Evaluation of cystatin C activities against HIV

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.