Detection and Quantification of Label-Free Infectious Adenovirus Using a Switch-On Cell-Based Fluorescent Biosensor.

Reliable and fast viral detection and quantification protocols are a requirement for the advance of basic research and clinical approaches with wild type or recombinant viruses. However, available cell-based assays are either time-consuming or require labeled viral particles, which may alter virus biology or pose safety issues in clinical applications. Since adenoviruses constitute a major healthcare burden but also, when engineered, widely used vectors in vaccination and gene and oncolytic therapies, herein we developed a genetically encoded switch-on fluorescent biosensor consisting of a cyclized Green fluorescent protein-cVisensor-with an adenoviral protease cleavable site as a switch. After initial sensor optimization (35% increase in performance), whole-cell biosensors were established-by stably expressing cVisensor in mammalian cells-and used for live-cell monitoring of adenovirus infection as the intracellular biosensor is specifically activated by the viral protease. A rapid flow cytometry-based bioassay using cVisensor cells was established 48 h postinfection, showing an estimated limit of detection of 105 infectious particles/mL, in-line with previously reported flow cytometry assays requiring labeled virus, and significantly faster than standard plaque-forming assays requiring up to 14 days. cVisensor was also successfully applied in the detection of HIV-1 protease activity, validating its wider potential for the detection of other viruses. Overall, this work presents a fast and easy method for detection and quantification of label-free infectious virus, allowing the establishment of new biosensing platforms for basic research in virology and biotechnological applications of recombinant virus biopharmaceuticals.

Graphene oxide-based biosensing platform for rapid and sensitive detection of HIV-1 protease.

HIV-1 protease is essential for the life cycle of the human immunodeficiency virus (HIV), and is one of the most important clinical targets for antiretroviral therapies. In this work, we developed a graphene oxide (GO)-based fluorescence biosensing platform for the rapid, sensitive, and accurate detection of HIV-1 protease, in which fluorescent-labeled HIV-1 protease substrate peptide molecules were covalently linked to GO. In the absence of HIV-1 protease, fluorescein was effectively quenched by GO. In contrast, in the presence of HIV-1 protease, it would cleave the substrate peptide into short fragments, thus producing fluorescence. Based on this sensing strategy, HIV-1 protease could be detected at as low as 1.18 ng/mL. More importantly, the sensor could successfully detect HIV-1 protease in human serum. Such GO-based fluorescent sensors may find useful applications in many fields, including diagnosis of protease-related diseases, as well as sensitive and high-throughput screening of drug candidates. Graphical abstract ᅟ.

A novel and rapid assay for HIV-1 protease detection using magnetic bead mediation.

A simple sensing assay was established for label-free detection of HIV-1 protease. HIV-1 protease peptide substrate conjugated to magnetic beads via its N-terminus is directly fixed onto the sensor gold surface through the sulphur atom of cysteine. Surface plasmon resonance (SPR) was used to study the peptide substrate cleavage efficiency of the protease with magnetic beads of different sizes (1 µm and 30 nm). Cyclic voltammetry and faradic impedance spectroscopy were employed in order to characterize the functionalized gold electrode. It was found that the nano-sized beads are a more efficient sensing probe for the protease. Electrochemical biosensing showed a gradual decrease in charge transfer resistance after injection of the HIV-1 protease. The experimental data established a detection limit of 10 pg/ml, as well as demonstrated a drug screening assay. This HIV-1 protease biosensor represents a new detection approach which will lead to low-cost point-of-care devices for sensitive HIV-1 diagnosis, as well as high-throughput drug screening platforms.

Picomolar detection of protease using peptide/single walled carbon nanotube/gold nanoparticle-modified electrode.

Picomolar electrochemical detection of human immunodeficiency virus type-1 protease (HIV-1 PR) using ferrocene (Fc)-pepstatin-modified surfaces has been presented. Gold electrode surface was modified with gold nanoparticles (AuNP) or thiolated single walled carbon nanotubes/gold nanoparticles (SWCNT/AuNP). Thiol-terminated Fc-pepstatin was then self-assembled on such surfaces as confirmed by Raman spectroscopy and scanning electron microscope. The interaction between the Fc-pepstatin-modified substrates and HIV-1 PR was studied by cyclic voltammetry and electrochemical impedance spectroscopy. Both electrode materials showed enhanced electrochemical responses to increasing concentrations of HIV-1 PR with shifting to higher potentials as well as decrease in the overall signal intensity. However, the sensing electrode modified with thiolated SWCNTs/AuNPs showed remarkable detection sensitivity with an estimated detection limit of 0.8 pM.

