and cyctochrome oxidase at the messenger RNA (mRNA) level in both regions. Further, we found a significant downregulation of Gstm5 in the NAc and upregulation of Cox1, Cox3, and Gsta6 in the VTA of HIV-1Tg rats. HIV-1Tg rats showed brain-region-specific responses to chronic nicotine treatment. This response resulted in a change in the expression of genes involved in antioxidant mechanisms including the downregulation of genes such as Atp5h, Calml1, Gpx7, Gstm5, Gsr, and Gsta6 and upregulation of Sod1 in the NAc, as well as downregulation of genes like Cox5a, Gpx4, Gpx6, Gpx7, Gstm5, and Sod1 in the VTA of HIV-1Tg rats. Together, we conclude that chronic nicotine treatment has a dual effect on the antioxidant defense system and oxidative-stress-induced apoptosis signaling in HIV-1Tg rats. These findings suggest that

  18. High Degree of HIV-1 Group M (HIV-1M) Genetic Diversity within Circulating Recombinant Forms: Insight into the Early Events of HIV-1M Evolution.

    PubMed

    Tongo, Marcel; Dorfman, Jeffrey R; Martin, Darren P

    2016-03-01

    The existence of various highly divergent HIV-1 lineages and of recombination-derived sequence tracts of indeterminate origin within established circulating recombinant forms (CRFs) strongly suggests that HIV-1 group M (HIV-1M) diversity is not fully represented under the current classification system. Here we used a fully exploratory screen for recombination on a set of 480 near-full-length genomes representing the full known diversity of HIV-1M. We decomposed recombinant sequences into their constituent parts and then used maximum-likelihood phylogenetic analyses of this mostly recombination-free data set to identify rare divergent sequence lineages that fall outside the major named HIV-1M taxonomic groupings. We found that many of the sequence fragments occurring within CRFs (including CRF04_cpx, CRF06_cpx, CRF11_cpx, CRF18_cpx, CRF25_cpx, CRF27_cpx, and CRF49_cpx) are in fact likely derived from divergent unclassified parental lineages that may predate the current subtypes, even though they are presently identified as derived from currently defined HIV-1M subtypes. Our evidence suggests that some of these CRFs are descended predominantly from what were or are major previously unidentified HIV-1M lineages that were likely epidemiologically relevant during the early stages of the HIV-1M epidemic. The restriction of these divergent lineages to the Congo basin suggests that they were less infectious and/or simply not present at the time and place of the initial migratory wave that triggered the global epidemic.IMPORTANCE HIV-1 group M (HIV-1M) likely spread to the rest of the world from the Congo basin in the mid-1900s (N. R. Faria et al., Science 346:56-61, 2014, http://dx.doi.org/10.1126/science.1256739) and is today the principal cause of the AIDS pandemic. Here, we show that large sequence fragments from several HIV-1M circulating recombinant forms (CRFs) are derived from divergent parental lineages that cannot reasonably be classified within the nine

  19. The Scaffolding Protein Dlg1 Is a Negative Regulator of Cell-Free Virus Infectivity but Not of Cell-to-Cell HIV-1 Transmission in T Cells

    PubMed Central

    Nzounza, Patrycja; Chazal, Maxime; Guedj, Chloé; Schmitt, Alain; Massé, Jean-Marc; Randriamampita, Clotilde; Pique, Claudine; Ramirez, Bertha Cecilia

    2012-01-01

    Background Cell-to-cell virus transmission of Human immunodeficiency virus type-1 (HIV-1) is predominantly mediated by cellular structures such as the virological synapse (VS). The VS formed between an HIV-1-infected T cell and a target T cell shares features with the immunological synapse (IS). We have previously identified the human homologue of the Drosophila Discs Large (Dlg1) protein as a new cellular partner for the HIV-1 Gag protein and a negative regulator of HIV-1 infectivity. Dlg1, a scaffolding protein plays a key role in clustering protein complexes in the plasma membrane at cellular contacts. It is implicated in IS formation and T cell signaling, but its role in HIV-1 cell-to-cell transmission was not studied before. Methodology/Principal Findings Kinetics of HIV-1 infection in Dlg1-depleted Jurkat T cells show that Dlg1 modulates the replication of HIV-1. Single-cycle infectivity tests show that this modulation does not take place during early steps of the HIV-1 life cycle. Immunofluorescence studies of Dlg1-depleted Jurkat T cells show that while Dlg1 depletion affects IS formation, it does not affect HIV-1-induced VS formation. Co-culture assays and quantitative cell-to-cell HIV-1 transfer analyses show that Dlg1 depletion does not modify transfer of HIV-1 material from infected to target T cells, or HIV-1 transmission leading to productive infection via cell contact. Dlg1 depletion results in increased virus yield and infectivity of the viral particles produced. Particles with increased infectivity present an increase in their cholesterol content and during the first hours of T cell infection these particles induce higher accumulation of total HIV-1 DNA. Conclusion Despite its role in the IS formation, Dlg1 does not affect the VS and cell-to-cell spread of HIV-1, but plays a role in HIV-1 cell-free virus transmission. We propose that the effect of Dlg1 on HIV-1 infectivity is at the stage of virus entry. PMID:22272285

  20. Fetal exposure to HIV-1 alters chemokine receptor expression by CD4+T cells and increases susceptibility to HIV-1.

    PubMed

    Bunders, Madeleine J; van Hamme, John L; Jansen, Machiel H; Boer, Kees; Kootstra, Neeltje A; Kuijpers, Taco W

    2014-01-01

    Absolute numbers of lymphocytes are decreased in uninfected infants born to HIV-1-infected women (HIV-1-exposed). Although the exact mechanism is unknown, fetal exposure to maternal HIV-1-infection could prime the immune system and affect T cell trafficking. We compared the expression of chemokine receptors on cord blood CD4(+) T cells from HIV-1-exposed children and healthy controls. At baseline CD4(+) T cells had a largely naïve phenotype. However, stimulation with cytokines resulted in an upregulation of inflammatory response-related chemokine receptors on CD4(+) T cells, with HIV-1-exposed infants having a significantly higher frequency of CD4(+) T cells expressing, in particularly Th2 associated chemokine receptors (CCR3 p < 0.01, CCR8 p = 0.03). Numbers of naive CCR7(+) CD4(+) T cells were reduced (p = 0.01) in HIV-1-exposed infants. We further assessed whether the inflammatory phenotype was associated with susceptibility to HIV-1 and detected higher levels of p24 upon in in vitro infection of stimulated CD4(+) T cells of HIV-1-exposed infants. In summary, fetal exposure to HIV-1 primes the immune system in the infant leading to an enhanced immune activation and altered T cell homing, with potential ramifications regarding T cell responses and the acquisition of HIV-1 as an infant. PMID:25341640

  1. New Class of HIV-1 Integrase (IN) Inhibitors with a Dual Mode of Action

    PubMed Central

    Tsiang, Manuel; Jones, Gregg S.; Niedziela-Majka, Anita; Kan, Elaine; Lansdon, Eric B.; Huang, Wayne; Hung, Magdeleine; Samuel, Dharmaraj; Novikov, Nikolai; Xu, Yili; Mitchell, Michael; Guo, Hongyan; Babaoglu, Kerim; Liu, Xiaohong; Geleziunas, Romas; Sakowicz, Roman

    2012-01-01

    tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3′-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction. PMID:22535962

  2. New class of HIV-1 integrase (IN) inhibitors with a dual mode of action.

    PubMed

    Tsiang, Manuel; Jones, Gregg S; Niedziela-Majka, Anita; Kan, Elaine; Lansdon, Eric B; Huang, Wayne; Hung, Magdeleine; Samuel, Dharmaraj; Novikov, Nikolai; Xu, Yili; Mitchell, Michael; Guo, Hongyan; Babaoglu, Kerim; Liu, Xiaohong; Geleziunas, Romas; Sakowicz, Roman

    2012-06-15

    tert-Butoxy-(4-phenyl-quinolin-3-yl)-acetic acids (tBPQA) are a new class of HIV-1 integrase (IN) inhibitors that are structurally distinct from IN strand transfer inhibitors but analogous to LEDGINs. LEDGINs are a class of potent antiviral compounds that interacts with the lens epithelium-derived growth factor (LEDGF) binding pocket on IN and were identified through competition binding against LEDGF. LEDGF tethers IN to the host chromatin and enables targeted integration of viral DNA. The prevailing understanding of the antiviral mechanism of LEDGINs is that they inhibit LEDGF binding to IN, which prevents targeted integration of HIV-1. We showed that in addition to the properties already known for LEDGINs, the binding of tBPQAs to the IN dimer interface inhibits IN enzymatic activity in a LEDGF-independent manner. Using the analysis of two long terminal repeat junctions in HIV-infected cells, we showed that the inhibition by tBPQAs occurs at or prior to the viral DNA 3'-processing step. Biochemical studies revealed that this inhibition operates by compound-induced conformational changes in the IN dimer that prevent proper assembly of IN onto viral DNA. For the first time, tBPQAs were demonstrated to be allosteric inhibitors of HIV-1 IN displaying a dual mode of action: inhibition of IN-viral DNA assembly and inhibition of IN-LEDGF interaction. PMID:22535962

  3. Differential contribution of basic residues to HIV-1 nucleocapsid protein’s nucleic acid chaperone function and retroviral replication

    PubMed Central

    Wu, Hao; Mitra, Mithun; Naufer, M. Nabuan; McCauley, Micah J.; Gorelick, Robert J.; Rouzina, Ioulia; Musier-Forsyth, Karin; Williams, Mark C.

    2014-01-01

    The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) protein contains 15 basic residues located throughout its 55-amino acid sequence, as well as one aromatic residue in each of its two CCHC-type zinc finger motifs. NC facilitates nucleic acid (NA) rearrangements via its chaperone activity, but the structural basis for this activity and its consequences in vivo are not completely understood. Here, we investigate the role played by basic residues in the N-terminal domain, the N-terminal zinc finger and the linker region between the two zinc fingers. We use in vitro ensemble and single-molecule DNA stretching experiments to measure the characteristics of wild-type and mutant HIV-1 NC proteins, and correlate these results with cell-based HIV-1 replication assays. All of the cationic residue mutations lead to NA interaction defects, as well as reduced HIV-1 infectivity, and these effects are most pronounced on neutralizing all five N-terminal cationic residues. HIV-1 infectivity in cells is correlated most strongly with NC’s NA annealing capabilities as well as its ability to intercalate the DNA duplex. Although NC’s aromatic residues participate directly in DNA intercalation, our findings suggest that specific basic residues enhance these interactions, resulting in optimal NA chaperone activity. PMID:24293648

  4. A study of the topoisomerase II activity in HIV-1 replication using the ferrocene derivatives as probes.

    PubMed

    Kondapi, Anand K; Satyanarayana, Nathamu; Saikrishna, A D

    2006-06-15

    Human Topoisomerase II is present in two isoforms, 170KDa alpha and 180KDa beta. Both the isoforms play a crucial role in maintenance of topological changes during DNA replication and recombination. It has been shown that Topoisomerase II activity is required for HIV-1 replication and the enzyme is phosphorylated during early time points of HIV-1 replication. In the present study, we have studied the molecular action of Topoisomerase II inhibitors, azalactone ferrocene (AzaFecp), Thiomorpholide amido methyl ferrocene (ThioFecp), and Ruthenium benzene amino pyridine (Ru(ben)Apy) on cell proliferation and also on various events of HIV-1 replication cycle. The Topoisomerase II beta over-expressing neuroblastoma cell line shows a higher sensitivity to these compounds compared to the Sup-T1 cell line. All the three Topoisomerase II inhibitors show significant anti-HIV activity at nanomolar concentrations against an Indian isolate of HIV-1(93IN101) in Sup-T1 cell line. An analysis of action of these compounds on proviral DNA synthesis at 5h of post-infection shows that they inhibit proviral DNA synthesis as well as the formation of pre-integration complexes completely. Further analysis, using polymerase chain reaction and western blot, showed that both the Topoisomerase II alpha and beta isoforms are present in the pre-integration complexes, suggesting their significant role in HIV-1 replication. PMID:16712776

  5. Potentiation of the immune response in HIV-1+ individuals.

    PubMed

    Schmitz, T; Underwood, R; Khiroya, R; Bachovchin, W W; Huber, B T

    1996-03-15

    T cells from HIV-1+ individuals have a defect in mounting an antigen specific response. HIV-1 Tat has been implicated as the causative agent of this immunosuppression. We have previously shown that HIV-1 Tat inhibits antigen specific proliferation of normal T cells in vitro by binding to the accessory molecule CD26, a dipeptidase expressed on the surface of activated T cells. We now demonstrate that the defective in vitro recall antigen response in HIV-1 infected individuals can be restored by the addition of soluble CD26, probably by serving as a decoy receptor for HIV-1 Tat. The restored response is comparable to that of an HIV-1- individual, suggesting that early in HIV infection there is a block in the memory cell response, rather than deletion of these cells. PMID:8617888

  6. Purinergic Receptors: Key Mediators of HIV-1 Infection and Inflammation.

    PubMed

    Swartz, Talia H; Dubyak, George R; Chen, Benjamin K

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) causes a chronic infection that afflicts more than 30 million individuals worldwide. While the infection can be suppressed with potent antiretroviral therapies, individuals infected with HIV-1 have elevated levels of inflammation as indicated by increased T cell activation, soluble biomarkers, and associated morbidity and mortality. A single mechanism linking HIV-1 pathogenesis to this inflammation has yet to be identified. Purinergic receptors are known to mediate inflammation and have been shown to be required for HIV-1 infection at the level of HIV-1 membrane fusion. Here, we review the literature on the role of purinergic receptors in HIV-1 infection and associated inflammation and describe a role for these receptors as potential therapeutic targets. PMID:26635799

  7. Purinergic Receptors: Key Mediators of HIV-1 Infection and Inflammation

    PubMed Central

    Swartz, Talia H.; Dubyak, George R.; Chen, Benjamin K.

