Investigation of the Antiviral Activity of Triterpenoid Compounds Against Retoviruses

Arabzadeh, Seyedali M (1996) Investigation of the Antiviral Activity of Triterpenoid Compounds Against Retoviruses. PhD thesis, University of Glasgow.

Abstract

The triterpenoid compound glycyrrhizin (GL) is widely used in Japan for the treatment of viral hepatitis. GL, carbenoxolone sodium (CBX), and cicloxolone sodium (CCX) exhibit broad range antiviral activity both in vivo and in vitro. Both in vitro and in vivo anti-HIV activity has also been reported for GL. This thesis investigates the effect of triterpenoid compounds on replication of two retrovirus in tissue cultures; Feline leukaemia virus type A (FeLV-A) and Feline immunodeficiency virus (FIV). Both of these viruses cause AIDS-like immunodeficiency diseases in cats. The anti-retrovirus activity of the triterpenoid compounds is contrasted with the effect of the drugs on the replication of bovine herpesvirus type 1 (BHV-1) in MDBK cells. Triterpenoid compounds are lipophilic and effect the host cell membrane functions. In order to determine drug concentrations in which cytotoxic effect of the drug could be uncoupled from the antiviral activity, cyotoxicity tests were performed. The effect of the drugs on cell culture growth and percentage cell viability were determined by trypan blue dye exclusion. The highest drug concentrations tolerated were; for FeA and CrFK cells 100muM CCX, or 100muM CBX and for MDBK cells 300muM CCX (used for BHV-1 studies). Drug removal experiments have shown that the effect of the drugs was reversible and that two days treatment with 100muM CCX, or 100muM CBX had no effect on the subsequent growth of FeA or CrFK cell cultures. The IC50 concentrations (concentration of the drug inhibiting cellular growth by 50%) determined for both CrFK and FeA cells after 24h or 48h drug treatments were > 300muM and 150muM respectively. Based on the data obtained from cytotoxicity tests, it was concluded that CrFK and FeA cells could be placed within the grouping of CCX-resistant cell lines classified by Galt et al., (1990). In order, to investigate the intracellular location of CCX molecules, an attempt was made to generate an anti-CCX antibody in immunise rabbits. Despite detection of an apparent anti-CCX antibody in an ELISA test, experiments designed to demonstrate the specific antibody tagging of CCX molecules in drug treated cells by immunofluorescence were unsuccessful. Previous studies have shown that CCX treatment impaired the glycosylation of HSV, VSV, and SFV glycoproteins, suggesting that the Golgi apparatus might be a target for triterpenoid compounds. A trans-Golgi specific monoclonal antibody (directed against the p58 protein) was employed to investigate the effect of CCX on the MDBK and CrFK cells Golgi apparatus. While perinuclear (Golgi) immunofluorescence was observed in drugfree cultures, little or no such staining was detected in cells treated with CCX, suggesting that the drug perturb the Golgi apparatus. It was further postulated that up-regulation of Na+/K+ATPase activity (a known effect of triterpenoid compounds) might play a role in the effect of CCX on the Golgi apparatus. To test this hypothesis, ouabain (a specific inhibitor of Na+/K+ATPase activity) was used in Golgi immunofluorescence labelling experiments. The effect of CCX on the Golgi apparatus of MDBK cells was impaired when cells were pre-treated with ouabain, confirming that the CCX-induced perturbation of the trans-Golgi compartment was associated with the up-regulation of Na+/K+ATPase activity. A model that can explain the effect of the triterpenoid compounds on Golgi apparatus has been given. CBX and GL exhibit mild virucidal activity against FeLV-A, FIV, and BHV-1. 300muM CCX treatment of virus particles in suspensions resulted in a 4 fold reduction in FeLV-A, and 10 fold reduction of FIV infectivity. Treatment of BHV-1 particles with 1500muM GL resulted in a 4.2 fold reduction infectivity. FeLV-A infectious virus yield, drug-dose-response experiments in which infected FeA cells were treated with increasing concentrations of CCX, CBX, or GL resulted in extracellular reductions of 155, 101, and 128 fold, when treated with 150muM CCX, 150muM CBX, or 2500muM GL respectively. The kinetics of the dose-response curves obtained for each drug were triphasic; an initial ~ 10 fold reduction in infectivity when cells were treated with 25muM CCX, 25muM CBX, or 500muM GL, followed by a near plateau in the curve up to 100muM CCX, 100muM CBX or 2000muM GL, and a sharp decrease in the curve at 150muM CCX, 150muM CBX, or 2500muM GL. The results suggest that the antiviral activity of triterpenoid compounds may operate by inhibiting two different target functions; the first, a function that enhances but is not essential for virus infectivity and which is sensitive to low drug concentrations, and the second, a function which is probably essential for virus replication and which is only sensitive to high concentrations of the drugs. (Abstract shortened by ProQuest.).

• Item Type: Thesis (PhD)
• Qualification Level: Doctoral
• Additional Information: Adviser: Derrick Dargan
• Keywords: Virology, Pharmacology
• Date of Award: 1996
• Depositing User: Enlighten Team
• Unique ID: glathesis:1996-75697
• Copyright: Copyright of this thesis is held by the author.
• Date Deposited: 19 Nov 2019 18:55
• Last Modified: 19 Nov 2019 18:55
• URI: https://theses.gla.ac.uk/id/eprint/75697