Evolution of human immunodeficiency virus type 1 protease genotypes and phenotypes in vivo under selective pressure of the protease inhibitor ritonavir.

We examined the population dynamics of human immunodeficiency virus type 1 pro variants during the evolution of resistance to the protease inhibitor ritonavir (RTV) in vivo. pro variants were followed in subjects who had added RTV to their previously failed reverse transcriptase inhibitor therapy using a heteroduplex tracking assay designed to detect common resistance-associated mutations. In most cases the initial variant appeared rapidly within 2 to 3 months followed by one or more subsequent population turnovers. Some of the subsequent transitions between variants were rapid, and some were prolonged with the coexistence of multiple variants. In several cases variants without resistance mutations persisted despite the emergence of new variants with an increasing number of resistance-associated mutations. Based on the rate of turnover of pro variants in the RTV-treated subjects we estimated that the mean fitness of newly emerging variants was increased 1.2-fold (range, 1.02 to 1.8) relative to their predecessors. A subset of pro genes was introduced into infectious molecular clones. The corresponding viruses displayed impaired replication capacity and reduced susceptibility to RTV. A subset of these clones also showed increased susceptibility to two nonnucleoside reverse transcriptase inhibitors and the protease inhibitor saquinavir. Finally, a significant correlation between the reduced replication capacity and reduced processing at the gag NC-p1 processing site was noted. Our results reveal a complexity of patterns in the evolution of resistance to a protease inhibitor. In addition, these results suggest that selection for resistance to one protease inhibitor can have pleiotropic effects that can affect fitness and susceptibility to other drugs.

Rapid enzymatic test for phenotypic HIV protease drug resistance.

A phenotypic resistance test based on recombinant expression of the active HIV protease in E. coli from patient blood samples was developed. The protease is purified in a rapid one-step procedure as active enzyme and tested for inhibition by five selected synthetic inhibitors (amprenavir, indinavir, nelfinavir, ritonavir, and saquinavir) used presently for chemotherapy of HIV-infected patients. The HPLC system used in a previous approach was replaced by a continuous fluorogenic assay suitable for high-throughput screening on microtiter plates. This reduces significantly the total assay time and allows the determination of inhibition constants (Ki). The Michaelis constant (Km) and the inhibition constant (Ki) of recombinant wild-type protease agree well with published data for cloned HIV protease. The enzymatic test was evaluated with recombinant HIV protease derived from eight HIV-positive patients scored from 'sensitive' to 'highly resistant' according to mutations detected by genotypic analysis. The measured Ki values correlate well with the genotypic resistance scores, but allow a higher degree of differentiation. The non-infectious assay enables a more rapid yet sensitive detection of HIV protease resistance than other phenotypic assays.

Enzyme kinetics determined using calorimetry: a general assay for enzyme activity?

Two techniques for determining enzyme kinetic constants using isothermal titration microcalorimetry are presented. The methods are based on the proportionality between the rate of a reaction and the thermal power (heat/time) generated. (i) An enzyme can be titrated with increasing amounts of substrate, while pseudo-first-order conditions are maintained. (ii) Following a single injection, the change in thermal power as substrate is depleted can be continuously monitored. Both methods allow highly precise kinetic characterization in a single experiment and can be used to measure enzyme inhibition. Applicability is demonstrated using a representative enzyme from each EC classification, including (i) oxidation-reduction activity of DHFR (EC 1.5.1.3); (ii) transferase activity of creatine phosphokinase (EC 2.7.3.2) and hexokinase (EC 2.7.1.1); (iii) hydrolytic activity of Helicobacter pylori urease (EC 3.5.1.5), trypsin (EC 3.4.21.4), and the HIV-1 protease (EC 3.4.21.16); (iv) lyase activity of heparinase (EC 4.1.1.7); and (v) ligase activity of pyruvate carboxylate (EC 6.4.1.1). This nondestructive method is completely general, enabling precise analysis of reactions in spectroscopically opaque solutions, using physiological substrates. Such a universal assay may have wide applicability in functional genomics.

Use of a phosphotyrosine-antibody pair as a general detection method in homogeneous time-resolved fluorescence: application to human immunodeficiency viral protease.