    2015-01-01

    Human immunodeficiency virus type 1 (HIV-1) causes a chronic infection that afflicts more than 30 million individuals worldwide. While the infection can be suppressed with potent antiretroviral therapies, individuals infected with HIV-1 have elevated levels of inflammation as indicated by increased T cell activation, soluble biomarkers, and associated morbidity and mortality. A single mechanism linking HIV-1 pathogenesis to this inflammation has yet to be identified. Purinergic receptors are known to mediate inflammation and have been shown to be required for HIV-1 infection at the level of HIV-1 membrane fusion. Here, we review the literature on the role of purinergic receptors in HIV-1 infection and associated inflammation and describe a role for these receptors as potential therapeutic targets. PMID:26635799

  8. Oligomerization Requirements for MX2-Mediated Suppression of HIV-1 Infection

    PubMed Central

    Dicks, Matthew D. J.; Goujon, Caroline; Pollpeter, Darja; Betancor, Gilberto; Apolonia, Luis; Bergeron, Julien R. C.

    2015-01-01

    ABSTRACT Human myxovirus resistance 2 (MX2/MXB) is an interferon-stimulated gene (ISG) and was recently identified as a late postentry suppressor of human immunodeficiency virus type 1 (HIV-1) infection, inhibiting the nuclear accumulation of viral cDNAs. Although the HIV-1 capsid (CA) protein is believed to be the viral determinant of MX2-mediated inhibition, the precise mechanism of antiviral action remains unclear. The MX family of dynamin-like GTPases also includes MX1/MXA, a well-studied inhibitor of a range of RNA and DNA viruses, including influenza A virus (FLUAV) and hepatitis B virus but not retroviruses. MX1 and MX2 are closely related and share similar domain architectures and structures. However, MX2 possesses an extended N terminus that is essential for antiviral function and confers anti-HIV-1 activity on MX1 [MX1(NMX2)]. Higher-order oligomerization is required for the antiviral activity of MX1 against FLUAV, with current models proposing that MX1 forms ring structures that constrict around viral nucleoprotein complexes. Here, we performed structure-function studies to investigate the requirements for oligomerization of both MX2 and chimeric MX1(NMX2) for the inhibition of HIV-1 infection. The oligomerization state of mutated proteins with amino acid substitutions at multiple putative oligomerization interfaces was assessed using a combination of covalent cross-linking and coimmunoprecipitation. We show that while monomeric MX2 and MX1(NMX2) mutants are not antiviral, higher-order oligomerization does not appear to be required for full antiviral activity of either protein. We propose that lower-order oligomerization of MX2 is sufficient for the effective inhibition of HIV-1. IMPORTANCE Interferon plays an important role in the control of virus replication during acute infection in vivo. Recently, cultured cell experiments identified human MX2 as a key effector in the interferon-mediated postentry block to HIV-1 infection. MX2 is a member of a family

  9. HSV-2- and HIV-1- permissive cell lines co-infected by HSV-2 and HIV-1 co-replicate HSV-2 and HIV-1 without production of HSV-2/HIV-1 pseudotype particles

    PubMed Central

    LeGoff, Jérôme; Bouhlal, Hicham; Lecerf, Maxime; Klein, Christophe; Hocini, Hakim; Si-Mohamed, Ali; Muggeridge, Martin; Bélec, Laurent

    2007-01-01

    Background Herpes simplex virus type 2 (HSV-2) is a major cofactor of human immunodeficiency virus type 1 (HIV-1) sexual acquisition and transmission. In the present study, we investigated whether HIV-1 and HSV-2 may interact at the cellular level by forming HIV-1 hybrid virions pseudotyped with HSV-2 envelope glycoproteins, as was previously reported for HSV type 1. Methods We evaluated in vitro the production of HSV-2/HIV-1 pseudotypes in mononuclear CEM cells and epithelial HT29 and P4P cells. We analyzed the incorporation into the HIV-1 membrane of HSV-2 gB and gD, two major HSV-2 glycoproteins required for HSV-2 fusion with the cell membrane, in co-infected cells and in HIV-1-infected P4P cells transfected by plasmids coding for gB or gD. Results We show that HSV-2 and HIV-1 co-replicated in dually infected cells, and gB and gD were co-localized with gp160. However, HIV-1 particles, produced in HIV-1-infected cells expressing gB or gD after transfection or HSV-2 superinfection, did not incorporate either gB or gD in the viral membrane, and did not have the capacity to infect cells normally non-permissive for HIV-1, such as epithelial cells. Conclusion Our results do not support the hypothesis of HSV-2/HIV-1 pseudotype formation and involvement in the synergistic genital interactions between HIV-1 and HSV-2. PMID:17207276

  10. Replication Competent Molecular Clones of HIV-1 Expressing Renilla Luciferase Facilitate the Analysis of Antibody Inhibition in PBMC

    PubMed Central

    Edmonds, Tara G.; Ding, Haitao; Yuan, Xing; Wei, Qing; Smith, Kendra S.; Conway, Joan A.; Wieczorek, Lindsay; Brown, Bruce; Polonis, Victoria; West, John T.; Montefiori, David C.; Kappes, John C.; Ochsenbauer, Christina

    2010-01-01

    Effective vaccine development for human immunodeficiency virus type 1 (HIV-1) will require assays that ascertain the capacity of vaccine immunogens to elicit neutralizing antibodies (NAb) to diverse HIV-1 strains. To facilitate NAb assessment in peripheral blood mononuclear cell (PBMC)-based assays, we developed an assay-adaptable platform based on a Renilla luciferase (LucR) expressing HIV-1 proviral backbone. LucR was inserted into pNL4-3 DNA, preserving all viral open reading frames. The proviral genome was engineered to facilitate expression of diverse HIV-1 env sequences, allowing analysis in an isogenic background. The resulting Env-IMC-LucR viruses are infectious, and LucR is stably expressed over multiple replications in PBMC. HIV-1 neutralization, targeting TZM-bl cells, was highly correlative comparing virus (LucR) and cell (firefly luciferase) readouts. In PBMC, NAb activity can be analyzed either within a single or multiple cycles of replication. These results represent advancement toward a standardizable PBMC-based neutralization assay for assessing HIV-1 vaccine immunogen efficacy. PMID:20863545

  11. Cellular HIV-1 Inhibition by Truncated Old World Primate APOBEC3A Proteins Lacking a Complete Deaminase Domain

    PubMed Central

    Katuwal, Miki; Wang, Yaqiong; Schmitt, Kimberly; Guo, Kejun; Halemano, Kalani; Santiago, Mario L.; Stephens, Edward B.

    2014-01-01

    The APOBEC3 (A3) deaminases are retrovirus restriction factors that were proposed as inhibitory components of HIV-1 gene therapy vectors. However, A3 mutational activity may induce undesired genomic damage and enable HIV-1 to evade drugs and immune responses. Here, we show that A3A protein from Colobus guereza (colA3A) can restrict HIV-1 replication in producer cells in a deaminase-independent manner without inducing DNA damage. Neither HIV-1 reverse transcription nor integration were significantly affected by colA3A, but capsid protein synthesis was inhibited. The determinants for colA3A restriction mapped to the N-terminal region. These properties extend to A3A from mandrills and De Brazza’s monkeys. Surprisingly, truncated colA3A proteins expressing only the N-terminal 100 amino acids effectively exclude critical catalytic regions but retained potent cellular restriction activity. These highlight a unique mechanism of cellular HIV-1 restriction by several Old World monkey A3A proteins that may be exploited for functional HIV-1 cure strategies. PMID:25262471

  12. HIV-1 Nef Is Transferred from Expressing T Cells to Hepatocytic Cells through Conduits and Enhances HCV Replication

    PubMed Central

    Park, In-Woo; Fan, Yan; Luo, Xiaoyu; Ryou, Myoung-Gwi; Liu, Jinfeng; Green, Linden; He, Johnny J.

    2014-01-01

    HIV-1 infection enhances HCV replication and as a consequence accelerates HCV-mediated hepatocellular carcinoma (HCC). However, the precise molecular mechanism by which this takes place is currently unknown. Our data showed that infectious HIV-1 failed to replicate in human hepatocytic cell lines. No discernible virus replication was observed, even when the cell lines transfected with HIV-1 proviral DNA were co-cultured with Jurkat T cells, indicating that the problem of liver deterioration in the co-infected patient is not due to the replication of HIV-1 in the hepatocytes of the HCV infected host. Instead, HIV-1 Nef protein was transferred from nef-expressing T cells to hepatocytic cells through conduits, wherein up to 16% (average 10%) of the cells harbored the transferred Nef, when the hepatocytic cells were co-cultured with nef-expressing Jurkat cells for 24 h. Further, Nef altered the size and numbers of lipid droplets (LD), and consistently up-regulated HCV replication by 1.5∼2.5 fold in the target subgenomic replicon cells, which is remarkable in relation to the initially indolent viral replication. Nef also dramatically augmented reactive oxygen species (ROS) production and enhanced ethanol-mediated up-regulation of HCV replication so as to accelerate HCC. Taken together, these data indicate that HIV-1 Nef is a critical element in accelerating progression of liver pathogenesis via enhancing HCV replication and coordinating modulation of key intra- and extra-cellular molecules for liver decay. PMID:24911518

  13. TIM-family proteins inhibit HIV-1 release

    PubMed Central

    Li, Minghua; Ablan, Sherimay D.; Miao, Chunhui; Zheng, Yi-Min; Fuller, Matthew S.; Rennert, Paul D.; Maury, Wendy; Johnson, Marc C.; Freed, Eric O.; Liu, Shan-Lu

    2014-01-01

    Accumulating evidence indicates that T-cell immunoglobulin (Ig) and mucin domain (TIM) proteins play critical roles in viral infections. Herein, we report that the TIM-family proteins strongly inhibit HIV-1 release, resulting in diminished viral production and replication. Expression of TIM-1 causes HIV-1 Gag and mature viral particles to accumulate on the plasma membrane. Mutation of the phosphatidylserine (PS) binding sites of TIM-1 abolishes its ability to block HIV-1 release. TIM-1, but to a much lesser extent PS-binding deficient mutants, induces PS flipping onto the cell surface; TIM-1 is also found to be incorporated into HIV-1 virions. Importantly, TIM-1 inhibits HIV-1 replication in CD4-positive Jurkat cells, despite its capability of up-regulating CD4 and promoting HIV-1 entry. In addition to TIM-1, TIM-3 and TIM-4 also block the release of HIV-1, as well as that of murine leukemia virus (MLV) and Ebola virus (EBOV); knockdown of TIM-3 in differentiated monocyte-derived macrophages (MDMs) enhances HIV-1 production. The inhibitory effects of TIM-family proteins on virus release are extended to other PS receptors, such as Axl and RAGE. Overall, our study uncovers a novel ability of TIM-family proteins to block the release of HIV-1 and other viruses by interaction with virion- and cell-associated PS. Our work provides new insights into a virus-cell interaction that is mediated by TIMs and PS receptors. PMID:25136083

