Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins

Life Sciences 69 (2001) 2217–2223

Inhibition of human immunodeficiency virus type 1 reverse transcriptase, protease and integrase by bovine milk proteins T.B. Ng*, T.L. Lam, T.K. Au, X.Y. Ye, C.C. Wan Department of Biochemistry, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China Received 9 January 2001; accepted 3 April 2001

Abstract Different proteins have been isolated from bovine milk including lactoferrin, lactoperoxidase, glycolactin, angiogenin-1, lactogenin, a-lactalbumin, lactoglobulin and casein. These proteins have been assayed for inhibitory activity against human immunodeficiency virus type 1 (HIV-1) reverse transcriptase, protease and integrase, enzymes crucial to the HIV-1 life cycle. It was found that different milk proteins inhibited the three aforementioned HIV enzymes to different extents. Lactoferrin strongly inhibited HIV-1 reverse transcriptase but only slightly inhibited HIV-1 protease and integrase. On the other hand, a-lactalbumin, b-lactoglobulin and casein inhibited HIV-1 protease and integrase to an appreciable extent but did not inhibit HIV-1 reverse transcriptase. Glycolactin and angiogenin-1 suppressed the activity of HIV-1 reverse transcriptase by a moderate extent but more powerfully inhibited HIV-1 protease and integrase. In comparison with the other milk proteins glycolactin was a strong inhibitor of HIV-1 protease and integrase and a moderate inhibitor of HIV-1 reverse transcriptase. Lactogenin was a strong inhibitor of HIV-1 integrase, a moderate inhibitor of HIV-1 reverse transcriptase and a weak inhibitor of HIV-1 protease. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Milk proteins; HIV-1; Reverse transcriptase; Protease; Integrase

Introduction A number of milk proteins such as lactoferrin have been reported to be inhibitory to HIV (1, 2). Chemical modification of these milk proteins, by reaction with succinic anhydride or aconitic anhydride which introduces negative charges into the milk proteins, usually increased the anti-HIV efficacy of these proteins (1, 3). * Corresponding author. Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Basic Medical Sciences Bldg., Shatin, NT, Hong Kong. Tel.: 852 2609-6875; fax: 852 2603 5123. E-mail address: [email protected] (T.B. Ng) 0024-3205/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 1 )0 1 3 1 1 -X

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Previously we have demonstrated that some of the bovine milk proteins were capable of inhibiting HIV-1 reverse transcriptase (HIV-1 RT). Succinylation of these milk proteins ensued in a marked enhancement of their potency to inhibit HIV-1 RT (4). We have extended this study by examining the milk proteins for possible inhibitory effects on all three HIV enzymes, reverse transcriptase, protease and integrase. The results furnish corroborative evidence for the suppressive action of the milk proteins on HIV-1 replication and evidence for the first time that different milk proteins inhibit HIV-1 protease and HIV-1 integrase to different extents. Materials and methods Materials a-Lactalbumin and b-lactoglobulin were purchased from Sigma Chemical Company Ltd. Casein was from BDH. Angiogenin-1, lactogenin, lactoperoxidase, glycolactin and lactoferrin were isolated in our laboratory from fresh unpasteurized bovine milk collected from a local dairy farm, in accordance with the following published procedures. Isolation of lactoferrin and lactoperoxidase For the isolation of lactoferrin and lactoperoxidase, the procedure of Yoshida and Ye (5) was utilized. Skimmed milk was prepared from whole milk by centrifugation, and then acidified to pH 4.5. The precipitated casein was removed by filtration and the acid whey was prepared from the filtrate. When the acid whey was applied on a sulfopropyl-Toyopearl column, which had previously been equilibrated with 50 mM phosphate buffer (pH 6.5), lactoferrin was adsorbed and eluted at nearly the end of a linear gradient of 0 – 0.5 M NaC1 in 50 mM phosphate buffer (pH 6.5). Lactoperoxidase was eluted at the beginning of the NaCl gradient. Isolation of angiogenin-l and lactogenin For the isolation of angiogenin-1 and lactogenin from bovine milk, acid whey (1 liter) was prepared by the procedure previously described (6). Globulin fraction was removed from acid whey by precipitation with 1.8 M ammonium sulfate (AS) followed by centrifugation. The 1.8 M AS whey collected was brought to 3.6 M with AS. Precipitate (3.6 M AS-PPT) was prepared by centrifugation. After dialysis, chromatography on a CM-Sepharose column was conducted in 50 mM sodium phosphate (pH 7.7). The adsorbed proteins were then desorbed with a linear gradient of NaC1. Fractions pooled from several runs on CM-Sepharose, were applied to a Mono S HR 5/5 column (1 ml). Angiogenin-1 and lactogenin were adsorbed on CM-Sepharose and eluted as two adjacent peaks (CM-1 and CM-2 respectively) when a linear NaC1 concentration gradient (0 – 0.55 M) was applied. Both proteins were adsorbed on Mono S. Isolation of glycolactin Globulin fraction was prepared from acid whey by precipitation with 1.8 M ammonium sulfate followed by centrifugation. After dialysis, chromatography on a CM-Sepharose col-

