Evaluation of cystatin C activities against HIV

Abstract

Background & objectives: Several host defense proteins known to possess antimicrobial activities are present on mucosal surfaces and are consequently found in body fluids of vertebrates. Naturally occurring protease inhibitors like cystatins, especially cystatin C (cys C), are abundantly present in human seminal plasma. Although its antiviral activity against herpes simplex virus (HSV) has been demonstrated, the role of this protein against HIV is not well studied. Therefore, the aim of the present study was to evaluate the anti-HIV activities of cys C, which is present innately in the male reproductive tract.

Methods: Protein-protein interaction of cys C with various HIV proteins was studied using a commercially available HIV blot and specific interaction with HIV protease was studied by dot-blot technique using commercially available cys C. To purify biologically active cys C from human seminal plasma to be used for subsequent experiments, gel-permeation chromatography followed by affinity chromatography was used. The HIV infectivity inhibition activity of the purified cystatin C was tested in TZM-bl cells. To study its activity on HIV protease, time-course enzyme kinetics studies were performed using spectrometric assay.

Results: Cystatin C reacted with some HIV proteins including HIV protease. Biologically active cys C was purified using gel permeation chromatography followed by affinity chromatography. When tested in TZM-bl cells, purified cystatin C demonstrated HIV-infectivity inhibitory activity (IC 50: 0.28 μM). Enzyme kinetic studies demonstrated that it abrogated the action of HIV protease on its substrate.

Interpretation & conclusions: The present data demonstrate that cystatin C possesses anti-HIV activities. Molecular models need to be designed with this protein which would assist towards prevention/ therapeutics against HIV.

Fig. 1

(A). Bands of HIV proteins (gp160, gp120, p31, p24) interacting with cys C. (B). No bands were seen with an unrelated protein treated similarly (C). Bands visualized with HIV-positive serum only are shown (D). Interaction of cys C with HIV protease by dot blot. No binding was observed with an unrelated protein.

Fig 2

(A). depicts the chromatogram of human seminal plasma proteins purified by gel permeation chromatography, the protein profile of which is shown in (B). The presence of cys C in Fr.4 is indicated in (C). (M, mol wt. marker; Control, blot treated with pre-immune serum; test, Blot treated with cys C antibody).

Fig. 3

(A). demonstrates the band(s) of purified cystatin C from fraction 4 of human seminal plasma proteins purified by gel-permeation chromatography and by western blotting using specific antibodies to cys C (B).

Fig. 4

A representative graph depicting the activity of 0.75 μM cys C (test), Darunavir (positive control) and an unrelated protein (negative control) on the action of HIV protease on its peptide substrate. A continuous decrease in absorbance at 310 nm was observed with unrelated protein whereas cys C and Darunavir did not show decrease in absorbance.

Fig. 5

Cys C exhibited a dose-dependant decrease of blue-stained HIV-infected cells as compared to virus only control.

Fig. 6

No significant difference observed in the absorbance with increasing concentrations of cys C as compared to control in MTT assay.