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

    PubMed

    Kumar, Amit; Coquard, Laurie; Herbein, Georges

    2016-01-01

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

  15. CRISPR-Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape

    PubMed Central

    Wang, Gang; Zhao, Na; Berkhout, Ben; Das, Atze T

    2016-01-01

    Several recent studies demonstrated that the clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 can be used for guide RNA (gRNA)-directed, sequence-specific cleavage of HIV proviral DNA in infected cells. We here demonstrate profound inhibition of HIV-1 replication by harnessing T cells with Cas9 and antiviral gRNAs. However, the virus rapidly and consistently escaped from this inhibition. Sequencing of the HIV-1 escape variants revealed nucleotide insertions, deletions, and substitutions around the Cas9/gRNA cleavage site that are typical for DNA repair by the nonhomologous end-joining pathway. We thus demonstrate the potency of CRISPR-Cas9 as an antiviral approach, but any therapeutic strategy should consider the viral escape implications. PMID:26796669

  16. Antiretroviral Therapy Fails to Restore Levels of HIV-1 Restriction miRNAs in PBMCs of HIV-1-infected MSM

    PubMed Central

    Liu, Man-Qing; Zhao, Min; Kong, Wen-Hua; Peng, Jin-Song; Wang, Fang; Qiu, Hong-Yan; Zhu, Ze-Rong; Tang, Li; Sang, Ming; Wu, Jian-Guo; Ho, Wen-Zhe; Zhou, Wang

    2015-01-01

    Abstract A number of cellular microRNAs (miRNAs) have been identified to have the ability to inhibit HIV-1 replication. In this study, we examined the impact of combination antiretroviral therapy (cART) on the expression of HIV-1 restriction miRNAs in peripheral blood mononuclear cells of HIV-1–infected men who have sex with men (MSM). Compared with male healthy donors, HIV-infected MSM had significantly lower levels of 9 HIV-1 restriction miRNAs. The treatment of HIV-1–infected MSM with cART, however, failed to restore the levels of these miRNAs in peripheral blood mononuclear cells. These observations suggest that the suppression of the cellular restriction miRNAs by HIV-1 may attribute to the virus latency during cART. PMID:26579828

  17. Antiretroviral Therapy Fails to Restore Levels of HIV-1 Restriction miRNAs in PBMCs of HIV-1-infected MSM.

    PubMed

    Liu, Man-Qing; Zhao, Min; Kong, Wen-Hua; Peng, Jin-Song; Wang, Fang; Qiu, Hong-Yan; Zhu, Ze-Rong; Tang, Li; Sang, Ming; Wu, Jian-Guo; Ho, Wen-Zhe; Zhou, Wang

    2015-11-01

    A number of cellular microRNAs (miRNAs) have been identified to have the ability to inhibit HIV-1 replication. In this study, we examined the impact of combination antiretroviral therapy (cART) on the expression of HIV-1 restriction miRNAs in peripheral blood mononuclear cells of HIV-1-infected men who have sex with men (MSM). Compared with male healthy donors, HIV-infected MSM had significantly lower levels of 9 HIV-1 restriction miRNAs. The treatment of HIV-1-infected MSM with cART, however, failed to restore the levels of these miRNAs in peripheral blood mononuclear cells. These observations suggest that the suppression of the cellular restriction miRNAs by HIV-1 may attribute to the virus latency during cART. PMID:26579828

  18. Transcriptional Bursting from the HIV-1 Promoter is a Significant Source of Stochastic Noise in HIV-1 Gene Expression

    SciTech Connect

    Singh, A; Razooky, B; Cox, Chris D.; Simpson, Michael L; Weinberger, Leor S.

    2010-01-01

    Analysis of noise in gene expression has proven a powerful approach for analyzing gene regulatory architecture. To probe the regulatory mechanisms controlling expression of HIV-1, we analyze noise in gene-expression from HIV-1 s long terminal repeat (LTR) promoter at different HIV-1 integration sites across the human genome. Flow cytometry analysis of GFP expression from the HIV-1 LTR shows high variability (noise) at each integration site. Notably, the measured noise levels are inconsistent with constitutive gene expression models. Instead, quantification of expression noise indicates that HIV-1 gene expression occurs through randomly timed bursts of activity from the LTR and that each burst generates an average of 2 10 mRNA transcripts before the promoter returns to an inactive state. These data indicate that transcriptional bursting can generate high variability in HIV-1 early gene products, which may critically influence the viral fate-decision between active replication and proviral latency.

  19. HIV-1 Treatment-as-Prevention

    PubMed Central

    Tang, Zhenzhu; Lan, Guanghua; Chen, Ying Qing; Zhu, Qiuying; Yang, Xiaoyi; Shen, Zhiyong; Chen, Yi; Zhang, Heng; Kan, Wei; Xing, Hui; Ruan, Yuhua; Shao, Yiming

    2015-01-01

    Abstract The Chinese national observational cohort study suggests that the treatment-as-prevention (TasP) approach can be an effective public health HIV-1 prevention strategy. However, results from that study may have been biased because the follow-up time of index patients prior to their initiation of antiretroviral therapy (ART) was excluded. In this study, we correct for such bias by using an extended time-dependent Cox regression model to conduct a cohort study analysis of serodiscordant couples in Guangxi of China, inclusive of all follow-up time. During the follow-up of this observational cohort study of HIV-1 sero-discordant couples, the positive index partners may have never be treated with ART, or enter untreated but subsequently began treatment, or may have been treated immediately upon entry into the public health system. The treatment effectiveness of ART in HIV-1 acquisition among HIV-negative partners is assessed by the extended Cox regression model with treatment status as a time-varying covariate. A total of 6548 sero-discordant couples were included in the cohort study analysis. Among them, 348 negative partners sero-converted. HIV seroincidence was significantly higher among the nontreated (4.3 per 100 person-years, 3.7–4.9) compared with those receiving ART (1.8 per 100 person-years, 1.5–2.0). An overall 35% reduction in risk of HIV transmission was associated with receiving ART (adjusted hazard ratio [AHR] 0.65, 95% confidence interval [CI] 0.51–0.83), and the yearly risk reduction was also significant in the first 3 consecutive years of follow-up. Moreover, ART was found to be significantly inversely associated with multiple baseline characteristics of index partners. TasP may be feasible on a national or regional scale. In addition to other proven preventive strategies such as the use of condoms, ART adherence to maintain viral suppression would then be the key challenge for successful TasP implementation.

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

    SciTech Connect

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

    2008-01-01

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

  1. DUAL AND OPPOSITE EFFECTS OF hRAD51 CHEMICAL MODULATION ON HIV-1 INTEGRATION

    PubMed Central

    Thierry, Sylvain; Benleulmi, Mohamed Salah; Sinzelle, Ludivine; Thierry, Eloise; Calmels, Christina; Chaignepain, Stephane; Waffo-Teguo, Pierre; Merillon, Jean-Michel; Budke, Brian; Pasquet, Jean-Max; Litvak, Simon; Ciuffi, Angela; Sung, Patrick; Connell, Philip; Hauber, Ilona; Hauber, Joachim; Andreola, Marie-Line; Delelis, Olivier; Parissi, Vincent

    2016-01-01

    SUMMARY The cellular DNA repair hRAD51 protein has been shown to restrict HIV-1 integration both in vitro and in vivo. To investigate its regulatory functions, we performed a pharmacological analysis of the retroviral integration modulation by hRAD51. We found that, in vitro, chemical activation of hRAD51 stimulates its integration inhibitory properties, whereas inhibition of hRAD51 decreases the integration restriction, indicating that the modulation of HIV-1 integration depends on the hRAD51 recombinase activity. Cellular analyses demonstrated that cells exhibiting high hRAD51 levels prior to de novo infection are more resistant to integration. On the other hand, when hRAD51 was activated during integration, cells were more permissive. Altogether, these data establish the functional link between hRAD51 activity and HIV-1 integration. Our results highlight the multiple and opposite effects of the recombinase during integration and provide new insights into the cellular regulation of HIV-1 replication. PMID:26051216

  2. Host Proteins Ku and HMGA1 As Participants of HIV-1 Transcription

    PubMed Central

    Shadrina, O. A.; Knyazhanskaya, E. S.; Korolev, S.P.; Gottikh, M. B.

    2016-01-01

    Human immunodeficiency virus type 1 is known to use the transcriptional machinery of the host cell for viral gene transcription, and the only viral protein that partakes in this process is Tat, the viral trans-activator of transcription. During acute infection, the binding of Tat to the hairpin at the beginning of the transcribed viral RNA recruits the PTEFb complex, which in turn hyperphosphorylates RNA-polymerase II and stimulates transcription elongation. Along with acute infection, HIV-1 can also lead to latent infection that is characterized by a low level of viral transcription. During the maintenance and reversal of latency, there are no detectable amounts of Tat protein in the cell and the mechanism of transcription activation in the absence of Tat protein remains unclear. The latency maintenance is also a problematic question. It seems evident that cellular proteins with a yet unknown nature or role regulate both transcriptional repression in the latent phase and its activation during transition into the lytic phase. The present review discusses the role of cellular proteins Ku and HMGA1 in the initiation of transcription elongation of the HIV-1 provirus. The review presents data regarding Ku-mediated HIV-1 transcription and its dependence on the promoter structure and the shape of viral DNA. We also describe the differential influence of the HMGA1 protein on the induced and basal transcription of HIV-1. Finally, we offer possible mechanisms for Ku and HMGA1 proteins in the proviral transcription regulation. PMID:27099783

  3. Potential of Radiation-Induced Cellular Stress for Reactivation of Latent HIV-1 and Killing of Infected Cells.

    PubMed

    Iordanskiy, Sergey; Kashanchi, Fatah

    2016-02-01

    The use of highly active antiretroviral therapy against HIV-1 for last two decades has reduced mortality of patients through extension of nonsymptomatic phase of infection. However, HIV-1 can be preserved in long-lived resting CD4(+) T cells, which form a viral reservoir in infected individuals, and potentially in macrophages and astrocytes. Reactivation of viral replication is critical since the host immune response in combination with antiretroviral therapy may eradicate the virus (shock and kill strategy). In this opinion piece, we consider potential application of therapeutic doses of irradiation, the well-known and effective stress signal that induces DNA damage and activates cellular stress response, to resolve two problems: activate HIV-1 replication and virion production in persistent reservoirs under cART and deplete infected cells through selective cell killing using DNA damage responses. PMID:26765533

  4. Gnidimacrin, a Potent Anti-HIV Diterpene, Can Eliminate Latent HIV-1 Ex Vivo by Activation of Protein Kinase C β.

    PubMed

    Lai, Weihong; Huang, Li; Zhu, Lei; Ferrari, Guido; Chan, Cliburn; Li, Wei; Lee, Kuo-Hsiung; Chen, Chin-Ho

    2015-11-12

    HIV-1-latency-reversing agents, such as histone deacetylase inhibitors (HDACIs), were ineffective in reducing latent HIV-1 reservoirs ex vivo using CD4 cells from patients as a model. This deficiency poses a challenge to current pharmacological approaches for HIV-1 eradication. The results of this study indicated that gnidimacrin (GM) was able to markedly reduce the latent HIV-1 DNA level and the frequency of latently infected cells in an ex vivo model using patients peripheral blood mononuclear cells. GM induced approximately 10-fold more HIV-1 production than the HDACI SAHA or romidepsin, which may be responsible for the effectiveness of GM in reducing latent HIV-1 levels. GM achieved these effects at low picomolar concentrations by selective activation of protein kinase C βI and βII. Notably, GM was able to reduce the frequency of HIV-1 latently infected cells at concentrations without global T cell activation or stimulating inflammatory cytokine production. GM merits further development as a clinical trial candidate for latent HIV-1 eradication. PMID:26509731

