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.

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.

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.

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.

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.

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.

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.

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.

Tunable aptamer capillary electrophoresis and its application to protein analysis.

A tunable aptamer electrophoretic assay enables highly sensitive fluorescence detection of multiple proteins and protein isomers. The electrophoretic mobility of proteins is tuned with DNA aptamers binding to the target proteins. Fluorescently labeled aptamers of varying nucleotide lengths serve as both charge modulators for capillary electrophoresis separation and as fluorescent affinity probes for ultrasensitive laser-induced fluorescence detection. Simultaneous determination of pM levels of human immunodeficiency virus reverse transcriptase, thrombin, human immunoglobulin E, and two isomers of platelet-derived growth factor in dilute human serum demonstrates the potential applications of this assay to biomarker development.

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.

Use of an HIV-1 reverse-transcriptase enzyme-activity assay to measure HIV-1 viral load as a potential alternative to nucleic acid-based assay for monitoring antiretroviral therapy in resource-limited settings.

An inexpensive and technically less-demanding methodology to quantify HIV-1 viral load would be of great value for resource-limited settings, where the nucleic-acid amplification technique (NAAT) is impractical and/or resource-prohibitive. In this study, an HIV-1 reverse-transcriptase enzyme-activity assay (ExaVir Load assay, version 1) was compared with the gold standard RT-PCR assay, Roche HIV-1 Amplicor Monitor, version 1.5. A total of 121 plasma specimens were used for the evaluation. ExaVir Load had a sensitivity of 97 % and a specificity of 71 % in identifying specimens with <400 copies ml(-1) in the Roche RT-PCR assay as being less than the detection limit of the assay (5500 copies ml(-1)). The mean difference (95 % limits of agreement) between Roche RT-PCR and ExaVir Load was -0.23 (-1.59 to 1.13) log(10)(copies ml(-1)) by Bland-Altman analysis. Significant negative correlations were seen between CD4(+) T-cell counts and the ExaVir Load assay (r=-0.32, P<0.05), and between CD4(+) T-cell counts and the Roche RT-PCR (r=-0.38, P<0.01). The present study with HIV-1 showed a strong correlation between the ExaVir Load assay and the RT-PCR assay. Hence, the ExaVir Load assay could be considered for use in resource-limited settings as an alternative viral-load assay to the standard NAAT-based assay after further evaluation with prospective specimens.

Superiority of infectivity-based over particle-based methods for quantitation of drug resistant HIV-1 as inocula for cell cultures.

Performance of phenotypic assays and replication capacity assays require normalization of virus input. Therefore, quantitation of HIV-1 in supernatants to inoculate cell cultures is an important step. Since the gold standard for the determination of infectivity, the tissue culture infectious dose 50% (TCID50) is time-consuming, several other methods are in use. This study evaluated methods for the quantitation of drug resistant viruses in cell culture supernatants. The compared methods were based on the detection of viral structural components like genomic RNA or p24 antigen (CA-p24) (particle-based), the determination of reverse transcriptase (RT) activity, and methods based on the detection of viral infectivity like LTR-induced beta-galactosidase (beta-gal) activity and the TCID50 (infectivity-based). Significant correlations were observed between beta-gal activity and TCID50, and between CA-p24 and viral RNA. RT activity did not correlate with any other method. However, RT activity correlated significantly with infectivity when non-resistant subtype-B isolates were analyzed. In contrast to viral infectivity, CA-p24 exhibited a long half life and accumulated in cell culture, resulting in decreasing ratios of infectious virions to CA-p24 over time. As a consequence, relative replication capacities of drug resistant viruses were only determined reliably if the input virus was normalized according to infectivity. In conclusion, RT activity seems to be feasible for non-resistant subtype-B viruses but may be of limited use for non-B subtypes and for drug resistant viruses. Methods determining infectivity are most suitable for quantitation of cell culture inocula, whereas particle-based assays are more appropriate for quantitation of virus production during an experiment.

Naltrexone inhibits alcohol-mediated enhancement of HIV infection of T lymphocytes.

