A highly sensitive aptamer-based HIV reverse transcriptase detection assay.

Although many new assays for HIV have been developed, several labs still use simple and reliable radioactivity-based reverse transcriptase (RT) nucleotide incorporation assays for detection and quantification. We describe here a new assay for detection and quantitation of HIV RT activity that is based on a high affinity DNA aptamer to RT. The aptamer is sequestered on 96-well plates where it can bind to RT and other constituents can be removed by extensive washing. Since the aptamer mimics a primer-template, upon radiolabeled nucleotide addition, bound RT molecules can extend the aptamer and the radioactive signal can be detected by standard methods. In addition to being procedurally simple, the assay demonstrated high sensitivity (detection limits for RT and virions were ≤6400 molecules (∼4 × 10-8 units) and ∼100-300 virions, respectively) and was essentially linear over a range of at least 104. Both wild type and drug-resistant forms of HIV-1 RT were detectable as was HIV-2 RT, although there were some modest differences in sensitivity.

Quantitative Resolution of Monomer-Dimer Populations by Inversion Modulated DEER EPR Spectroscopy.

A simple method, based on inversion modulated double electron-electron resonance electron paramagnetic resonance (DEER EPR) spectroscopy, is presented for determining populations of monomer and dimer in proteins (as well as any other biological macromolecules). The method is based on analysis of modulation depth versus electron double resonance (ELDOR) pulse flip angle. High accuracy is achieved by complete deuteration, extensive sampling of a large number of ELDOR pulse flip angle values, and combined analysis of differently labeled spin samples. We demonstrate the method using two different proteins: an obligate monomer exemplified by the small immunoglobulin binding B domain of protein A, and the p66 subunit of HIV-1 reverse transcriptase which exists as an equilibrium mixture of monomer and dimer species whose relative populations are affected by glycerol content. This information is crucial for quantitative analysis of distance distributions involving proteins that may exist as mixtures of monomer, dimer and high order multimers under the conditions of the DEER EPR experiment.

Differential isotopic enrichment to facilitate characterization of asymmetric multimeric proteins using hydrogen/deuterium exchange mass spectrometry.

Hydrogen/deuterium exchange (HDX) coupled to mass spectrometry has emerged as a powerful tool for analyzing the conformational dynamics of protein-ligand and protein-protein interactions. Recent advances in instrumentation and methodology have expanded the utility of HDX for the analysis of large and complex proteins; however, asymmetric dimers with shared amino acid sequence present a unique challenge for HDX because assignment of peptides with identical sequence to their subunit of origin remains ambiguous. Here we report the use of differential isotopic labeling to facilitate HDX analysis of multimers using HIV-1 reverse transcriptase (RT) as a model. RT is an asymmetric heterodimer of 51 kDa (p51) and 66 kDa (p66) subunits. The first 440 residues of p51 and p66 are identical. In this study differentially labeled RT was reconstituted from isotopically enriched ((15)N-labeled) p51 and unlabeled p66. To enable detection of (15)N-deuterated RT peptides, the software HDX Workbench was modified to follow a 100% (15)N model. Our results demonstrated that (15)N enrichment of p51 did not affect its conformational dynamics compared to unlabeled p51, but (15)N-labeled p51 did show different conformational dynamics than p66 in the RT heterodimer. Differential HDX-MS of isotopically labeled RT in the presence of the non-nucleoside reverse transcriptase inhibitor (NNRTI) efavirenz (EFV) showed subunit-specific perturbation in the rate of HDX consistent with previously published results and the RT-EFV cocrystal structure.

RIG-I activation inhibits HIV replication in macrophages.

The RIG-I signaling pathway is critical in the activation of the type I IFN-dependent antiviral innate-immune response. We thus examined whether RIG-I activation can inhibit HIV replication in macrophages. We showed that the stimulation of monocyte-derived macrophages with 5'ppp-dsRNA, a synthetic ligand for RIG-I, induced the expression of RIG-I, IFN-α/ß, and several IRFs, key regulators of the IFN signaling pathway. In addition, RIG-I activation induced the expression of multiple intracellular HIV-restriction factors, including ISGs, several members of the APOBEC3 family, tetherin and CC chemokines, the ligands for HIV entry coreceptor (CCR5). The inductions of these factors were associated with the inhibition of HIV replication in macrophages stimulated by 5'ppp-dsRNA. These observations highlight the importance of RIG-I signaling in macrophage innate immunity against HIV, which can be beneficial for the treatment of HIV disease, where intracellular immune defense is compromised by the virus.