  5. HIV-1 Assembly, Budding, and Maturation

    PubMed Central

    Sundquist, Wesley I.; Kräusslich, Hans-Georg

    2012-01-01

    A defining property of retroviruses is their ability to assemble into particles that can leave producer cells and spread infection to susceptible cells and hosts. Virion morphogenesis can be divided into three stages: assembly, wherein the virion is created and essential components are packaged; budding, wherein the virion crosses the plasma membrane and obtains its lipid envelope; and maturation, wherein the virion changes structure and becomes infectious. All of these stages are coordinated by the Gag polyprotein and its proteolytic maturation products, which function as the major structural proteins of the virus. Here, we review our current understanding of the mechanisms of HIV-1 assembly, budding, and maturation, starting with a general overview and then providing detailed descriptions of each of the different stages of virion morphogenesis. PMID:22762019

  6. Evaluation of Immune Survival Factors in Pediatric HIV-1 Infection

    PubMed Central

    SHEARER, WILLIAM T.; EASLEY, KIRK A.; GOLDFARB, JOHANNA; JENSON, HAL B.; ROSENBLATT, HOWARD M.; KOVACS, ANDREA; MCINTOSH, KENNETH

    2015-01-01

    Peripheral blood CD4+ and CD8+ T cells, CD19+/20+ B cells, and serum immunoglobulins (Igs) have been implicated as survival factors for pediatric HIV-1 infection. To determine which of these immune factors might be important in predicting survival, we studied HIV-1 vertically infected (HIV-1+) children over a 5-year period. Peripheral blood lymphocytes and Igs were measured in 298 HIV-1+ children, who were classified as survivors or nonsurvivors, and in 463 HIV-1 vertically exposed and noninfected (HIV-1–) children. Measurements of other possible survival factors were included in this study: albumin, hemoglobin, lactic dehydrogenase (LDH), and HIV-1 RNA levels. Survivors had significantly higher CD4+ T-cell, CD8+ T-cell, and CD19+/CD20+ B-cell counts and serum IgG levels, but lower serum IgA and IgM levels than nonsurvivors. Serum albumin and blood hemoglobin levels were higher, but serum LDH and HIV-1 RNA levels were lower in the survivors compared to non-survivors. In univariable analysis, factors affecting survival were baseline CD4+ T-cell and CD8+ T-cell counts, IgG, albumin, hemoglobin, LDH, and HIV-1 RNA (all p < 0.001). In multivariable analysis, high baseline CD4+ T-cell count, IgG and albumin levels, and low baseline HIV-1 RNA load remained important factors for survival. Serum IgG level has been identified as an immune factor that independently predicts survival, in addition to the already established CD4+ T-cell count. The HIV-1 RNA and serum albumin levels also predicted survival. PMID:11144332

  7. Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs

    PubMed Central

    Malatinkova, Eva; Spiegelaere, Ward De; Bonczkowski, Pawel; Kiselinova, Maja; Vervisch, Karen; Trypsteen, Wim; Johnson, Margaret; Verhofstede, Chris; de Looze, Danny; Murray, Charles; Loes, Sabine Kinloch-de; Vandekerckhove, Linos

    2015-01-01

    Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure. DOI: http://dx.doi.org/10.7554/eLife.09115.001 PMID:26439007

  8. Impact of a decade of successful antiretroviral therapy initiated at HIV-1 seroconversion on blood and rectal reservoirs.

    PubMed

    Malatinkova, Eva; De Spiegelaere, Ward; Bonczkowski, Pawel; Kiselinova, Maja; Vervisch, Karen; Trypsteen, Wim; Johnson, Margaret; Verhofstede, Chris; de Looze, Danny; Murray, Charles; Kinloch-de Loes, Sabine; Vandekerckhove, Linos

    2015-01-01

    Persistent reservoirs remain the major obstacles to achieve an HIV-1 cure. Prolonged early antiretroviral therapy (ART) may reduce the extent of reservoirs and allow for virological control after ART discontinuation. We compared HIV-1 reservoirs in a cross-sectional study using polymerase chain reaction-based techniques in blood and tissue of early-treated seroconverters, late-treated patients, ART-naïve seroconverters, and long-term non-progressors (LTNPs) who have spontaneous virological control without treatment. A decade of early ART reduced the total and integrated HIV-1 DNA levels compared with later treatment initiation, but not reaching the low levels found in LTNPs. Total HIV-1 DNA in rectal biopsies did not differ between cohorts. Importantly, lower viral transcription (HIV-1 unspliced RNA) and enhanced immune preservation (CD4/CD8), reminiscent of LTNPs, were found in early compared to late-treated patients. This suggests that early treatment is associated with some immunovirological features of LTNPs that may improve the outcome of future interventions aimed at a functional cure. PMID:26439007

  9. HIV-1 Reverse Transcriptase Structure with RNase H Inhibitor dihydroxy benzoyl naphthyl Hydrazone Bound at a Novel Site

    SciTech Connect

    Himmel,D.; Sarafianos, S.; Dharmasena, S.; Hossain, M.; McCoy-Simandle, K.; Ilina, T.; Clark, A.; Knight, J.; Julias, J.; et al.

    2007-01-01

    The rapid emergence of drug-resistant variants of human immunodeficiency virus, type 1 (HIV-1), has limited the efficacy of anti-acquired immune deficiency syndrome (AIDS) treatments, and new lead compounds that target novel binding sites are needed. We have determined the 3.15 {angstrom} resolution crystal structure of HIV-1 reverse transcriptase (RT) complexed with dihydroxy benzoyl naphthyl hydrazone (DHBNH), an HIV-1 RT RNase H (RNH) inhibitor (RNHI). DHBNH is effective against a variety of drug-resistant HIV-1 RT mutants. While DHBNH has little effect on most aspects of RT-catalyzed DNA synthesis, at relatively high concentrations it does inhibit the initiation of RNA-primed DNA synthesis. Although primarily an RNHI, DHBNH binds >50 {angstrom} away from the RNH active site, at a novel site near both the polymerase active site and the non-nucleoside RT inhibitor (NNRTI) binding pocket. When DHBNH binds, both Tyr181 and Tyr188 remain in the conformations seen in unliganded HIV-1 RT. DHBNH interacts with conserved residues (Asp186, Trp229) and has substantial interactions with the backbones of several less well-conserved residues. On the basis of this structure, we designed substituted DHBNH derivatives that interact with the NNRTI-binding pocket. These compounds inhibit both the polymerase and RNH activities of RT.

  10. Striking HIV-1 Entry by Targeting HIV-1 gp41. But, Where Should We Target?

    PubMed Central

    Teixeira, Cátia; Barbault, Florent; Couesnon, Thierry; Gomes, José R. B.; Gomes, Paula; Maurel, François

    2016-01-01

    HIV-1 gp41 facilitates the viral fusion through a conformational switch involving the association of three C-terminal helices along the conserved hydrophobic grooves of three N-terminal helices coiled-coil. The control of these structural rearrangements is thought to be central to HIV-1 entry and, therefore, different strategies of intervention are being developed. Herewith, we describe a procedure to simulate the folding of an HIV-1 gp41 simplified model. This procedure is based on the construction of plausible conformational pathways, which describe protein transition between non-fusogenic and fusogenic conformations. The calculation of the paths started with 100 molecular dynamics simulations of the non-fusogenic conformation, which were found to converge to different intermediate states. Those presenting defined criteria were selected for separate targeted molecular dynamics simulations, subjected to a force constant imposing a movement towards the gp41 fusogenic conformation. Despite significant diversity, a preferred sequence of events emerged when the simulations were analyzed in terms of the formation, breakage and evolution of the contacts. We pointed out 29 residues as the most relevant for the movement of gp41; also, 2696 possible interactions were reduced to only 48 major interactions, which reveals the efficiency of the method. The analysis of the evolution of the main interactions lead to the detection of four main behaviors for those contacts: stable, increasing, decreasing and repulsive interactions. Altogether, these results suggest a specific small cavity of the HIV-1 gp41 hydrophobic groove as the preferred target to small molecules. PMID:26785380

  11. The fate of HIV-1 capsid: a biochemical assay for HIV-1 uncoating.

    PubMed

    Yang, Yang; Luban, Jeremy; Diaz-Griffero, Felipe

    2014-01-01

    The uncoating process of HIV-1 is a poorly understood process, so the development of a reliable assay to study uncoating is critical for moving the field forward. Here we describe an uncoating assay that currently represents the state-of-the-art biochemical procedure for monitoring uncoating and core stability during infection. This assay is based on the biochemical separation of soluble capsid protein from particulate capsid cores and provides information about the fate of the capsid during infection. PMID:24158811

  12. Iron chelators ICL670 and 311 inhibit HIV-1 transcription

    SciTech Connect

    Debebe, Zufan; Ammosova, Tatyana; Jerebtsova, Marina; Kurantsin-Mills, Joseph; Niu, Xiaomei; Charles, Sharroya; Richardson, Des R.; Ray, Patricio E.; Gordeuk, Victor R.; Nekhai, Sergei

    2007-10-25

    HIV-1 replication is induced by an excess of iron and iron chelation by desferrioxamine (DFO) inhibits viral replication by reducing proliferation of infected cells. Treatment of cells with DFO and 2-hydroxy-1-naphthylaldehyde isonicotinoyl hydrazone (311) inhibit expression of proteins that regulate cell-cycle progression, including cycle-dependent kinase 2 (CDK2). Our recent studies showed that CDK2 participates in HIV-1 transcription and viral replication suggesting that inhibition of CDK2 by iron chelators might also affect HIV-1 transcription. Here we evaluated the effect of a clinically approved orally effective iron chelator, 4-[3,5-bis-(hydroxyphenyl)-1,2,4-triazol-1-yl]-benzoic acid (ICL670) and 311 on HIV-1 transcription. Both ICL670 and 311 inhibited Tat-induced HIV-1 transcription in CEM-T cells, 293T and HeLa cells. Neither ICL670 nor 311 induced cytotoxicity at concentrations that inhibited HIV-1 transcription. The chelators decreased cellular activity of CDK2 and reduced HIV-1 Tat phosphorylation by CDK2. Neither ICL670A or 311 decreased CDK9 protein level but significantly reduced association of CDK9 with cyclin T1 and reduced phosphorylation of Ser-2 residues of RNA polymerase II C-terminal domain. In conclusion, our findings add to the evidence that iron chelators can inhibit HIV-1 transcription by deregulating CDK2 and CDK9. Further consideration should be given to the development of iron chelators for future anti-retroviral therapeutics.

  13. Tetraspanin CD63 is a regulator of HIV-1 replication

    PubMed Central

    Fu, Enqing; Pan, Lei; Xie, Yonghong; Mu, Deguang; Liu, Wei; Jin, Faguang; Bai, Xuefan

    2015-01-01

    Macrophages and CD4+ T-cells are the major reservoirs for HIV-1 infection. CD63 is a tetraspanin transmembrane protein, which has been shown to play an essential role during HIV-1 replication in macrophages. In this study, we further confirm the requirement of CD63 in HIV-1 replication events in primary human CD4+ T-cells, dendritic cells, and a CD4+ cell line. Most interestingly, we also show the evidences for the co-localization and internalization of CD63 and HIV-1 major receptor CD4 in primary human macrophages and CD4+ cell line by confocal microscopy and Co-Immunoprecipitation assay. Analysis revealed that CD63-depleted CD4+ T-cells, dendritic cells, and a cell line showed significant decrease in HIV-1 production. Further analysis showed that CD63 down regulation reduced production of the early HIV protein Tat, and affected HIV protein Gag by CD63-Gag interaction. In agreement, CD63 silencing also inhibited production of the late protein p24. Furthermore, we revealed that CD63 silencing has no effect on HIV-1 replication with extensive viral challenge (MOI > 0.2). These findings suggest that CD63 plays a dual-role both in early and late HIV-1 life cycle with a range of HIV-1 infection (MOI < 0.2). PMID:25973004

  14. [A new unique HIV-1 recombinant form detected in Belarus].