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umn was performed in 50 mM sodium phosphate (pH 7.7). The adsorbed proteins were then eluted with a linear gradient of NaCl (0 – 0.55 M), dialyzed and loaded on a Mono S HR 5/5 column. Glycolactin was adsorbed on both CM-Sepharose and Mono S (7). Chemical modification of milk proteins with succinic anhydride The procedure of Schoen et al (8) was followed. To a solution of the milk protein (10 mg) in 10 mL 0.2M K2HPO4 (pH8) was added solid succinic anhydride (10 mg). The solution was kept at pH8–8.5 with 3M NaOH and left in the dark at 258C for 1 h. Free succinic anhydride was removed using Centricon centrifugation. The reaction products were washed with distilled water and finally lyophilized. Assay of milk proteins for ability to inhibit-human immunodeficiency virus type 1 (HIV-1) reverse transcriptase activity The assay of milk proteins for ability to inhibit human immunodeficiency virus (HIV) reverse transcriptase activity was the same as that described by Collins et al (9) using a nonradioactive ELISA kit. The detection and quantification of synthesized DNA as a parameter to RT activity follows a sandwich ELISA protocol. Biotin-labeled DNA binds to the surface of microtiter plate modules precoated with strepavidin. An antibody to digoxigenin, conjugated to peroxidase (anti-DIG-POD), binds to the digoxigenin-labeled DNA. The peroxidase substrate is then added. The peroxidase enzyme catalyzes substrate cleavage to produce a colored reaction product. The absorbance of the samples at 405 nm is directly correlated to the level of RT activity. A fixed amount (4 – 6 ng) of recombinant HIV-1 reverse transcriptase was used. The inhibitory activity of milk protein was calculated as percent inhibition as compared to a control without the protein. Assay of milk proteins for ability to inhibit HIV-1 protease Expression and purification of recombinant HIV-1 protease The expression clone for recombinant human HIV-1 protease was a generous gift from Dr. J.N. Tang (Oklahoma Medical Research Foundtion, USA). HIV-1 protease cDNA was cloned in pET3b and transformed into Escherichia coli BL21(DE3)pLysS. HIV-1 protease expression was induced by IPTG. The expressed proteins, which were found predominantly as inclusion bodies, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bacterial colony which expressed a high level of the 11 kDa HIV-1 protease was picked and chosen for preparation of large-scale cultures. The procedure for purification of HIV-1 protease from cultures was carried out as described in (10). Fluorometric method for HIV-1 protease assay The activity of HIV-1 protease was assayed by cleavage of a fluorogenic substrate, Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)-Arg (Molecular Probes, USA), in a microtiter plate format for rapid screening of activity [16]. Briefly, 6.5 mg HIV-1 protease was added to reaction buffer (0.1 M sodium acetate, 1 M NaCl, 1 mM EDTA, 1 mM dithiothreitol, 10% dimethyl sulfoxide, 1 mg/ml bovine serum albumin (BSA), pH 4.7) containing 10 mM substrate in the presence or absence of milk proteins. After incubation at 378C for 2 h, the fluorescence intensity in each well was measured using a Cytofluor 2350 fluores-