Understanding providers' offering and patients' acceptance of HIV screening in emergency departments: a multilevel analysis. ANRS 95008, Paris, France.

OBJECTIVE: We assessed the EDs' characteristics associated with the offer and acceptance rates of a nontargeted HIV rapid-test screening in 29 Emergency Departments (EDs) in the metropolitan Paris region (11.7 million inhabitants), where half of France's new HIV cases are diagnosed annually. METHODS: EDs nurses offered testing to all patients 18-64-year-old, able to provide consent, either with or without supplemental staff (hybrid staff model or indigenous staff model). The EDS' characteristics collected included structural characteristics (location, type, size), daily workload (patients' number and severity, length of stay in hours), staff's participation (training, support to the intervention, leadership), type of week day (weekends vs weekdays) and time (in days). Associations between these variables and the staff model, the offer and acceptance rates were studied using multilevel modeling. RESULTS: Indigenous staff model was more frequent in EDs with a lower daily patient flow and a higher staff support score to the intervention. In indigenous-model EDs, the offer rate was associated with the patient flow (OR = 0.838, 95% CI = 0.773-0.908), was lower during weekends (OR = 0.623, 95% CI = 0.581-0.667) and decreased over time (OR = 0.978, 95% CI = 0.975-0.981). Similar results were found in hybrid-model EDs. Acceptance was poorly associated with EDs characteristics in indigenous-model EDs while in hybrid-model EDs it was lower during weekends (OR = 0.713, 95% CI = 0.623-0.816) and increased after the first positive test (OR = 1.526, 95% CI = 1.142-2.038). The EDs' characteristics explained respectively 38.5% and 15% of the total variance in the offer rate across indigenous model-EDs and hybrid model-EDs vs 12% and 1% for the acceptance rate. CONCLUSION: Our findings suggest the need for taking into account EDs' characteristics while considering the implementation of an ED-based HIV screening program. Strategies allowing the optimization of human resources' utilization such as HIV targeted screening in the EDs might be privileged.

Involvement of human topoisomerase II isoforms in HIV-1 reverse transcription.

HIV-1 reverse transcription (RTn) involves synthesis of double strand DNA (dsDNA) from viral genomic RNA. Topoisomerase II (Topo II) alpha and beta maintains topological reorganization of dsDNA regions and catalytic inhibition of these isoforms repressed viral replicative cycle. Present study is aimed to understand the role of Topo II isoforms in HIV-1 early replication. Topo IIα and ß showed differential expression in SupT1 cells and PBMCs during early hours of HIV-1 infection where Topo IIα expression increased after 4h, while Topo IIß showed relatively higher expression at 1 and 4h. In Topo IIα and/or ß down regulated cells, transcription of viral genes gag, pol and env as well as proviral DNA synthesis was abolished. In Topo IIα and/or ß down regulated cells, strong stop DNA synthesis was unaffected while other downstream events of reverse transcription such as first strand transfer, full length minus strand synthesis, and second strand transfer were completely inhibited, which affects HIV-1 replication. Further, co-localization of Topo II isoforms with HIV-1 reverse transcriptase was observed in SupT1 cells and PBMCs by immunofluorescence. These results collectively suggest a role of Topo II isoforms during HIV-1 RTn probably by promoting the alignment of viral RNA-DNA hybrids.

Maraviroc and other HIV-1 entry inhibitors exhibit a class-specific redistribution effect that results in increased extracellular viral load.

HIV entry inhibitors, such as maraviroc (MVC), prevent cell-free viruses from entering the cells. In clinical trials, patients who were treated with MVC often displayed viral loads that were above the limit of conventional viral load detection compared to efavirenz-based regimens. We hypothesize that viruses blocked by entry inhibitors may be redistributed to plasma, where they artificially increase viral load measurements compared to those with the use of antiretroviral drugs (ARVs) that act intracellularly. We infected PM-1 cells with CCR5-tropic HIV-1 BaL or CXCR4-tropic HIV-1 NL4-3 in the presence of inhibitory concentrations of efavirenz, raltegravir, enfuvirtide, maraviroc, and AMD3100, the latter three being entry inhibitors. Supernatant viral load, reverse transcriptase enzyme activity, and intracellular nucleic acid levels were measured at times up to 24 h postinfection. Infectivity of redistributed dual-tropic HIV-1 was assessed using TZM-bl cells. Extracellular viral load analysis revealed that entry inhibitor-treated cells had higher levels of virus in the supernatant than the cells treated with other ARVs at 8 h postinfection. By 24 h, the supernatant viral load was still higher for entry inhibitors than other ARVs. We observed a correlation between viral load and the step of entry inhibition. Dual-tropic virus infectivity was undiminished utilizing the CCR5 coreceptor following redistribution by CXCR4 entry inhibition. This in vitro model indicates that entry inhibitors exhibit a redistribution effect unseen with intracellular ARV drugs. Based on these results, the effectiveness of some entry inhibitors may be underestimated if plasma viral load is used as a sole indicator of clinical success.