    PubMed

    Eremin, V F; Gasich, E L; Sosinovich, S V

    2012-01-01

    Republican Research-and-Practical Center for Epidemiology and Microbiology, Ministry of Health of Belarus, Minsk The paper presents data on the molecular genetic characteristics of a new HIV-1 recombinant form. The study has shown that the virus is referred to as HIV-1 subtype B in terms of the gag gene and HIV-1 subtype A in terms of the pol and env genes. At the same time the new isolate is closer, in terms of the gag gene, to the HIV-1 DQ207943 strain isolated in Georgia, in terms of the pol gene, to the HIV-1 AF413987.1 strain isolated in Ukraine and, in terms of the env gene to the HIV-1 AY500393 strain isolated in Russia. Thus, the described new HIV-1 recombinant form has the following structure: BgagApolAenv. The gag, pol, and env gene sequences from the new unique HIV-1 recombinant form have been registered in the international database EMBL/Genbank/DDBJ under accession numbers FR775442.1, FN995656.1, and FR775443.1. PMID:22905420

  15. 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

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

    PubMed Central

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

    2015-01-01

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

  17. KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection

    PubMed Central

    Dharan, Adarsh; Talley, Sarah; Tripathi, Abhishek; Mamede, João I.; Majetschak, Matthias; Campbell, Edward M.

    2016-01-01

    Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection. PMID:27327622

  18. 5-Azacytidine Enhances the Mutagenesis of HIV-1 by Reduction to 5-Aza-2'-Deoxycytidine.

    PubMed

    Rawson, Jonathan M O; Daly, Michele B; Xie, Jiashu; Clouser, Christine L; Landman, Sean R; Reilly, Cavan S; Bonnac, Laurent; Kim, Baek; Patterson, Steven E; Mansky, Louis M

    2016-04-01

    5-Azacytidine (5-aza-C) is a ribonucleoside analog that induces the lethal mutagenesis of human immunodeficiency virus type 1 (HIV-1) by causing predominantly G-to-C transversions during reverse transcription. 5-Aza-C could potentially act primarily as a ribonucleotide (5-aza-CTP) or as a deoxyribonucleotide (5-aza-2'-deoxycytidine triphosphate [5-aza-dCTP]) during reverse transcription. In order to determine the primary form of 5-aza-C that is active against HIV-1, Illumina sequencing was performed using proviral DNA from cells treated with 5-aza-C or 5-aza-dC. 5-Aza-C and 5-aza-dC were found to induce highly similar patterns of mutation in HIV-1 in terms of the types of mutations observed, the magnitudes of effects, and the distributions of mutations at individual sequence positions. Further, 5-aza-dCTP was detected by liquid chromatography-tandem mass spectrometry in cells treated with 5-aza-C, demonstrating that 5-aza-C was a substrate for ribonucleotide reductase. Notably, levels of 5-aza-dCTP were similar in cells treated with equivalent effective concentrations of 5-aza-C or 5-aza-dC. Lastly, HIV-1 reverse transcriptase was found to incorporate 5-aza-CTPin vitroat least 10,000-fold less efficiently than 5-aza-dCTP. Taken together, these data support the model that 5-aza-C enhances the mutagenesis of HIV-1 primarily after reduction to 5-aza-dC, which can then be incorporated during reverse transcription and lead to G-to-C hypermutation. These findings may have important implications for the design of new ribonucleoside analogs directed against retroviruses. PMID:26833151

  19. In Vivo HIV-1 Hypermutation and Viral Loads Among Antiretroviral-Naive Brazilian Patients

    PubMed Central

    de Lima-Stein, Mariana Leão; Alkmim, Wagner Tadeu; Bizinoto, Maria Clara de Souza; Lopez, Luis Fernandez; Burattini, Marcelo Nascimento; Maricato, Juliana Terzi; Giron, Leila; Sucupira, Maria Cecília Araripe; Diaz, Ricardo Sobhie

    2014-01-01

    Abstract Hypermutation alludes to an excessive number of specific guanine-to-adenine (G- >A) substitutions in proviral DNA and this phenomenon is attributed to the catalytic activity of cellular APOBECs. Population studies relating hypermutation and the progression of infection by human immunodeficiency virus type 1 (HIV-1) have been performed to elucidate the effect of hypermutation on the natural course of HIV-1 infection. However, the many different approaches employed to assess hypermutation in nucleotide sequences render the comparison of results difficult. This study selected 157 treatment-naive patients and sought to correlate the hypermutation level of the proviral sequences in clinical samples with demographic variables, HIV-1 RNA viral load, and the level of CD4+ T cells. Nested touchdown polymerase chain reaction (PCR) was performed with specific primers to detect hypermutation in the region of HIV-1 integrase, and the amplified sequences were run in agarose gels with HA-Yellow. The analysis of gel migration patterns using the k-means clustering method was validated by its agreement with the results obtained with the software Hypermut. Hypermutation was found in 31.2% of the investigated samples, and a correlation was observed between higher hypermutation levels and higher viral load levels. These findings suggest a high frequency of hypermutation detection in a Brazilian cohort, which can reflect a particular characteristic of this population, but also can result from the method approach by aiming at hypermutation-sensitive sites. Furthermore, we found that hypermutation events are pervasive during HIV-1 infection as a consequence of high viral replication, reflecting its role during disease progression. PMID:25065371

  20. KIF5B and Nup358 Cooperatively Mediate the Nuclear Import of HIV-1 during Infection.

    PubMed

    Dharan, Adarsh; Talley, Sarah; Tripathi, Abhishek; Mamede, João I; Majetschak, Matthias; Hope, Thomas J; Campbell, Edward M

    2016-06-01

    Following envelope mediated fusion, the HIV-1 core is released into the cytoplasm of the target cell and undergoes a series of trafficking and replicative steps that result in the nuclear import of the viral genome, which ultimately leads to the integration of the proviral DNA into the host cell genome. Previous studies have found that disruption of microtubules, or depletion of dynein or kinesin motors, perturb the normal uncoating and trafficking of the viral genome. Here, we show that the Kinesin-1 motor, KIF5B, induces a relocalization of the nuclear pore component Nup358 into the cytoplasm during HIV-1 infection. This relocalization of NUP358 is dependent on HIV-1 capsid, and NUP358 directly associates with viral cores following cytoplasmic translocation. This interaction between NUP358 and the HIV-1 core is dependent on multiple capsid binding surfaces, as this association is not observed following infection with capsid mutants in which a conserved hydrophobic binding pocket (N74D) or the cyclophilin A binding loop (P90A) is disrupted. KIF5B knockdown also prevents the nuclear entry and infection by HIV-1, but does not exert a similar effect on the N74D or P90A capsid mutants which do not rely on Nup358 for nuclear import. Finally, we observe that the relocalization of Nup358 in response to CA is dependent on cleavage protein and polyadenylation factor 6 (CPSF6), but independent of cyclophilin A. Collectively, these observations identify a previously unappreciated role for KIF5B in mediating the Nup358 dependent nuclear import of the viral genome during infection. PMID:27327622

  1. Mucosal Correlates of Protection in HIV-1-Exposed Seronegative Persons

    PubMed Central

    Shen, Ruizhong; Smith, Phillip D.

    2014-01-01

    Resistance to HIV-1 infection in HIV-1-exposed seronegative (HESN) persons offers a promising opportunity to identify mechanisms of “natural” protection. Unique features of the mucosa in particular may contribute to this protection. Here we highlight several key issues pertaining to the mucosal correlates of protection in HESN persons, including humoral immune responses, mechanisms of mucosal HIV-1-neutralization, immune cell activation, and role of the microbiota in mucosal responses. We also discuss mucosal model systems that can be used to investigate the mechanisms of resistance in HESN subjects. A clear understanding of the mucosal correlates of protection against HIV-1 in HESN persons will provide critical new insights for the development of effective vaccine and microbicide strategies for the prevention of HIV-1 transmission. PMID:24428610

  2. Multifarious immunotherapeutic approaches to cure HIV-1 infection

    PubMed Central

    Imami, Nesrina; Herasimtschuk, Anna A

    2015-01-01

    Immunotherapy in the context of treated HIV-1 infection aims to improve immune responses to achieve better control of the virus. To date, multifaceted immunotherapeutic approaches have been shown to reduce immune activation and increase CD4 T-lymphocyte counts, further to the effects of antiretroviral therapy alone, in addition to improving HIV-1-specific T-cell responses. While sterilizing cure of HIV-1 would involve elimination of all replication-competent virus, a functional cure in which the host has long-lasting control of viral replication may be more feasible. In this commentary, we discuss novel strategies aimed at targeting the latent viral reservoir with cure of HIV-1 infection being the ultimate goal, an achievement that would have considerable impact on worldwide HIV-1 infection. PMID:26048144

  3. HIV-1 infection, microenvironment and endothelial cell dysfunction.

    PubMed

    Mazzuca, Pietro; Caruso, Arnaldo; Caccuri, Francesca

    2016-09-01

    HIV-1 promotes a generalized immune activation that involves the main targets of HIV-1 infection but also cells that are not sensitive to viral infection. ECs display major dysfunctions in HIV+ patients during long-standing viral infection that persist even in the current cART era, in which new-generation drugs have reduced dysmetabolic side effects and successfully impeded viral replication. In vivo studies have failed to demonstrate the presence of replicating virus in ECs suggesting that a direct role of the virus is unlikely, and implying that the mechanism accounting for vascular dysfunction may rely on the indirect action of molecules released in the microenvironment by HIV-1-infected cells. This article reviews the current understanding of how HIV-1 infection can contribute to vascular dysfunction. In particular, we discuss the emerging role played by different HIV-1 proteins in driving inflammation and EC dysregulation, and highlight the need to target them for therapeutic benefit. PMID:27602413

  4. The role of human dendritic cells in HIV-1 infection.

    PubMed

    Ahmed, Zahra; Kawamura, Tatsuyoshi; Shimada, Shinji; Piguet, Vincent

    2015-05-01

    Dendritic cells (DCs) and their subsets have multifaceted roles in the early stages of HIV-1 transmission and infection. DC studies have led to remarkable discoveries, including identification of restriction factors, cellular structures promoting viral transmission including the infectious synapse or the interplay of the C-type lectins, Langerin on Langerhans cells (LCs), and dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin on other DC subsets, limiting or facilitating HIV transmission to CD4(+) T cells, respectively. LCs/DCs are also exposed to encountering HIV-1 and other sexually transmitted infections (herpes simplex virus-2, bacteria, fungi), which reprogram HIV-1 interaction with these cells. This review will summarize advances in the role of DCs during HIV-1 infection and discuss their potential involvement in the development of preventive strategies against HIV-1 and other sexually transmitted infections. PMID:25407434

  5. HIV-1 Group O Integrase Displays Lower Enzymatic Efficiency and Higher Susceptibility to Raltegravir than HIV-1 Group M Subtype B Integrase

    PubMed Central

    Depatureaux, Agnès; Quashie, Peter K.; Mesplède, Thibault; Han, Yingshan; Koubi, Hannah; Plantier, Jean-Christophe; Oliveira, Maureen; Moisi, Daniela; Brenner, Bluma

    2014-01-01

    HIV-1 group O (HIV-O) is a rare HIV-1 variant characterized by a high number of polymorphisms, especially in the integrase coding region. As HIV-O integrase enzymes have not previously been studied, our aim was to assess the impact of HIV-O integrase polymorphisms on enzyme function and susceptibility to integrase inhibitors. Accordingly, we cloned and purified integrase proteins from each of HIV-1 group O clades A and B, an HIV-O divergent strain, and HIV-1 group M (HIV-M, subtype B), used as a reference. To assess enzymatic function of HIV-O integrase, we carried out strand transfer and 3′ processing assays with various concentrations of substrate (DNA target and long terminal repeats [LTR], respectively) and characterized these enzymes for susceptibility to integrase strand transfer inhibitors (INSTIs) in cell-free assays and in tissue culture, in the absence or presence of various concentrations of several INSTIs. The inhibition constant (Ki) and 50% effective concentration (EC50) values were calculated for HIV-O integrases and HIV-O viruses, respectively, and compared with those of HIV-M. The results showed that HIV-O integrase displayed lower activity in strand transfer assays than did HIV-M enzyme, whereas 3′ processing activities were similar to those of HIV-M. HIV-O integrases were more susceptible to raltegravir (RAL) in competitive inhibition assays and in tissue culture than were HIV-M enzymes and viruses, respectively. Molecular modeling suggests that two key polymorphic residues that are close to the integrase catalytic site, 74I and 153A, may play a role in these differences. PMID:25224008

