Detection of inhibition of HIV-1 protease activity by an enzyme-linked immunosorbent assay (ELISA)

Journal oflmmunological Methods, 161 (1993) 151-155 © 1993 Elsevier Science Publishers B.V. All rights reserved 0022-1759/93/$06.00

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JIM 06677

Detection of inhibition of HIV-1 protease activity by an enzyme-linked immunosorbent assay (ELISA) H a n s - W e r n e r Mansfeld

a

Stefan Schulz a Gerald Griitz and Siegfried Ansorge a

b

Riidiger von Baehr b

a Division of Experimental Immunology, Department of Internal Medicine, MedicalAcademy of Magdeburg, Magdeburg, Germany, and b Institute of Medical Immunology, Department of Medicine (Charitg), HumboMt University of Berlin, Berlin, Germany (Received 28 July 1992, revised received 18 November 1992, accepted 30 November 1992)

An ELISA is described for the detection of HIV-1 protease activity using an immobilized gag-related polyprotein as substrate. Proteolytic activity was demonstrated with either bacterial lysates expressing HIV-1 protease or purified protease. No cleavage was observed with a protein preparation from control bacteria not expressing HIV-1 protease. Under these conditions the aspartyl-type protease inhibitor, pepstatin A, was found to inhibit HIV-1 protease cleavage by > 90% at a concentration of 0.1 mM. This assay may be a useful tool for the study of both synthetic and natural inhibitors of HIV-1 protease. Key words: HIV-1 protease; ELISA; Pepstatin A

Introduction

The genome of HIV-1 encodes a highly specific aspartyl-type protease, which is responsible for proteolytic processing of gag and gag-pol precursor polyproteins (Debouck et al. 1987; Seelmeier et al., 1988). Since the protease-dependent processing is essential to the retroviral life cycle (Katoh et al. 1985; Kohl et al., 1988; Von der Helm et al., 1989), the protease may be a suitable target for antiviral therapy against HIV-1, the causative agent of the aquired immunodeficiency syndrome (AIDS) and related diseases. Correspondence to: S. Ansorge, Division of Experimental Immunology, Department of Internal Medicine, Medical Academy of Magdeburg, Leipziger Str. 44, 0-3090 Magdeburg, Germany. Abbreviations: ELISA, enzyme-linked immunosorbent assay, HIV, human immunodeficiency virus, HRP, horseradish peroxidase, oPD, orthophenylenediamine, PBS, phosphatebuffered saline, PBST, phosphate-buffered saline-0.05% Tween 20.

To date several HIV-1 proteinase assays have been described which are based on HPLC analysis of the cleavage of peptide substrates. However, such assays have several limitations. For example, they are relatively laborious and time consuming and, more significantly, the data obtained using peptide substrates must be confirmed with natural protease substrates (Kotler et al., 1988; Billich et al., 1988). In this paper we report a simple and fast method for the detection of HIV-1 protease activity by an ELISA using, as substrate, a gag-related polyprotein immobilized on a solid phase.

Materials and methods

Plasmids and bacterial strain In order, to construct a protease expressing vector, a Bal I fragment containing most of the revertase gene was deleted from the primary construct pUC19BEE7 of the pol reading frame. The

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remaining part of the primary construct containing the protease and part of the endonuclease was inserted into the SalI site of the pUR278 vector using a EcoRI-HindIII linker. The resulting plasmid, named pUR278-Prot15, expresses a protease-/3-galactosidase fusion protein from which the protease is cleaved out autocatalytically (Fig. 1; Griitz et al., 1989). For the expression of gp41-p24 fusion protein as protease substrate a PvuII-EcoRI fragment from the primary construct pUC9gag of the gag reading frame was inserted into pUC19 using the Sinai and EcoRI sites of pUC19. In addition, a RsaI fragment of gp41 was cloned into the HincII site of pUC19. The resulting plasmid was named pUC19gp41p24 and contains part of the gp41, part of the p17, and all of the p24 and p5 regions (Fig. 2). The primary constructs of the pol reading frame pUC19BEE7 and the gag reading frame pUC9gag were a kind gift of H. Wolf (Pettenkofer Institute, Munich, Germany). Recombinant plasmids were transfected into the Escherichia coil strain JM109. All DNA manipulations were carried out as previously described (Maniatis et al., 1982).