A BioDVD media with multilayered structure is suitable for analyzing biomolecular interactions.

A biomolecular interactive analysis with antibody-antigen and aptamer-protein was evaluated on Au-over layers deposited on the BioDVD surface. BioDVD consists of multilayered structures with Au layer on the top and it detects analytes by monitoring the changes in reflected light intensity due to analyte adsorption to the sensor surface, on which functional biomolecules are immobilized to bind specifically to the analytes. The BioDVD sensing instrument is based on a commercial digital versatile disc system, which allows the instrument to be small and inexpensive. The BioDVD platform can be fabricated utilizing mass production techniques with additional functional phase change layers that can serve both to enhance sensitivity by optimization of the interferometric cavity optical properties and also as a possible medium for the storage of test related information.

Continuous signal enhancement for sensitive aptamer affinity probe electrophoresis assay using electrokinetic concentration.

We describe an electrokinetic concentration-enhanced aptamer affinity probe electrophoresis assay to achieve highly sensitive and quantitative detection of protein targets in a microfluidic device. The key weaknesses of aptamer as a binding agent (weak binding strength/fast target dissociation) were counteracted by continuous injection of fresh sample while band-broadening phenomena were minimized due to self-focusing effects. With 30 min of continuous signal enhancement, we can detect 4.4 pM human immunoglobulin E (IgE) and 9 pM human immunodeficiency virus 1 reverse transcriptase (HIV-1 RT), which are among the lowest limits of detection (LOD) reported. IgE was detected in serum sample with a LOD of 39 pM due to nonspecific interactions between aptamers and serum proteins. The method presented in this paper also has broad applicability to improve sensitivities of various other mobility shift assays.

Aptamer beacons for visualization of endogenous protein HIV-1 reverse transcriptase in living cells.

Direct visualization of endogenous proteins in living cells remains a challenge. Aptamer beacon is a promising technique to resolve this problem by combining the excellent protein binding specificity of the aptamer with the sensitive signal transduction mechanism of the molecular beacon. In this study, aptamer 93 del against HIV-1 reverse transcriptase (RT) was engineered into aptamer beacons to recognize and image HIV-1 RT. The constructed aptamer beacons could specifically bind to HIV-1 RT and the beacon-RT binding showed effective fluorescence signal transduction in homogeneous solution. In solutions with 1 µM of the aptamer beacon, the effective fluorescence signal increased with increasing concentration of HIV-1 RT from 0.5 µM to 5 µM. When the aptamer beacons were delivered into the living cells that transiently expressed HIV-1 RT, HIV-1 RT could be specifically labeled and imaged. The designed aptamer beacons were further successfully applied for RT imaging in HIV-1 integrated U1 cells. The method developed here may be extended to visualize many other endogenous proteins in living cells using appropriate aptamer beacons.

Identification of alternative binding sites for inhibitors of HIV-1 ribonuclease H through comparative analysis of virtual enrichment studies.

The ribonuclease H (RNase H) domain on the p66 monomer of HIV-1 reverse transcriptase enzyme has become a target for inhibition. The active site is one potential binding site, but other RNase H sites can accommodate inhibitors. Using a combination of experimental and computational studies, potential new binding sites and binding modes have been identified. Libraries of compounds were screened with an experimental assay to identify actives without knowledge of the binding site. The compounds were computationally docked at putative binding sites. Based on positive enrichment of natural-product actives relative to the database of compounds, we propose that many inhibitors bind to an alternative, potentially allosteric, site centered on Q507 of p66. For a series of hydrazone compounds, a small amount of positive enrichment was obtained when active compounds were bound by induced-fit docking at the interface between the DNA:RNA substrate and the RNase H domain near residue Q500.