Acute and chronic alcohol abuse impairs various functions of the immune system and thus, has been implicated as a cofactor in the immunopathogenesis of human immunodeficiency virus (HIV) disease progression. We determined whether naltrexone, an opioid receptor antagonist widely used in the treatment of alcoholism, inhibits alcohol-mediated enhancement of HIV infection of T cells. Alcohol enhanced HIV infection of peripheral blood lymphocytes (PBL) and a human lymphoid cell line (CEMX174). Alcohol increased HIV X4 envelope (Env), not murine leukemia virus Env-pseudotyped infection of CEMX174 cells. Naltrexone antagonized the enhancing effect of alcohol on HIV infection of PBL and CEMX174 cells. The specific mu-opioid receptor antagonist, Cys2, Tyr3, Arg5, Pen7 (CTAP) amide, also blocked the enhancing effect of alcohol on HIV infection. Investigation of the underlying mechanism for the alcohol action showed that alcohol significantly increased endogenous beta-endorphin production and induced mu-opioid receptor mRNA expression in PBL and CEMX174 cells. The role of beta-endorphin in alcohol-mediated enhancement of HIV infection was indicated by the observations that naltrexone and CTAP antagonized ether alcohol- or exogenous beta-endorphin-mediated enhancement of HIV infection. These findings suggest a biological mechanism for the potential therapeutic benefit of naltrexone in treating HIV-infected alcoholics.

Reactivation of latent human immunodeficiency virus 1 by human herpesvirus 6 infection.

Infection of the ACH-2 line of human leukemic T cells carrying latent Human immunodeficiency virus 1 (HIV-1) with Human herpesvirus 6 (HHV-6) resulted in an increase in reverse transcriptase (RT) activity, a marker of HIV-1 activation, in the culture supernatant. A similar effect was obtained with 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The RT activity reached a peak at 24 hrs post infection (p.i.) and then declined, suggesting that the cells underwent lysis. The HIV-1 antigen was co-expressed with an early-late HHV-6 product, but not always with an immediate-early (IE) HHV-6 product, suggesting that one or more IE gene products were involved in the activation of latent HIV-1 in ACH-2 cells.

In vitro and in vivo anti-retroviral activity of the substance purified from the aqueous extract of Chelidonium majus L.

We have isolated a substance with anti-retroviral activity from the freshly prepared crude extract of Chelidonium majus L. (greater celandine) by 9-aminoacridine precipitation method and ion exchange chromatography using Dowex-50W/H+ resin followed by the gel filtration on Sephadex-75 column. Elemental and phenol/sulfuric acid method analyses as well as the mass spectrometry of the purified substance indicated that it may represent a low-sulfated poly-glycosaminoglycan moiety with molecular weight of approximately 3800 Da. The substance prevented infection of human CD4+ T-cell lines AA2 and H9 with HIV-1 at concentration of 25 microg/mL as well as the cell-to-cell virus spread in H9 cells continuously infected with HIV-1, as determined by the measurement of reverse transcriptase activity and p24 content in cell cultures. Furthermore, we have shown in a murine AIDS model that the treatment with purified substance significantly prevented splenomegaly and the enlargement of cervical lymph nodes in C57Bl/6 mice chronically infected with the pool of murine leukemia retroviruses. The mechanism(s) of anti-retroviral activity of this substance have to be elucidated.

Sequences within Pr160gag-pol affecting the selective packaging of primer tRNA(Lys3) into HIV-1.