Preparation of gp41-p24 fusion protein JM109 cells harboring the plasmid pUC19 gp41p24 were grown overnight in 100 ml LB

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broth containing 100 /xg/ml ampicilline at 37°C. Cultures were then diluted with 100 ml LB broth and expression of the gp41-p24 fusion protein was induced by addition of'isopropyl-fl-o-thiogalactopyranoside at a final concentration of 0.4 mM. After 2 h, cells were harvested by centrifugation, and the cell pellet was suspended in 10 ml of buffer A (50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 50 mM NaC1). Cells were lysed by sonication at 4°C and centrifuged at 15 000 × g for 30 min. The pellet was resuspended in 10 ml buffer B (50 mM Tris-HC1, pH 8.0, 10 mM EDTA, 50 mM NaCl, 0.5% Triton X-100). After 15 min, the lysate was centrifuged at 15 000 × g for 30 min. The majority of the gp41-p24 fusion proteins were produced as inclusion bodies and found in the pellet. The inclusion bodies were then dissolved in 8 M urea, and the solution was stepwise diluted with PBS to a protein concentration of 20 /~g/ml, which was used as protease substrate.

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Fig. 1. Construction of the protease expression vector pUR278-Prot15. A Bali fragment was deleted from the primary construct pUC19BEE7 containing the protease and part of the endonuclease. The remaining part of the primary construct was inserted into the SalI site of the pUR278 vector using a EcoRI-HindlII linker.

Preparation of H1V-1 protease The recombinant bacterial clone containing the plasmid pUR278-Prot15 was grown and induced as described above. Cell paste from a 200 ml culture was suspended in 10 ml buffer A, lysed by

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sonication, and centrifuged at 15000 x g for 30 min. Supernatant was diluted in assay buffer (0.1 M sodium-phosphate buffer, pH 6.0, 1 mM EDTA, 200 mM NaC1) to a protein concentration of 100 /xg/ml and used as a crude HIV-1 protease extract. A corresponding extract from JM109 cells not expressing HIV-1 protease was employed for controls. Purified HIV-1 protease in its dimeric form was kindly provided by B.D. Korant (DuPont, Wilmington, Delaware, USA).

Proteolytic assay The described assay was based on decreased

p24 immunoreactivity of the solid-phase-immobilized substrate after proteolytic cleavage. Ninetysix-well microtiter plates (Greiner, Niitringen, Germany) were coated overnight with 50 ~I/well of 20 ~zg/ml gp41-p24 fusion protein solution in PBS at 4°C. Plates were washed three times with assay buffer and then incubated (50 /xl/well) with either HIV-1 protease extracts or control extracts for time periods of 30 rain to 3 h at 37°C. Purified HIV-1 protease was used at a concentration of 4 / x g / m l . In experiments studying inhibition of the HIV-1 protease, the protease inhibitors pepstatin A, aprotinin, antipain, leupeptin (all purchased from Sigma, Deisenhofen, Germany), puromycin (Serva, Heidelberg, Germany), or actinonin (kind gift of T. Aoyagi, Microbial Chemistry Foundation, Tokyo, Japan) were included at the stated concentrations. Plates were washed three times with PBST, and mouse monoclonal anti-p24 4/1 antibody (Kiittner et al., 1992) was added at 50/zl/well, and incubated for 2 h at 37°C. Anti-p24 4/1 antibody was kindly provided by R. Grunow (Humboldt University, Berlin, Germany) and used as culture supernatant diluted 1/2 with PBST. After washing three times with PBST, plates were incubated with HRP-labeled goat-anti-mouse immunoglobulin antibody (SIFIN, Berlin, Germany, diluted 1/250 with PBST) for 1 h at 37°C. Wells were washed extensively with PBS, then a colorimetric reaction was performed by the addition of 50 /zl/well of a solution of 1 mg/ml oPD in 0.1 M sodium-citrate buffer (pH 5.0) containing 0.006% H 2 0 2. After 20 min, the reaction was stopped by adding 50 /.tl of 2 M sulphuric acid containing 0.05 M sodium sulphite. Absorbance values at

492 nm were measured using an automated microtiterplate reader (model 2001, Anthos, Siegburg, Germany).