  6. IL-8 Alterations in HIV-1 Infected Children With Disease Progression

    PubMed Central

    Pananghat, Ambili Nair; Aggarwal, Heena; Prakash, Somi Sankaran; Makhdoomi, Muzamil Ashraf; Singh, Ravinder; Lodha, Rakesh; Ali, Shakir; Srinivas, Maddur; Das, Bimal Kumar; Pandey, Ravindra Mohan; Kabra, Sushil Kumar; Luthra, Kalpana

    2016-01-01

    Abstract Disease progression in HIV-1 infected children is faster than in adults. Less than 5% of the infected children maintain stable CD4 counts beyond 7 years of infection and are termed long-term nonprogressors (LTNPs). Delineating the host immune response in antiretroviral naïve (ART) and treated HIV-1 infected children at different disease stages will help in understanding the immunopathogenesis of the disease. A total of 79 asymptomatic, perinatally HIV-1 infected children (50 ART naïve and 29 ART treated) and 8 seronegative donors were recruited in this study. T- and B-cell activation PCR arrays were performed from the cDNA, using total RNA extracted from the peripheral blood mononuclear cells (PBMCs) of 14 HIV-1 infected children at different stages of the disease. The differentially expressed genes were identified. Quantitative RT-PCR was performed for the (interleukin-8) IL-8 gene and its transcriptional mediators, that is, SHP2, GRB2, and IL-8R (IL-8 receptor/CXCR1). Plasma levels of IL-8 were measured by flow cytometry. Gene array data revealed a higher expression of IL-8 in the ART naïve HIV-1 infected progressors and in ART nonresponders than LTNPs and ART responders, respectively. Quantitative RT-PCR analysis demonstrated a significant higher expression of IL-8 (P < 0.001), its receptor CXCR1 (P = 0.03) and the upstream signaling molecule SHP2 (P = 0.04) in the progressors versus LTNPs. Plasma levels of IL-8 were significantly higher in progressors versus LTNPs (P < 0.001), and ART nonresponders versus ART responders (P < 0.001). A significant negative correlation of plasma levels of IL-8 with CD4 counts (cells/μL) was observed in HIV-1 infected ART naïve subjects (r = −0.488; P < 0.001), while the IL-8 levels positively correlated with viral load in the ART treated children (r = 0.5494; P < 0.001). ART naïve progressors on follow up demonstrated a significant reduction in the mRNA expression (P = 0

  7. Biochemical and virological analysis of the 18-residue C-terminal tail of HIV-1 integrase

    PubMed Central

    Dar, Mohd J; Monel, Blandine; Krishnan, Lavanya; Shun, Ming-Chieh; Di Nunzio, Francesca; Helland, Dag E; Engelman, Alan

    2009-01-01

    Background The 18 residue tail abutting the SH3 fold that comprises the heart of the C-terminal domain is the only part of HIV-1 integrase yet to be visualized by structural biology. To ascertain the role of the tail region in integrase function and HIV-1 replication, a set of deletion mutants that successively lacked three amino acids was constructed and analyzed in a variety of biochemical and virus infection assays. HIV-1/2 chimers, which harbored the analogous 23-mer HIV-2 tail in place of the HIV-1 sequence, were also studied. Because integrase mutations can affect steps in the replication cycle other than integration, defective mutant viruses were tested for integrase protein content and reverse transcription in addition to integration. The F185K core domain mutation, which increases integrase protein solubility, was furthermore analyzed in a subset of mutants. Results Purified proteins were assessed for in vitro levels of 3' processing and DNA strand transfer activities whereas HIV-1 infectivity was measured using luciferase reporter viruses. Deletions lacking up to 9 amino acids (1-285, 1-282, and 1-279) displayed near wild-type activities in vitro and during infection. Further deletion yielded two viruses, HIV-11-276 and HIV-11-273, that displayed approximately two and 5-fold infectivity defects, respectively, due to reduced integrase function. Deletion mutant HIV-11-270 and the HIV-1/2 chimera were non-infectious and displayed approximately 3 to 4-fold reverse transcription in addition to severe integration defects. Removal of four additional residues, which encompassed the C-terminal β strand of the SH3 fold, further compromised integrase incorporation into virions and reverse transcription. Conclusion HIV-11-270, HIV-11-266, and the HIV-1/2 chimera were typed as class II mutant viruses due to their pleiotropic replication defects. We speculate that residues 271-273 might play a role in mediating the known integrase-reverse transcriptase interaction, as

  8. Slower uncoating is associated with impaired replicative capability of simian-tropic HIV-1.

    PubMed

    Kono, Ken; Takeda, Eri; Tsutsui, Hiromi; Kuroishi, Ayumu; Hulme, Amy E; Hope, Thomas J; Nakayama, Emi E; Shioda, Tatsuo

    2013-01-01

    Human immunodeficiency virus type 1 (HIV-1) productively infects only humans and chimpanzees, but not Old World monkeys, such as rhesus and cynomolgus (CM) monkeys. To establish a monkey model of HIV-1/AIDS, several HIV-1 derivatives have been constructed. We previously generated a simian-tropic HIV-1 that replicates efficiently in CM cells. This virus encodes a capsid protein (CA) with SIVmac239-derived loops between α-helices 4 and 5 (L4/5) and between α-helices 6 and 7 (L6/7), along with the entire vif from SIVmac239 (NL-4/5S6/7SvifS). These SIVmac239-derived sequences were expected to protect the virus from HIV-1 restriction factors in monkey cells. However, the replicative capability of NL-4/5S6/7SvifS in human cells was severely impaired. By long-term cultivation of human CEM-SS cells infected with NL-4/5S6/7SvifS, we succeeded in partially rescuing the impaired replicative capability of the virus in human cells. This adapted virus encoded a G-to-E substitution at the 116(th) position of the CA (NL-4/5SG116E6/7SvifS). In the work described here, we explored the mechanism by which the replicative capability of NL-4/5S6/7SvifS was impaired in human cells. Quantitative analysis (by real-time PCR) of viral DNA synthesis from infected cells revealed that NL-4/5S6/7SvifS had a major defect in nuclear entry. Mutations in CA are known to affect viral core stability and result in deleterious effects in HIV-1 infection; therefore, we measured the kinetics of uncoating of these viruses. The uncoating of NL-4/5S6/7SvifS was significantly slower than that of wild type HIV-1 (WT), whereas the uncoating of NL-4/5SG116E6/7SvifS was similar to that of WT. Our results suggested that the lower replicative capability of NL-4/5S6/7SvifS in human cells was, at least in part, due to the slower uncoating of this virus. PMID:23967315

  9. The Depsipeptide Romidepsin Reverses HIV-1 Latency In Vivo

    PubMed Central

    Søgaard, Ole S.; Graversen, Mette E.; Leth, Steffen; Olesen, Rikke; Brinkmann, Christel R.; Nissen, Sara K.; Kjaer, Anne Sofie; Schleimann, Mariane H.; Denton, Paul W.; Hey-Cunningham, William J.; Koelsch, Kersten K.; Pantaleo, Giuseppe; Krogsgaard, Kim; Sommerfelt, Maja; Fromentin, Remi; Chomont, Nicolas; Rasmussen, Thomas A.; Østergaard, Lars; Tolstrup, Martin

    2015-01-01

    Pharmacologically-induced activation of replication competent proviruses from latency in the presence of antiretroviral treatment (ART) has been proposed as a step towards curing HIV-1 infection. However, until now, approaches to reverse HIV-1 latency in humans have yielded mixed results. Here, we report a proof-of-concept phase Ib/IIa trial where 6 aviremic HIV-1 infected adults received intravenous 5 mg/m2 romidepsin (Celgene) once weekly for 3 weeks while maintaining ART. Lymphocyte histone H3 acetylation, a cellular measure of the pharmacodynamic response to romidepsin, increased rapidly (maximum fold range: 3.7–7.7 relative to baseline) within the first hours following each romidepsin administration. Concurrently, HIV-1 transcription quantified as copies of cell-associated un-spliced HIV-1 RNA increased significantly from baseline during treatment (range of fold-increase: 2.4–5.0; p = 0.03). Plasma HIV-1 RNA increased from <20 copies/mL at baseline to readily quantifiable levels at multiple post-infusion time-points in 5 of 6 patients (range 46–103 copies/mL following the second infusion, p = 0.04). Importantly, romidepsin did not decrease the number of HIV-specific T cells or inhibit T cell cytokine production. Adverse events (all grade 1–2) were consistent with the known side effects of romidepsin. In conclusion, romidepsin safely induced HIV-1 transcription resulting in plasma HIV-1 RNA that was readily detected with standard commercial assays demonstrating that significant reversal of HIV-1 latency in vivo is possible without blunting T cell-mediated immune responses. These finding have major implications for future trials aiming to eradicate the HIV-1 reservoir. Trial Registration clinicaltrials.gov NTC02092116 PMID:26379282

  10. HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin.

    PubMed

    Romani, Bizhan; Kamali Jamil, Razieh; Hamidi-Fard, Mojtaba; Rahimi, Pooneh; Momen, Seyed Bahman; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2016-01-01

    HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter. PMID:27550312

  11. HIV-1 Vpr reactivates latent HIV-1 provirus by inducing depletion of class I HDACs on chromatin

    PubMed Central

    Romani, Bizhan; Kamali Jamil, Razieh; Hamidi-Fard, Mojtaba; Rahimi, Pooneh; Momen, Seyed Bahman; Aghasadeghi, Mohammad Reza; Allahbakhshi, Elham

    2016-01-01

    HIV-1 Vpr is an accessory protein that induces proteasomal degradation of multiple proteins. We recently showed that Vpr targets class I HDACs on chromatin for proteasomal degradation. Here we show that Vpr induces degradation of HDAC1 and HDAC3 in HIV-1 latently infected J-Lat cells. Degradation of HDAC1 and HDAC3 was also observed on the HIV-1 LTR and as a result, markers of active transcription were recruited to the viral promoter and induced viral activation. Knockdown of HDAC1 and HDAC3 activated the latent HIV-1 provirus and complementation with HDAC3 inhibited Vpr-induced HIV-1 reactivation. Viral reactivation and degradation of HDAC1 and HDAC3 was conserved among Vpr proteins of HV-1 group M. Serum Vpr isolated from patients or the release of virion-incorporated Vpr from viral lysates also activated HIV-1 in latently infected cell lines and PBMCs from HIV-1 infected patients. Our results indicate that Vpr counteracts HIV-1 latency by inducing proteasomal degradation of HDAC1 and 3 leading to reactivation of the viral promoter. PMID:27550312

  12. Specific interactions between HIV-1 nucleocapsid protein and the TAR element.

    PubMed

    Kanevsky, Igor; Chaminade, Françoise; Ficheux, Damien; Moumen, Abdeladim; Gorelick, Robert; Negroni, Matteo; Darlix, Jean-Luc; Fossé, Philippe

    2005-05-20

    During retroviral reverse transcription, the minus-strand strong-stop DNA (ss-cDNA) is transferred to the 3' end of the genomic RNA and this requires the repeat (R) sequences present at both ends of the genome. In vitro, the human immunodeficiency virus type 1 (HIV-1) R sequence can promote DNA strand transfer when present in ectopic internal positions. Using HIV-1 model systems, the R sequences and nucleocapsid protein (NC) were found to be key determinants of ss-cDNA transfer. To gain insights into specific interactions between HIV-1 NC and RNA and the influence of NC on R folding, we investigated the secondary structures of R in two natural contexts, namely at the 5' or 3' end of RNAs representing the terminal regions of the genome, and in two ectopic internal positions that also support efficient minus-strand transfer. To investigate the roles of NC zinc fingers and flanking basic domains in the NC/RNA interactions, we used NC mutants. Analyses of the viral RNA/NC complexes by chemical and enzymatic probings, and gel retardation assays were performed under conditions allowing ss-cDNA transfer by reverse transcriptase. We report that NC binds the TAR apical loop specifically in the four genetic contexts without changing the folding of the TAR hairpin and R region significantly, and this requires the NC zinc fingers. In addition, we show that efficient annealing of cTAR DNA to the 3' R relies on sequence complementarities between TAR and cTAR terminal loops. These findings suggest that the TAR apical loop in the acceptor RNA is the initiation site for the annealing reaction that is chaperoned by NC during the minus-strand transfer. PMID:15854644