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cence plate reader (Millipore Corp., USA) with an excitation wavelength at 340 nm and an emission wavelength at 490 nm (10). Assay of milk proteins for ability to inhibit HIV-1 integrase Expression and purification of recombinant HIV-1 integrase The plasmid that expressed His-tagged wild-type HIV-1 integrase, pT7-7-His(Y|TX)-HIV1-IN, was a generous gift from Dr. S.A. Chow (School of Medicine, UCLA). To express the protein, a 1-liter culture of E. coli BL21(DE3) cells containing the expression plasmid was grown at 378C until OD600 reached 0.7 – 0.8. Cells were induced by addition of 0.8 mM IPTG and harvested after a 4 h incubation by centrifugation at 6000 3 g for 10 min at 48C. Cells were suspended at a concentration of 10 ml/g wet cell paste in 20 mM Tris-HCl (pH 8.0), containing 0,1 mM EDTA, 2 mM b-mercaptoethanol, 0.5 M NaCl and 5 mM imidazole. Lysozyme was added to a concentration of 0.2 mg/ml. After 1 h incubation at 48C, the lysate was sonicated and centrifuged at 40,000 3 g at 48C for 20 min. The pellet was homogenized in 50 ml buffer A (20 mM Tris-HCl, pH 8.0, 2 M NaCl, 2 mM b-mercapto-ethanol) containing 5 mM imidazole. The suspension was rotated at 48C for 1 h and cleared by centrifugation at 40,000 3 g at 48C for 20 min. The supernatant was loaded onto a 1 ml chelating Sepharose column charged with 50 mM imidazole. The column was washed with five column volumes of buffer A containing 5 mM imidazole and the protein was eluted with three column volumes of buffer A containing 200 mM and 400 mM imidazole, respectively. Protein-containing fractions were pooled and EDTA was added to a final concentration of 5 mM. The protein was dialyzed against buffer B (20 mM HEPES, pH 7.5, 1 mM EDTA, 1 M NaCl, 20% glycerol) containing 2 mM b-mercaptoethanol and then against buffer B containing 1 mM dithiothreitol. Aliquots of the protein were stored at 2708C (11). HIV-1 integrase assays A non-radioactive ELISA-based HIV-1 integrase assay was performed according to the DNA-coated plates method. In this study, 1 mg of Smal-linearized p Bluescript SK was coated onto each well in the presence of 2 M NaCl as target DNA. The donor DNA was prepared by annealing VU5BR (59-biotin-GTGTGGAAAATCTCTAGCAGT-39) and VU5 (59-ACTGCTAGAGATTTTCCACAC-39) in 10 mM Tris-HC1, pH 8.0, 1 mM EDTA and 0.1 M NaCl at 808C followed by 30 min at room temperature. Integrase reaction was performed in 20 mM HEPES (pH 7.5), containing 10 mM MnCl2, 30 mM NaCl, 10 mM dithiothreitol and 0.05% Nonidet-P40 (Sigma). After the integrase reaction, the biotinylated DNA immobilized on the wells was detected by incubation with streptavidin-conjugated alkaline phosphatase (Boehringer Mannheim) followed by colorimetric detection with 1 mg/ml p-nitrophenyl phosphate in 10% diethanolamine buffer, pH 9.8, containing 0.5 mM MgCl2. The absorbance due to the alkaline phosphatase reaction was measured at 415 nm (11). Results Lactoferrin manifested the highest potency in inhibiting HIV-1 reverse transcriptase. Lactoperoxidase exhibited the next highest inhibitory potency, followed by lactogenin, angiogenin-1 and glycolactin. a-Lactalbumin, b-lactoglobulin and casein did not elicit any inhibition (Table 1).

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Table 1 Effect of various bovine milk proteins on activities of HIV-1 reverse transcriptase, HIV-1 protease and HIV-1 integrase

MW (kDa) Lactoferrin Lactoperoxidase Glycolactin Angiogenin-1 Lactogenin a-Lactalbumin b-Lactoglobulin Casein

8.6 8.2 64 15 17 14.4 18.3 22

% Inhibition of HIV-1 % Inhibition of % Inhibition of HIV-1 integrase activity HIV-1 reverse protease activity transcriptase Unsuccinylated Succinylated by 5 mg 20 mg milk 200 mg milk milk protein milk protein Conc. protein/ml (50 mg/ml) (50 mg/ml) in milk milk protein/ml protein/ml , 0.1a , 0.03a 1.9b 0.8b 0.7b 0.6–1a 2–4a z10a

88.6 6 6.3 78.2 6 6.7 39.8 6 3,3 48.3 6 3.2 58.7 6 4.9 N.D. N.D. N.D.

0.62 6 0.27 3.17 6 0.41 10.62 6 0.19 11.64 6 0.63 5.44 6 0.39 4.50 6 0.21 3.29 6 0.09 5.51 6 0.37

8.77 6 0.11 9.47 6 0.12 32.15 6 0.27 44.46 6 0.16 13.95 6 0.73 21.40 6 0.93 21.93 6 1.82 40.59 6 0.37

11.3 6 0.2 63.2 6 3.6 78.8 6 3.4 80.1 6 3.8 84.1 6 3.7 98.4 6 1.6 80.6 6 5.9 98.9 6 0.4

74.5 6 2.5 70.4 6 3.7 71.0 6 2.3 74.2 6 4.6 91.9 6 1.8 100 6 0.2 91.7 6 1.5 93.0 6 1.6

N.D. 5 no inhibition observed. Results represent means 6 SD (N 5 3). Concentration in mg/ml milk. b Concentration in mg/ml milk. a