Results and discussion

The gp41-p24 fusion protein was expressed in Escherichia coli at high levels resulting in the production of inclusion bodies, which were then solubilized by a denaturation/refolding step. Soluble gp41-p24 fusion protein was immobilized on a solid phase and used as substrate for hydrolysis by the HIV-1 protease. The gp41-p24 fusion protein comprises about half of gp41, part of p17 and all of p24 containing the p17-p24 junction, which is a natural HIV protease cleavage site of the gag precursor polyptotein. Proteolytic activity was demonstrated by incubation with either crude protease extracts or adequate control extracts for time periods of 30 min to 3 h. A specific antibody to p24 was then added, and proteolytic cleavage was determined as the decrease in absorbance at 492 nm after the colorimetric reaction (Fig. 3). Among different gag-related polyproteins, which were tested for their suitability as substrate under these conditions, the gp41-p24 fusion protein gave the best results and was therefore used throughout the study (data not shown). Correct process2.4 2.0

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Time (rnin) Fig. 3. Time-dependent cleavage of gp41-p24 fusion protein by crude HIV-1 protease extract. Microtiter plates coated with gp41-p24 fusion protein were incubated with crude HIV-1 protease extract (©) or control extract (o) for either 0, 30, 60, 90, 120, 150 or 180 min at 37°C. Monoclonal mouse anti-p24 antibody and a second HRP-labeled goat-anti-mouse immunoglobulin antibody were then added, and proteolytic cleavage was determined as the decrease in absorbance at 492 nm. Each data point represents a mean_+ SEM of four replicates. The experiment was repeated five times.

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hibitors such as aprotinin, actinonin, antipain, leupeptin or puromycin had no or very little effect. In summary, we report a solid-phase immunoassay for the detection of inhibitors of HIV1 protease activity. This detection-system may be a useful tool to screen large numbers of samples for both synthetic and natural inhibitors of HIV-1 protease. Moreover, it may also be possible to develop similar immunoassays for studies of the activity and inhibition of other proteases.

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Fig. 4. Inhibition of HIV-1 protease activity. Microtiter plates were coated with the gp41-p24 fusion protein. In A, plates were incubated with crude HIV-1 protease extract for 60 min at 37°C in the presence of either 0.1 mM pepstatin A, 14× 106 U / I aprotinin, 14/~M actinonin, 0.14 mM antipain, 0.14 mM leupeptin or 1.4/~M puromycin. In B, plates were incubated with purified HIV-1 protease in the presence of either 0.17 mM pepstatin A, 1.4 × 106 U/1 aprotinin, 14 /.~M actinonin, 0.14 mM antipain, 0.14 mM leupeptin or 1.4/~M puromycin. Monoclonal mouse anti-p24 antibody and a second HRPlabeled goat-anti-mouse immunoglobulin antibody were then added. The decrease in absorbance at 492 nm observed without the addition of protease inhibitors was used as a control. Each bar shows the m e a n + S E M of four replicates. The experiment was repeated three times.

ing of the gp41-p24 fusion protein by the HIV-1 protease was confirmed by immunoblot analysis using a pooled human anti-HIV-1 antibody-positive serum to detect the cleavage products (Griitz et al., 1989). A number of protease inhibitors were tested for their ability to inhibit the proteolytic activity of either crude HIV-1 protease extracts or purified HIV-1 protease (Fig. 4). The specific aspartyl-type protease inhibitor pepstatin A inhibited HIV-1 protease cleavage by > 90% at a concentration of 0.1 mM. This result is comparable to previously reported concentrations of pepstatin A for inhibiting HIV-1 protease (Hansen et al., 1988; Seelmeier et al., 1988) and other retroviral proteases (Katoh et al., 1987) using a gag precursor cleavage reaction. Other protease in-

Acknowledgements H.W.M. and S.S. contributed equally to this study. The authors are grateful to Dr. H. Wolf (Pettenkofer Institute, Munich, Germany) for kindly providing the primary constructs of the pol reading frame pUC19BEE7 and the gag reading frame pUC9gag, to Dr. B.D. Korant (DuPont, Wilmington, Delaware, USA) for his gift of purified HIV-1 protease, to Dr. R. Grunow (Humboldt University, Berlin, Germany) for kindly providing mouse monoclonal anti-p24 4/1 antibody, and to Dr. T. Aoyagi (Microbial Chemistry Foundation, Tokyo, Japan) for his kind gift of actinonin. Furthermore, the authors would like to thank Mrs. U. Thiel for technical assistance and Mrs. B. Schotte for typing the manuscript.

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Baehr, R. (1992) Immunoglobulin V regions and epitope mapping of a murine monoclonal antibody against p24 core protein of HIV-1. Molec. Immunol. 29, 561. Maniates, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. Seelmeier, S., Schmidt, H., Turk, V. and Von der Helm, K. (1988) Human immunodeficiency virus has an aspartic-type protease that can be inhibited by pepstatin A. Proc. Natl. Acad. Sci. USA 85, 6612. Von der Helm, K., Giirtler, L., Eberle, J. and Deinhardt, F. (1989) Inhibition of HIV replication in cell culture by the specific aspartic protease inhibitor pepstatin A. FEBS Lett. 247, 349.