  13. Mechanistic evaluation of new plant-derived compounds that inhibit HIV-1 reverse transcriptase.

    PubMed

    Pengsuparp, T; Cai, L; Constant, H; Fong, H H; Lin, L Z; Kinghorn, A D; Pezzuto, J M; Cordell, G A; Ingolfsdóttir, K; Wagner, H

    1995-07-01

    Swertifrancheside [1], a new flavonone-xanthone glucoside isolated from Swertia franchetiana, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2], a triterpene isolated from the roots of Maprounea africana, and protolichesterinic acid [3], an aliphatic alpha-methylene-gamma-lactone isolated from the lichen Cetraria islandica, were found to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT), with 50% inhibitory doses (IC50 values) of 43, 3.7, and 24 microM, respectively. They were not cytotoxic with cultured mammalian cells. The kinetic mechanisms by which compounds 1-3 inhibited HIV-1 RT were studied as was their potential to inhibit other nucleic acid polymerases. Swertifrancheside [1] bound to DNA and was shown to be a competitive inhibitor with respect to template-primer, but a mixed-type competitive inhibitor with respect to TTP. On the other hand, 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] were mixed-type competitive inhibitors with respect to template-primer and noncompetitive inhibitors with respect to TTP. Therefore, the mechanism of action of 1 beta-hydroxyaleuritolic acid 3-p-hydroxybenzoate [2] and protolichesterinic acid [3] as HIV-1 RT inhibitors involves nonspecific binding to the enzyme at nonsubstrate binding sites, whereas swertifrancheside [1] inhibits enzyme activity by binding to the template-primer. PMID:7561895

  14. TNPO3 Is Required for HIV-1 Replication after Nuclear Import but prior to Integration and Binds the HIV-1 Core

    PubMed Central

    Valle-Casuso, Jose Carlos; Di Nunzio, Francesca; Yang, Yang; Reszka, Natalia; Lienlaf, Maritza; Arhel, Nathalie; Perez, Patricio; Brass, Abraham L.

    2012-01-01

    TNPO3 is a nuclear importer required for HIV-1 infection. Here, we show that depletion of TNPO3 leads to an HIV-1 block after nuclear import but prior to integration. To investigate the mechanistic requirement of TNPO3 in HIV-1 infection, we tested the binding of TNPO3 to the HIV-1 core and found that TNPO3 binds to the HIV-1 core. Overall, this work suggests that TNPO3 interacts with the incoming HIV-1 core in the cytoplasm to assist a process that is important for HIV-1 infection after nuclear import. PMID:22398280

  15. Anti-HIV-1 Activity of Flavonoid Myricetin on HIV-1 Infection in a Dual-Chamber In Vitro Model

    PubMed Central

    Pasetto, Silvana; Pardi, Vanessa; Murata, Ramiro Mendonça

    2014-01-01

    HIV infection by sexual transmission remains an enormous global health concern. More than 1 million new infections among women occur annually. Microbicides represent a promising prevention strategy that women can easily control. Among emerging therapies, natural small molecules such as flavonoids are an important source of new active substances. In this study we report the in vitro cytotoxicity and anti-HIV-1 and microbicide activity of the following flavonoids: Myricetin, Quercetin and Pinocembrin. Cytotoxicity tests were conducted on TZM-bl, HeLa, PBMC, and H9 cell cultures using 0.01–100 µM concentrations. Myricetin presented the lowest toxic effect, with Quercetin and Pinocembrin relatively more toxic. The anti-HIV-1 activity was tested with TZM-bl cell plus HIV-1 BaL (R5 tropic), H9 and PBMC cells plus HIV-1 MN (X4 tropic), and the dual tropic (X4R5) HIV-1 89.6. All flavonoids showed anti-HIV activity, although Myricetin was more effective than Quercetin or Pinocembrin. In TZM-bl cells, Myricetin inhibited ≥90% of HIV-1 BaL infection. The results were confirmed by quantification of HIV-1 p24 antigen in supernatant from H9 and PBMC cells following flavonoid treatment. In H9 and PBMC cells infected by HIV-1 MN and HIV-1 89.6, Myricetin showed more than 80% anti-HIV activity. Quercetin and Pinocembrin presented modest anti-HIV activity in all experiments. Myricetin activity was tested against HIV-RT and inhibited the enzyme by 49%. Microbicide activities were evaluated using a dual-chamber female genital tract model. In the in vitro microbicide activity model, Myricetin showed promising results against different strains of HIV-1 while also showing insignificant cytotoxic effects. Further studies of Myricetin should be performed to identify its molecular targets in order to provide a solid biological foundation for translational research. PMID:25546350

  16. Elimination of helminth infection restores HIV-1C vaccine specific T cell responses independent of helminth-induced IL-10

    PubMed Central

    Da’dara, Akram A.; Harn, Donald A.

    2009-01-01

    HIV-1 prevalence is highest in developing countries; similarly helminth parasites are often highly endemic in these same areas. Helminths are strong immune modulators, and negatively impact the ability of the infected hosts to mount protective vaccine specific T cell immune responses for HIV-1 and other pathogens. Indeed, previously we found that Schistosoma mansoni infected mice had significantly impaired HIV-1C vaccine specific T cell responses. Anthelminthics are available and inexpensive; therefore, in this study, we evaluated whether elimination of schistosome infection prior to vaccination with an HIV-1C DNA vaccine would increase recipients vaccine specific responses. As expected, splenocytes from S. mansoni infected mice produced significantly elevated amounts of interleukin (IL)-4 and IL-10, and significantly lower amounts of interferon (IFN)-γ than splenocytes from naïve mice. Following elimination of parasites by praziquantel (PZQ) treatment, splenomegaly was significantly reduced, though splenocytes produced similar or higher levels of IL-10 than splenocytes from infected mice. However, we found that PZQ treatment significantly increased levels of IFN-γ in response to Concanavalin A or SEA compared to splenocytes from untreated mice. Importantly, PZQ treatment resulted in complete restoration of HIV-1C vaccine specific T cell responses at 8 weeks post PZQ treatment. Restoration of HIV-1C vaccine specific T cell responses following elimination of helminth infection was time dependent, but surprisingly independent of the levels of IL-4 and IL-10 induced by parasite antigens. Our study shows that elimination of worms offers an affordable and a simple means to restore immune responsiveness to T cell based vaccines for HIV-1 and other infectious diseases in helminth endemic settings. PMID:19941988

  17. Polymorphisms of CUL5 are associated with CD4+ T cell loss in HIV-1 infected individuals.

    PubMed

    An, Ping; Duggal, Priya; Wang, Li Hua; O'Brien, Stephen J; Donfield, Sharyne; Goedert, James J; Phair, John; Buchbinder, Susan; Kirk, Gregory D; Winkler, Cheryl A

    2007-01-26

    Human apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like 3 (Apobec3) antiretroviral factors cause hypermutation of proviral DNA leading to degradation or replication-incompetent HIV-1. However, HIV-1 viral infectivity factor (Vif) suppresses Apobec3 activity through the Cullin 5-Elongin B-Elongin C E3 ubiquitin ligase complex. We examined the effect of genetic polymorphisms in the CUL5 gene (encoding Cullin 5 protein) on AIDS disease progression in five HIV-1 longitudinal cohorts. A total of 12 single nucleotide polymorphisms (SNPs) spanning 93 kb in the CUL5 locus were genotyped and their haplotypes inferred. A phylogenetic network analysis revealed that CUL5 haplotypes were grouped into two clusters of evolutionarily related haplotypes. Cox survival analysis and mixed effects models were used to assess time to AIDS outcomes and CD4(+) T cell trajectories, respectively. Relative to cluster I haplotypes, the collective cluster II haplotypes were associated with more rapid CD4(+) T cell loss (relative hazards [RH] = 1.47 and p = 0.009), in a dose-dependent fashion. This effect was mainly attributable to a single cluster II haplotype (Hap10) (RH = 2.49 and p = 0.00001), possibly due to differential nuclear protein-binding efficiencies of a Hap10-specifying SNP as indicated by a gel shift assay. Consistent effects were observed for CD4(+) T cell counts and HIV-1 viral load trajectories over time. The findings of both functional and genetic epidemiologic consequences of CUL5 polymorphism on CD4(+) T cell and HIV-1 levels point to a role for Cullin 5 in HIV-1 pathogenesis and suggest interference with the Vif-Cullin 5 pathway as a possible anti-HIV-1 therapeutic strategy. PMID:17257057

  18. Stochastic extinction dynamics of HIV-1

    NASA Astrophysics Data System (ADS)

    Schwartz, Ira; Forgoston, Eric; Weinberger, Leor

    2012-02-01

    We consider an HIV-1 within host model in which T cells are infected by the virus. Due to small numbers of molecules, stochastic effects play an important role in the dynamical outcomes in that two states are observed experimentally: a replication state in which the virus is active, or a dormant state leading to latency in which the virus becomes active after a delay. The two states are conjectured to be governed by the Tat gene protein transcription process, which does not possess two stable attractors. Rather, the active state is stable, while the dormant state is unstable. Therefore the dormant state can only be achieved through the dynamics of stochastic fluctuations in which noise organizes a path to dormancy. Here we use optimal path theory applied to a Tat gene stochastic model to show how random fluctuations generate the dormant state by deriving a path which optimizes the probability of achieving the dormant state. We explicitly show how the probability of achieving dormancy scales with the transition rate parameters.

  19. Bifunctional Inhibition of HIV-1 Reverse Transcriptase: A First Step in Designing a Bifunctional Triphosphate‡

    PubMed Central

    Piao, Dongyuan; Basavapathruni, Aravind; Iyidogan, Pinar; Dai, Guangxiu; Hinz, Wolfgang; Ray, Adrian S.; Murakami, Eisuke; Feng, Joy Y.; You, Fei; Dutschman, Ginger E.; Austin, David J.; Parker, Kathlyn A.; Anderson, Karen S.

    2013-01-01

    The onset of resistance to approved anti-AIDS drugs by HIV necessitates the search for novel inhibitors of HIV-1 reverse transcriptase (RT). Developing single molecular agents concurrently occupying the nucleoside and nonnucleoside binding sites in RT is an intriguing idea but the proof-of-concept has so far been elusive. As a first step, we describe molecular modeling to guide focused chemical syntheses of conjugates having nucleoside (d4T) and nonnucleoside (TIBO) moieties tethered by a flexible polyethylene glycol (PEG) linker. A triphosphate of d4T-6PEG-TIBO conjugate was successfully synthesized that is recognized as a substrate by HIV-1 RT and incorporated into a double-stranded DNA. PMID:23380374

  20. HIV-1-inhibiting activity of the essential oil of Ridolfia segetum and Oenanthe crocata.

    PubMed

    Bicchi, Carlo; Rubiolo, Patrizia; Ballero, Mauro; Sanna, Cinzia; Matteodo, Maura; Esposito, Francesca; Zinzula, Luca; Tramontano, Enzo

    2009-10-01

    The essential oils of Ridolfia segetum (L.) Moris and Oenanthe crocata L. (Apiaceae), collected in Sardinia (Italy), have been assayed for two enzyme-associated activities of the HIV-1 reverse transcriptase (RT): RNA-dependent DNA polymerase (RDDP) activity and ribonuclease H (RNase H) activity. In biochemical assays, the essential oils inhibited HIV-1 RT RDDP activity in a dose-dependent manner, while they were inactive towards RNase H activity. Furthermore, the oils were cytotoxic towards K (562) cell replication. GC-MS analysis of the essential oils obtained by steam distillation of the aerial parts showed that the main components of R. segetum were alpha-phellandrene, alpha-terpinolene, beta-phellandrene, and dillapiol and those of O. crocata were sabinene, TRANS-beta-ocimene, CIS-beta-ocimene, and beta-pinene. PMID:19347799