A homogeneous time-resolved fluorescence (HTRF) assay has been developed for human immunodeficiency viral (HIV) protease. The assay utilizes a peptide substrate, differentially labeled on either side of the scissile bond, to bring two detection components, streptavidin-cross-linked XL665 (SA/XL665) and a europium cryptate (Eu(K))-labeled antiphosphotyrosine antibody, into proximity allowing fluorescence resonance energy transfer (FRET) to occur. Cleavage of the doubly labeled substrate by HIV protease precludes complex formation, thereby decreasing FRET, and allowing enzyme activity to be measured. Potential substrates were evaluated by HTRF with the best results being obtained using (LCB)K4AVSQNbeta-NapPIVpYA(NH2) and Eu(K)-pY20 where the peptide titrated with an EC50 of 7.7 +/- 0.3 nM under optimized detection conditions. Using these HTRF detection conditions, HIV protease cleaved the substrate in 50 mM NaOAc, 150 mM KF, 0.05% Tween 20, pH 5.5, with apparent first-order kinetics with a Km of 37.8 +/- 8.7 microM and a kcat of 0.95 +/- 0.07 s-1. Examination of the first-order rate constant versus enzyme concentration suggested a Kd of 9.4 +/- 2.7 nM for the HIV protease monomer-dimer equilibrium. The HTRF assay was also utilized to measure the inhibition of the enzyme by two known inhibitors.

Development and standardization of an immuno-quantified solid phase assay for HIV-1 aspartyl protease activity and its application to the evaluation of inhibitors.

The catELISA technique was modified and standardized for measuring HIV-1 aspartyl protease activity and evaluating the potency of synthetic peptide inhibitors. This immuno-quantified solid phase assay combines the use of an immobilized C-terminal biotinylated peptide as substrate, a crude enzyme preparation, and a highly specific antiserum elicited against the C-terminal product of the enzyme reaction. A standard curve of this C-terminal product was constructed to determine the enzyme activity. This assay, which requires less enzyme and substrate, is more sensitive than the conventional HPLC method. The amounts of C-terminal peptide produced in solution as determined from ELISA and HPLC standard curves were comparable. Analogues of peptidomimetics designed in our laboratory were assayed for their potency to inhibit the enzyme. One of them, H4, which is a hydroxyethylamine isostere of the Phe-Pro peptide bond, was a powerful inhibitor.

Assay of HIV-1 protease activity by use of crude preparations of enzyme and biotinylated substrate.

An enzyme immunoassay was developed for monitoring protease reactions of human immunodeficiency virus (HIV). The protease and its substrate, the gag precursor, were generated separately in Escherichia coli. The HIV-1 protease was generated with a glutathione-S-transferase expression system and the gag substrate, named Pin17/24, was prepared with a PinPoint expression system. Pin17/24 consists of an N-terminal peptide, which is biotinylated in E. coli, fused with a C-terminal peptide that contains a protease cleavage site flanked by p17 and p24 segments. Through its biotin in the N-terminal region, Pin17/24 bound to ELISA plates coated with avidin, whereas through its C-terminal region, the same molecule of Pin17/24 could be recognized by an anti-p24 monoclonal antibody. When the protease was added to Pin17/24, the p24 fragment was released from the biotinylated fusion protein and could no longer be retained on the avidin plates, and as a result, binding of the anti-p24 monoclonal antibody decreased. The binding was specific and the reaction was inhibited by a known HIV protease inhibitor. Due to the specific interactions between avidin and biotin, monoclonal antibody and antigen, and the HIV protease and the gag substrate, crude preparations of these reagents can be used readily in the assay. The simplicity and feasibility of this method should be useful for simultaneous monitoring of many enzyme reactions, particularly for screening possible HIV protease inhibitors.

A protease assay using time-resolved lanthanide luminescence from an engineered calcium binding protein substrate.

OBJECTIVES: The objective of this work was to demonstrate the utility of luminescence from lanthanides bound to a mutant of the Ca2+ binding protein, oncomodulin, to monitor protease activity. DESIGN AND METHODS: A mutant of oncomodulin with a cysteine residue at position 57 located in the CD binding loop was conjugated to a salicylic acid group. The luminescence of Tb3+ resulting from electronic energy transfer from the salicylic acid group was monitored using time resolved lanthanide luminescence in the presence of proteolytic enzymes. RESULTS: Low detection limits for subtilisin (150 pg), chymotrypsin (2.5 ng), cathepsin B (3.5 ng), and HIV-1 protease (25 ng) were found. CONCLUSION: The simplicity of the assay coupled with its high level of sensitivity make it useful for the detection of protease at very low concentrations.