Towards an early diagnosis of HIV infection: an electrochemical approach for detection of HIV-1 reverse transcriptase enzyme.

Oriented for the rapid diagnosis of HIV infection, highly sensitive and facile electrochemical assays for HIV-1 reverse transcriptase (HIV1-RT) are presented in this article. A non-labeled and a labeled assay format were based on the formation of self-assembled monolayers (SAMs) of either lipoic acid active ester or a newly synthesized ferrocene (Fc)-labeled cystamine derivative on electrode surfaces, respectively. A short RT-specific peptide, VEAIIRILQQLLFIH, was covalently attached to the surface of the formed SAMs. Electrochemical impedance spectroscopy (EIS) allowed a sensitive interrogation of RT in the non-labeled assay format. Furthermore, square wave voltammetry (SWV) offered a two-dimensional measurement of RT based on the anodic shift and reduction of current density of the Fc redox signal upon binding of RT to its specific peptide. These techniques allowed a linear quantification of the target RT in the range of 75 to 750 pg mL(-1), with a limit of detection of 50 pg mL(-1). Furthermore, the developed biosensors showed a good specificity and allowed a proper discrimination between RT and other HIV enzymes.

Short communication: Molecular epidemiology of HIV type 1 in the Republic of Dagestan, Russian Federation: virtually uniform circulation of subtype A, former Soviet Union variant, with predominance of the V77I(PR) subvariant.

We examine the distribution of viral genetic forms and the presence of antiretroviral drug resistance mutations in HIV-1 infections in the Republic of Dagestan, in the North Caucasus area of Russia, where a recent large increase in HIV-1 infections has been documented. Samples were collected from 41 HIV-1-infected individuals from Dagestan, most of them from the cities of Derbent (n = 21) and Mahachkala (n = 18). Thirty six were injecting drug users and five were infected by heterosexual contact. None was on antiretroviral drug treatment. HIV-1 protease and a segment of reverse transcriptase were amplified by RT-PCR from plasma RNA and sequenced, and phylogenetic trees were constructed via maximum likelihood. Forty (97.6%) of 41 samples were of subtype A, former Soviet Union variant (A(FSU)), of which 27 (67.5%) clustered with the subvariant containing the V77I substitution in protease (V77I(PR)). Within this cluster, 13 viruses formed a local subcluster, 10 of which were from Derbent. Four viruses clustered with the A(SP2) subcluster, recently identified in St. Petersburg, two with a virus from Georgia and one with a virus from Azerbaijan. No mutations associated with antiretroviral drug resistance were detected. The results, therefore, show the relationship of the HIV-1 epidemic in Dagestan with that of other areas of Russia and of neighboring countries, and reveal the spread of the A(FSU) V77I(PR) variant in the North Caucasus area.

HIV contamination of commercial PCR enzymes raises the importance of quality control of low-cost in-house genotypic HIV drug resistance tests.

BACKGROUND: Low-cost in-house technologies for genotypic drug resistance testing use reagents with quality labels for research only. Here, we report on the results of PCR amplifications in negative-controls that were observed in two independent laboratories. METHODS: Positive PCR amplifications of protease and reverse transcriptase fragments for genotypic drug resistance testing of HIV on dried blood and/or plasma spots were observed on negative-control samples and were analysed in detail by PCR and sequence and phylogenetic analyses to identify the origin of the PCR contamination. RESULTS: Detailed analysis revealed that the RT-PCR enzymes were contaminated with an HIV-based vector commercialized by the same company. CONCLUSIONS: These observations show the need to implement quality control steps that verify for the absence of HIV in new reagent batches because this can significantly compromise molecular diagnosis of HIV and genotypic drug resistance tests using in-house protocols.

A novel non-radioactive assay for HIV-RT (RdDp) based on pyrosequencing for high-throughput drug screening.

Current in vitro assays for the activity of HIV-RT (reverse transcriptase) require radio-labeled or chemically modified nucleotides to detect reaction products. However, these assays are inherently end-point measurements and labor intensive. Here we describe a novel non-radioactive assay based on the principle of pyrosequencing coupled-enzyme system to monitor the activity of HIV-RT by indirectly measuring the release of pyrophosphate (PP(i)), which is generated during nascent strand synthesis. The results show that our assay could monitor HIV-RT activity with high sensitivity and is suitable for rapid high-throughput drug screening targeting anti-HIV therapies due to its high speed and convenience. Moreover, this assay can be used to measure primase activity in an easy and sensitive manner, which suggests that this novel approach could be wildly used to analyze the activity of PP(i)-generated and ATP-free enzyme reactions.