Angiogenin-1 and casein were the most potent in inhibiting HIV-1 protease, followed by glycolactin. a-Lactalbumin and b-lactoglobulin demonstrated a weaker inhibitory activity. Lactogenin exhibited an even weaker activity. Lactoferrin showed the weakest activity (Table 1). a-Lactalbumin and casein demonstrated the strongest inhibitory activity toward HIV-1 integrase. Glycolactin, angiogenin-1, lactogenin and b-lactoglobulin formed a group with the next highest inhibiting activity, followed by lactoperoxidase, and lactoferrin was the weakest inhibitor (Table 1). Succinylation enhanced the ability of lactoferrin and b-lactoglobulin to inhibit HIV-1 integrase (Table 1). Discussion The results of the present investigation regarding the inhibition of HIV-1 reverse transcriptase were in general similar to those previously reported (4). The experiment was repeated in the present study to make the study more complete and three HIV-1 enzymes were included in one investigation. Our earlier investigation (4) revealed that lactoferrin, lactoperoxidase, angiogenin-1 and the novel milk proteins lactogenin and glycolactin were able to inhibit HIV-1 reverse transcriptase. The inhibition of HIV-1 reverse transcriptase by lactoferrin followed a dose-dependent relationship. The dose responses for the other milk proteins were not examined because it would appear from the dose-responses for lactoferrin that a 10-fold dilution of the other milk proteins would result in only about 10% inhibition remaining. The present study further disclosed that, lactoferrin only weakly inhibited HIV-1 protease and HIV-1 integrase, lactoperoxidase weakly inhibited HIV-1 protease, and a-lactalbumin, b-lactoglobulin and casein did not inhibit HIV-1 reverse transcriptase. All other milk proteins examined including glycolactin, angiogenin-1 and lactogenin were capable of inhibiting HIV-1 reverse transcriptase, protease and integrase to an appreciable extent.

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The effect of succinylation in augmenting the ability of lactoferrin to inhibit HIV-1 integrase was analogous to the previous observation that after succinylation casein, a-lactalbumin, b-lactoglobulin, glycolactin and lactoferrin acquired a higher potency in inhibiting HIV-1 reverse transcriptase (4). Previously it has been reported that a-lactalbumin,casein and b-lactoglobulin acquired enhanced anti-HIV potency after chemical modification with succinic anhydride, aconitic anhydride or 3-hydroxyphthalic anhydride (1, 3, 13, 14,). Hence succinylation not only enhanced the anti-HIV potency of the milk proteins at the level of the virus but also at the level of the viral reverse transcriptase and integrase. Chemical modification such as reaction with 3-hydroxyphthalic anhydride causes a milk protein to acquire negatively charged carboxyl groups along the polypeptide backbone. Electrostatic repulsion within the protein occurs thereby affecting specific tertiary and probably also the secondary structure of the protein (14). The use of biophysical techniques has disclosed that the chemically modified protein is largely unstructured. It is also devoid of cytotoxicity (14) but blocks the CD4 cell receptor for HIV (14). Swart et al (3) carried out peptide-scanning studies to show that the native lactoferrin and the charge modified proteins bound strongly to the V3 loop of the gp 120 envelope protein and thereby inhibiting virus-cell fusion and viral entry. It is interesting to note that succinylation of the milk proteins led to, in some cases, heightened potency in inhibiting HIV-1 reverse transcriptase. Succinylation also changed the binding of the milk proteins with HIV-1 reverse transcriptase and integrase. The chemically modified milk proteins have some probable practical uses. It has been shown that 3-hydroxyphthaloylb-lactoglobulin prevents vaginal transmission of simian immunodeficiency virus in rhesus monkeys (15). Some of the milk proteins e.g. a-lactalbumin, b-lactoglobulin and casein were able to inhibit HIV-1 protease and HIV-1 integrase at concentrations found in bovine milk. Lactoferrin was able to inhibit HIV-1 integrase at concentrations found in bovine milk. However, none of the milk proteins tested was unable to inhibit HIV-1 reverse transcriptase at concentrations found in bovine milk. In general, the bovine milk proteins inhibited HIV-1 reverse transcriptase, HIV-1 protease and HIV-1 integrase at micromolar concentrations. Some anti-HIV natural products of plant origin also inhibit HIV-1 reverse transcriptase at similar concentrations (16). However, the anti-HIV potency of milk proteins are considerably heightened by chemical modifications such as aconitylation and succinylation (3, 4, 8, 12). Acknowledgments We thank the Research Grants Council for award of an earmarked grant and a direct grant and Ms. Fion Yung for excellent secretarial assistance. References 1. 2.

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