The selective packaging of the primer tRNA(Lys3) into HIV-1 particles is dependent upon the viral incorporation of the Pr160gag-pol precursor protein. In order to map a tRNA(Lys3) binding site within this precursor, we have studied the effects of mutations in Pr160gag-pol upon the selective incorporation of tRNA(Lys3). Many of these mutations were placed in a protease-negative HIV-1 proviral DNA to prevent viral protease degradation of the mutant Gag-Pol protein. C-terminal deletions of protease-negative Gag-Pol that removed the entire integrase sequence and the RNase H and connection subdomains of reverse transcriptase did not inhibit the incorporation of either the truncated Gag-Pol or the tRNA(Lys3), indicating that these regions are not required for tRNA(Lys3) binding. On the other hand, larger C-terminal deletions, which also remove the thumb subdomain sequence, did prevent tRNA(Lys3) packaging, without inhibiting viral incorporation of the truncated Gag-Pol, indicating a possible interaction between thumb subdomain sequences and tRNA(Lys3). While point mutations K249E, K249Q, and R307E in the primer grip region of the thumb subdomain have been reported to inhibit the in vitro interaction of mature reverse transcriptase with the anticodon loop of tRNA(Lys3), we find that these mutations do not inhibit tRNA(Lys3) packaging into the virus, which supports other work indicating that the anticodon loop of tRNA(Lys3) is not involved in interactions with Pr160gag-pol during tRNA(Lys3) packaging.

An experimental model for study of sialoglycoproteins of human immunodeficiency virus 1 epitope structures.

Sialic acid (SA) molecules located terminally on retrovirus glycoproteins (gps) play a key role in virus-cell interactions. The specificity of sialylation of Human immunodeficiency virus 1 (HIV-1) gps has not yet been studied. Looking for a convenient and reproducible experimental virus-cell model for studying the problem mentioned above we compared viral sialoglycoprotein (Sgp) patterns in H9/HTLV III B cells chronically infected with laboratory-adapted HIV-1LAI and MT-2 cells acutely infected with the same virus. Cytosols (CSs) and supernatant concentrates (SNs) from these cells and cell cultures, respectively, following N-acetyl-D-[U-14C]-mannosamine ([14C]NAcMan) labeling were subjected to preparative isoelectrofocusing and the obtained fractions were assayed for 14C-incorporation, reverse transcriptase (RT) activity and protein content. Sgp patterns in CSs from the two types of infection were similar. Highly sialylated peaks clustered mainly in the acidic region where the highest 14C-incorporation, RT activity and protein content were found. The 14C-incorporation was higher in CS than in SN. Analysis of CS from MT-2 cells infected with HIV-1 for the markers described above seems to be the experimental approach and model of choice for clinical isolates of HIV-1.

Use of HIV-1 reverse transcriptase recovered from human plasma for phenotypic drug susceptibility testing.

OBJECTIVE: To demonstrate the use of HIV-1 reverse transcriptase (RT) recovered directly from plasma for phenotypic drug susceptibility testing. METHODS: Plasma from HIV-1 infected individuals with and without drug resistance-associated mutations were selected for the study. The blind coded plasmas were treated to inactivate cellular enzymes. The virions were immobilized on a gel and washed to remove antiretroviral drugs and RT activity blocking antibodies. The immobilized virions were lysed; the viral RT eluted and quantified, all according to the ExaVir Load procedure. The drug sensitivity profiles of each RT were determined using serially diluted drugs and modified Cavidi HS Lenti RT kits. RESULTS: The phenotypic drug sensitivity profiles of the RT and the patterns of drug resistance mutations were highly concordant. Plasma RT from virions devoid of mutations associated with drug resistance had average 50% inhibitory concentrations (IC(50)) of 1.5 +/- 0.93 microM for nevirapine, 0.21 +/- 0.099 microM for efavirenz, 7.1 +/- 3.2 microM for delavirdine, 0.42 +/- 0.15 microM for azidothymidine triphosphate and 0.059 +/- 0.018 microM for didehydrothymidine triphosphate. The increase in IC(50) value for RT with drug resistance associated substitutions was from 3- to more than 65-fold for non-nucleoside inhibitors and between 2- and 30-fold for thymidine analogue drugs. CONCLUSION: RT derived from virions recovered from the plasma of HIV infected individuals can be used for analysis of phenotypic drug susceptibility. The methods presented provide rapid alternatives for analysing phenotypic drug susceptibility especially when the therapy is based on non-nucleoside RT inhibitors and thymidine-analogue drugs.