Use of a prenylation inhibitor as a novel antiviral agent

Abstract

No specific therapy exists for hepatitis delta virus (HDV), which can cause severe liver disease. Molecular genetic studies have implicated the prenylation site of large delta antigen as a critical determinant of HDV particle assembly. We have established a cell culture model which produces HDV-like particles, and we show that delta antigen prenylation can be pharmacologically inhibited by the prenylation inhibitor BZA-5B. Furthermore, BZA-5B specifically abolishes particle production in a dose-dependent manner. These results demonstrate that the use of such a prenylation inhibitor-based antiviral therapy may be feasible and identify a novel class of potential antiviral agents.

Figures

FIG. 1

LH cells produce HDV-like particles. (A) HDV-like particles (lane 1) were immunoprecipitated from clarified medium supernatants (Sup.) of LH cells with a monoclonal antibody to HBsAg and subjected to immunoblot analysis for the presence of large delta antigen. LH cells remaining on the dish were harvested in cell lysis buffer and an aliquot (lane 2) was included in the immunoblot analysis. (B) Particles (lane 1) were isolated by ultracentrifugation of clarified medium supernatants of LH cells and an aliquot was subjected to immunoblot analysis, along with a sample of LH cell lysate (lane 2). L, large delta antigen. The locations of prestained molecular mass markers are shown at the left (in kilodaltons).

FIG. 2

BZA-5B inhibits prenylation of large delta antigen. Combined in vitro transcription-translation reactions were performed with rabbit reticulocyte lysates programmed with water (lanes 1) or a plasmid encoding large delta antigen (lanes 2 to 7) in the presence of [5-3H]mevalonate and either water (lanes 2), a carrier (0.5 mM DTT and 0.05% DMSO) (lanes 3), or a carrier with 5, 10, 25, or 50 μM BZA-5B, as indicated. Aliquots (1 μl) were subjected to SDS-PAGE and either fluorography (A) or immunoblot analysis (B). L, large delta antigen.

FIG. 3

BZA-5B inhibits HDV-like-particle production. Duplicate dishes of LH cells were grown in media containing a carrier (0.5 mM DTT and 0.05% DMSO) alone (lanes 1) or a carrier with either 10, 25, or 50 μM BZA-5B. Clarified medium supernatants were analyzed for the presence of HDV-like particles by quantitative immunoblot analysis (A). The underlying cells were harvested and counted, and the presence of large delta antigen was analyzed by immunoblotting (B). Only one of the duplicate set of blots is shown. (C) HBsAg was quantitated in duplicate aliquots of each medium supernatant sample, and the percentage of control HBsAg per cell (light bars) and of control particles per cell (dark bars), determined from the experiment whose results are shown in panel A, at each concentration of BZA-5B is plotted. Error bars represent the average deviations.