HIV-I reverse transcriptase variation in plasma and genital secretion of antiretroviral-naive females.

The reverse transcriptase (RT) enzyme of HIV type 1 (HIV-1) is largely targeted by the host immune selection pressure and would differ in the anatomical compartments, thereby having a drastic impact on viral quasi-species evolution. The HIV-1 RT region sequenced from plasma and genital secretions of 8 antiretroviral treatment (ART)-naive females was analyzed for the pattern of amino acid mutations and the ratio of synonymous and nonsynonymous substitutions to determine whether it is under different selection pressure in both the compartments. Phylogenetic and mutational analysis of the HIV-1 RT in plasma and genital secretions of HIV-1-infected ART-naive females showed limited variation likely reflecting the absence of differential selection pressure and therefore genetic variation in these compartments.

Comparison of HIV type 1 sequences from plasma, cell-free breast milk, and cell-associated breast milk viral populations in treated and untreated women in Mozambique.

We analyzed the sequences of the HIV viral populations obtained from plasma, cell-free breast milk, and breast milk cells of HAART-treated (23) and untreated (30) HIV-infected women to obtain information about the origin of the breast milk virus. Sequence analyses of viruses were performed using the TruGene HIV-1 assay. Direct sequences of the reverse transcriptase (RT) and protease (PR) genes were analyzed using the Phylip 3.68 suite of sequence analysis program and pairwise evolutionary distances were calculated with the Kimura two parameter model for estimation of distances. We found that the genetic distances between the plasma and the cell-free breast milk viruses and between the cell-free and cell-associated breast milk viruses for RT were higher in HAART-receiving women than in untreated women, suggesting viral evolution under selective drug pressure in breast milk. Our data support the hypothesis of the presence of an actively replicating viral population in the breast milk compartment, distinct from that present in plasma.

Aptamers that recognize drug-resistant HIV-1 reverse transcriptase.

Drug-resistant variants of HIV-1 reverse transcriptase (RT) are also known to be resistant to anti-RT RNA aptamers. In order to be able to develop diagnostics and therapies that can focus on otherwise drug-resistant viruses, we have isolated two aptamers against a well-known, drug-resistant HIV-1 RT, Mutant 3 (M3) from the multidrug-resistant HIV-1 RT panel. One aptamer, M302, bound M3 but showed no significant affinity for wild-type (WT) HIV-1 RT, while another aptamer, 12.01, bound to both M3 and WT HIV-1 RTs. In contrast to all previously selected anti-RT aptamers, neither of these aptamers showed observable inhibition of either polymerase or RNase H activities. Aptamers M302 and 12.01 competed with one another for binding to M3, but they did not compete with a pseudoknot aptamer for binding to the template/primer cleft of WT HIV-1 RT. These results represent the surprising identification of an additional RNA-binding epitope on the surface of HIV-1 RT. M3 and WT HIV-1 RTs could be distinguished using an aptamer-based microarray. By probing protein conformation as a correlate to drug resistance we introduce an additional and useful measure for determining HIV-1 drug resistance.

Development of aptamer-based affinity assays using temperature gradient focusing: minimization of the limit of detection.

A method is described for an aptamer-based affinity assay using a combination of two nonconventional techniques, temperature gradient focusing (TGF) and field-amplified continuous sample injection TGF (FACSI-TGF), with fluorescence detection. Human immunodeficiency virus reverse transcriptase (HIVRT) is used as the protein target for the assay. The TGF and FACSI-TGF assays are compared to similar results obtained with conventional CE. A range of starting aptamer concentrations are used to determine the optimal LOD for human immunodeficiency virus reverse transcriptase (HIVRT) using each approach. The results indicate that the LODs for HIVRT obtained with TGF and FACSI-TGF are comparable to or even lower than the LODs obtained with conventional CE in spite of the inferior detector used for the TGF and FACSI-TGF assays (arc lamp and low-cost CCD for TGF versus LIF with PMT for CE). It is hypothesized that this is due to the greater reproducibility of the TGF and FACSI-TGF techniques since they do not employ a defined sample injection. The lowest LOD achieved with the new aptamer assay approach is more than an order of magnitude lower than that reported for a similar CE-based aptamer assay for the same target.