Potency and selectivity of inhibition of human immunodeficiency virus protease by a small nonpeptide cyclic urea, DMP 323.

DMP 323 is a potent inhibitor of the protease of human immunodeficiency virus (HIV), with antiviral activity against both HIV type 1 and HIV type 2. This compound is representative of a class of small, novel, nonpeptide cyclic urea inhibitors of HIV protease that were designed on the basis of three-dimensional structural information and three-dimensional database searching. We report here studies of the kinetics of DMP 323 inhibition of the cleavage of peptide and HIV-1 gag polyprotein substrates. DMP 323 acts as a rapidly binding, competitive inhibitor of HIV protease. DMP 323 is as potent against both peptide and viral polyprotein substrates as A-80987, Q8024, and Ro-31-8959, which are among the most potent inhibitors of HIV protease described in the literature to date. Incubation with human plasma or serum did not decrease the effective potency of DMP 323 for HIV protease, suggesting that plasma protein binding is of a low affinity relative to that of HIV protease. DMP 323 was also assessed for its ability to inhibit the mammalian proteases renin, pepsin, cathepsin D, cathepsin G, and chymotrypsin. No inhibition of greater than 12% was observed for any of these enzymes at concentrations of DMP 323 that were 350 to 40,000 times higher than that required to inhibit the viral protease 50%.

Synthetic approaches to continuous assays of retroviral proteases.

This chapter has described a number of approaches for continuous assay of retroviral proteases using either chromogenic or fluorogenic synthetic substrates. The significant progress in this area has been catalyzed by the intense interest in HIV protease as a therapeutic target, but these versatile methods will be used widely in future for studies of many other proteases.

Kinetic assay for HIV proteinase subunit dissociation.

The kinetics and thermodynamics of the monomer--dimer equilibrium for HIV-1 proteinase are investigated in a concentration jump experiment, at a concentration of the substrate that is substantially lower than the Michaelis constant. Under these conditions the substrate-induced stabilization of the active dimer is suppressed, and the integral rate equation can be obtained in a closed form. Both the monomer--dimer bimolecular association rate constant and the corresponding equilibrium dissociation constant are obtained directly by nonlinear regression analysis of the reaction time-course. In buffers of low ionic strength and in the absence of external ligands (substrates and inhibitors), the equilibrium dissociation constant at 37 degrees C is 440 +/- 52 nM, a value significantly higher than previous estimates obtained at a comparatively high concentration of substrates.

A solid phase assay for the protease of human immunodeficiency virus.

A solid phase assay for human immunodeficiency virus (HIV) protease using an immobilized substrate, Affi Gel 10-Gly-Gly-Gly-Gly-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[3H]Gly-OH has been devised. The Tyr-Pro bond of the substrate was hydrolyzed by the protease, releasing the radiolabeled cleavage product, Pro-Ile-Val-Gln-[3H]Gly-OH, into the supernatant. The pH optimum was found to be 6.0, and a high ionic strength was required for maximal activity. The solid phase assay is usable for convenient monitoring of purification procedures, and rapid screening of inhibitors of HIV protease.

[The properties of HIV protease synthesized in E. coli cells]. / Izuchenie svoistv proteazy VICh, sintezirovannoi v kletkakh bakterii E. coli.

A hybrid plasmid pPR6 was constructed containing BgII-EcoRI fragment of the pol region of HIV (strain IIIB) genome which determined the synthesis of virus-specific protease. Extracts of E. coli DN5/pPR6 bacteria provided for specific hydrolysis of hybrid protein p165 (the N-terminus of which is presented by complete beta-galactosidase and the C-terminus by duplicated area of virus-specific precursor p55 containing a site for virus-specific protease located at the border of proteins p17 and p24) with formation of products having molecular weights of 19, 42, 28, 23, and 19 kD. Polypeptides 119K, 23K, and 19K are products of complete hydrolysis, and 42K a result of partial cleavage. The kinetics of hydrolysis in relation to pH values of the reaction mixture was analysed. It is suggested that the reported system of HIV protease activity determination be used for screening of potential inhibitors of this enzyme.