  1. Identification of Siglec-1 null individuals infected with HIV-1

    PubMed Central

    Martinez-Picado, Javier; McLaren, Paul J.; Erkizia, Itziar; Martin, Maureen P.; Benet, Susana; Rotger, Margalida; Dalmau, Judith; Ouchi, Dan; Wolinsky, Steven M.; Penugonda, Sudhir; Günthard, Huldrych F.; Fellay, Jacques; Carrington, Mary; Izquierdo-Useros, Nuria; Telenti, Amalio

    2016-01-01

    Siglec-1/CD169 is a myeloid-cell surface receptor critical for HIV-1 capture and infection of bystander target cells. To dissect the role of SIGLEC1 in natura, we scan a large population genetic database and identify a loss-of-function variant (Glu88Ter) that is found in ∼1% of healthy people. Exome analysis and direct genotyping of 4,233 HIV-1-infected individuals reveals two Glu88Ter homozygous and 97 heterozygous subjects, allowing the analysis of ex vivo and in vivo consequences of SIGLEC1 loss-of-function. Cells from these individuals are functionally null or haploinsufficient for Siglec-1 activity in HIV-1 capture and trans-infection ex vivo. However, Siglec-1 protein truncation does not have a measurable impact on HIV-1 acquisition or AIDS outcomes in vivo. This result contrasts with the known in vitro functional role of Siglec-1 in HIV-1 trans-infection. Thus, it provides evidence that the classical HIV-1 infectious routes may compensate for the lack of Siglec-1 in fuelling HIV-1 dissemination within infected individuals. PMID:27510803

  2. Macrophage Internal HIV-1 Is Protected from Neutralizing Antibodies

    PubMed Central

    Koppensteiner, Herwig; Banning, Carina; Schneider, Carola; Hohenberg, Heinrich

    2012-01-01

    In macrophages, HIV-1 accumulates in intracellular vesicles designated virus-containing compartments (VCCs). These might play an important role in the constitution of macrophages as viral reservoirs and allow HIV-1 to evade the immune system by sequestration in an internal niche, which is difficult to access from the exterior. However, until now, evidence of whether internal virus accumulations are protected from the host's humoral immune response is still lacking. In order to be able to study the formation and antibody accessibility of VCCs, we generated HIV-1 with green fluorescent protein (GFP)-tagged Gag replicating in primary macrophages. Live-cell observations revealed faint initial cytosolic Gag expression and subsequent large intracellular Gag accumulations which stayed stable over days. Taking advantage of the opportunity to study the accessibility of intracellular VCCs via the cell surface, we demonstrate that macrophage internal HIV-1-containing compartments cannot be targeted by neutralizing antibodies. Furthermore, HIV-1 was efficiently transferred from antibody-treated macrophages to T cells. Three-dimensional reconstruction of electron microscopic slices revealed that Gag accumulations correspond to viral particles within enclosed compartments and convoluted membranes. Thus, although some VCCs were connected to the plasma membrane, the complex membrane architecture of the HIV-1-containing compartment might shield viral particles from neutralizing antibodies. In sum, our study provides evidence that HIV-1 is sequestered into a macrophage internal membranous web, posing an obstacle for the elimination of this viral reservoir. PMID:22205742

  3. Sex and gender differences in HIV-1 infection.

    PubMed

    Griesbeck, Morgane; Scully, Eileen; Altfeld, Marcus

    2016-08-01

    The major burden of the human immunodeficiency (HIV) type 1 pandemic is nowadays carried by women from sub-Saharan Africa. Differences in the manifestations of HIV-1 infection between women and men have been long reported, and might be due to both socio-economic (gender) and biological (sex) factors. Several studies have shown that women are more susceptible to HIV-1 acquisition than men. Following HIV-1 infection, women have lower viral loads during acute infection and exhibit stronger antiviral responses than men, which may contribute to differences in the size of viral reservoirs. Oestrogen receptor signalling could represent an important mediator of sex differences in HIV-1 reservoir size and may represent a potential therapeutic target. Furthermore, immune activation, a hallmark of HIV-1 infection, is generally higher in women than in men and could be a central mechanism in the sex difference observed in the speed of HIV-1 disease progression. Here, we review the literature regarding sex-based differences in HIV-1 infection and discuss how a better understanding of the underlying mechanisms could improve preventive and therapeutic strategies. PMID:27389589

  4. Identification of Siglec-1 null individuals infected with HIV-1.

    PubMed

    Martinez-Picado, Javier; McLaren, Paul J; Erkizia, Itziar; Martin, Maureen P; Benet, Susana; Rotger, Margalida; Dalmau, Judith; Ouchi, Dan; Wolinsky, Steven M; Penugonda, Sudhir; Günthard, Huldrych F; Fellay, Jacques; Carrington, Mary; Izquierdo-Useros, Nuria; Telenti, Amalio

    2016-01-01

    Siglec-1/CD169 is a myeloid-cell surface receptor critical for HIV-1 capture and infection of bystander target cells. To dissect the role of SIGLEC1 in natura, we scan a large population genetic database and identify a loss-of-function variant (Glu88Ter) that is found in ∼1% of healthy people. Exome analysis and direct genotyping of 4,233 HIV-1-infected individuals reveals two Glu88Ter homozygous and 97 heterozygous subjects, allowing the analysis of ex vivo and in vivo consequences of SIGLEC1 loss-of-function. Cells from these individuals are functionally null or haploinsufficient for Siglec-1 activity in HIV-1 capture and trans-infection ex vivo. However, Siglec-1 protein truncation does not have a measurable impact on HIV-1 acquisition or AIDS outcomes in vivo. This result contrasts with the known in vitro functional role of Siglec-1 in HIV-1 trans-infection. Thus, it provides evidence that the classical HIV-1 infectious routes may compensate for the lack of Siglec-1 in fuelling HIV-1 dissemination within infected individuals. PMID:27510803

  5. Engineered Bispecific Antibodies with Exquisite HIV-1-Neutralizing Activity.

    PubMed

    Huang, Yaoxing; Yu, Jian; Lanzi, Anastasia; Yao, Xin; Andrews, Chasity D; Tsai, Lily; Gajjar, Mili R; Sun, Ming; Seaman, Michael S; Padte, Neal N; Ho, David D

    2016-06-16

    While the search for an efficacious HIV-1 vaccine remains elusive, emergence of a new generation of virus-neutralizing monoclonal antibodies (mAbs) has re-ignited the field of passive immunization for HIV-1 prevention. However, the plasticity of HIV-1 demands additional improvements to these mAbs to better ensure their clinical utility. Here, we report engineered bispecific antibodies that are the most potent and broad HIV-neutralizing antibodies to date. One bispecific antibody, 10E8V2.0/iMab, neutralized 118 HIV-1 pseudotyped viruses tested with a mean 50% inhibitory concentration (IC50) of 0.002 μg/mL. 10E8V2.0/iMab also potently neutralized 99% of viruses in a second panel of 200 HIV-1 isolates belonging to clade C, the dominant subtype accounting for ∼50% of new infections worldwide. Importantly, 10E8V2.0/iMab reduced virus load substantially in HIV-1-infected humanized mice and also provided complete protection when administered prior to virus challenge. These bispecific antibodies hold promise as novel prophylactic and/or therapeutic agents in the fight against HIV-1. PMID:27315479

  6. 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

  7. Defining the roles for Vpr in HIV-1-associated neuropathogenesis.

    PubMed

    James, Tony; Nonnemacher, Michael R; Wigdahl, Brian; Krebs, Fred C

    2016-08-01

    It is increasingly evident that the human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) has a unique role in neuropathogenesis. Its ability to induce G2/M arrest coupled with its capacity to increase viral gene transcription gives it a unique role in sustaining viral replication and aiding in the establishment and maintenance of a systemic infection. The requirement of Vpr for HIV-1 infection and replication in cells of monocytic origin (a key lineage of cells involved in HIV-1 neuroinvasion) suggests an important role in establishing and sustaining infection in the central nervous system (CNS). Contributions of Vpr to neuropathogenesis can be expanded further through (i) naturally occurring HIV-1 sequence variation that results in functionally divergent Vpr variants; (ii) the dual activities of Vpr as a intracellular protein delivered and expressed during HIV-1 infection and as an extracellular protein that can act on neighboring, uninfected cells; (iii) cell type-dependent consequences of Vpr expression and exposure, including cell cycle arrest, metabolic dysregulation, and cytotoxicity; and (iv) the effects of Vpr on exosome-based intercellular communication in the CNS. Revealing that the effects of this pleiotropic viral protein is an essential part of a greater understanding of HIV-1-associated pathogenesis and potential approaches to treating and preventing disease caused by HIV-1 infection. PMID:27056720

  8. Recent Advances in Lentiviral Vaccines for HIV-1 Infection

    PubMed Central

    Norton, Thomas D.; Miller, Elizabeth A.

    2016-01-01

    The development of an effective HIV vaccine to prevent and/or cure HIV remains a global health priority. Given their central role in the initiation of adaptive immune responses, dendritic cell (DC)-based vaccines are being increasingly explored as immunotherapeutic strategies to enhance HIV-specific T cells in infected individuals and, thus, promote immune responses that may help facilitate a functional cure. HIV-1-based lentiviral (LV) vectors have inherent advantages as DC vaccine vectors due to their ability to transduce non-dividing cells and integrate into the target cell genomic DNA, allowing for expression of encoded antigens over the lifespan of the cell. Moreover, LV vectors may express additional immunostimulatory and immunoregulatory proteins that enhance DC function and direct antigen-specific T cells responses. Recent basic and clinical research efforts have broadened our understanding of LV vectors as DC-based vaccines. In this review, we provide an overview of the pre-clinical and clinical LV vector vaccine studies for treating HIV to date. We also discuss advances in LV vector designs that have enhanced DC transduction efficiency, target cell specificity, and immunogenicity, and address potential safety concerns regarding LV vector-based vaccines. PMID:27446074

  9. Extreme Genetic Fragility of the HIV-1 Capsid

    PubMed Central

    Rihn, Suzannah J.; Wilson, Sam J.; Loman, Nick J.; Alim, Mudathir; Bakker, Saskia E.; Bhella, David; Gifford, Robert J.; Rixon, Frazer J.; Bieniasz, Paul D.

    2013-01-01

    Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses. Although CA participates in several steps in HIV-1 replication, analysis of conditionally (temperature sensitive) and constitutively non-viable mutants revealed that the biological basis for its genetic fragility was primarily the need to coordinate the accurate and efficient assembly of mature virions. All mutations that exist in naturally occurring HIV-1 subtype B populations at a frequency >3%, and were also present in the mutant library, had fitness levels that were >40% of WT. However, a substantial fraction of mutations with high fitness did not occur in natural populations, suggesting another form of selection pressure limiting variation in vivo. Additionally, known protective CTL epitopes occurred preferentially in domains of the HIV-1 CA that were even more genetically fragile than HIV-1 CA as a whole. The extreme genetic fragility of HIV-1 CA may be one reason why cell-mediated immune responses to Gag correlate with better prognosis in HIV-1 infection, and suggests that CA is a good target for therapy and vaccination strategies

  10. Extreme genetic fragility of the HIV-1 capsid.

    PubMed

    Rihn, Suzannah J; Wilson, Sam J; Loman, Nick J; Alim, Mudathir; Bakker, Saskia E; Bhella, David; Gifford, Robert J; Rixon, Frazer J; Bieniasz, Paul D

    2013-01-01

    Genetic robustness, or fragility, is defined as the ability, or lack thereof, of a biological entity to maintain function in the face of mutations. Viruses that replicate via RNA intermediates exhibit high mutation rates, and robustness should be particularly advantageous to them. The capsid (CA) domain of the HIV-1 Gag protein is under strong pressure to conserve functional roles in viral assembly, maturation, uncoating, and nuclear import. However, CA is also under strong immunological pressure to diversify. Therefore, it would be particularly advantageous for CA to evolve genetic robustness. To measure the genetic robustness of HIV-1 CA, we generated a library of single amino acid substitution mutants, encompassing almost half the residues in CA. Strikingly, we found HIV-1 CA to be the most genetically fragile protein that has been analyzed using such an approach, with 70% of mutations yielding replication-defective viruses.