Mechanism of the antiviral action of 1-beta-D-arabinofuranosylcytosine on Borna disease virus

Abstract

Borna disease virus (BDV) is a nonsegmented, negative-stranded RNA virus that causes neurological diseases in a variety of warm-blooded animal species. Recently, we showed that the nucleoside analog 1-beta-D-arabinofuranosylcytosine (Ara-C) was a potent inhibitor of BDV. This finding was surprising for an RNA virus, since Ara-C is a DNA polymerase inhibitor. Thus, we sought to better define the mechanism of action of Ara-C on BDV. Here, we show that (i) this effect is specific for an arabinoside ring carrying a cytosine base, (ii) it requires phosphorylation of the nucleotide, and (iii) it can be reversed by an excess of cytidine. Using the recently described minigenome assay for BDV, we provide evidence suggesting that Ara-C may act as a competitive inhibitor of the BDV replication complex.

FIG. 1.

Representative examples of the inhibitory effects of compounds used in this study. (Top panels) Analysis by immunofluorescence of the subcellular localization of the BDV nucleocapsid protein (N) following treatment with different compounds. Vero-BV cells were treated for 5 days with the different compounds and stained with a rabbit anti-N polyclonal antibody, followed by a fluorescein isothiocyanate-conjugated anti-rabbit antibody. Of all compounds tested (only examples are shown here), only Ara-C resulted in nuclear retention of BDV N protein. Magnification, ×120 (original magnification, ×200). (Bottom panels) FACS analysis of BDV cell-to-cell spread. Confluent Vero cells were labeled with 5- (and 6-)carboxyfluorescein diacetate (CFDA) and were subsequently cocultivated for 5 days at a ratio of 1:1 with unlabeled Vero-BV cells. Cocultivation took place under daily treatment with 4 μM Ara-C or with various drugs (e.g., 50 μM Ara-A). Thereafter, cells were analyzed by flow cytometry. The percentage of viral dissemination during the cocultivation period was calculated as indicated in Table 1 and is shown in each case. Note that inhibition of BDV cell-to-cell spread is specific to Ara-C. The negative control consisted of a 1:1 mixture of CFDA-labeled Vero cells with Vero-BV cells, which were fixed directly after mixing.

FIG. 2.

Metabolic phosphorylation pathways of cytidine and its derivatives. The mode of action of hydroxyurea is shown.

FIG. 3.

Dose-dependent, inhibitory effect of Ara-C in the BDV minireplicon assay. Analysis of reporter gene expression by a CAT ELISA. BSR-T7 cells (treated or not with the different compounds) were transfected with the minigenome construct together with optimal quantities of the expression plasmids for the BDV L, N, and P proteins. In the negative control (−), the plasmid encoding BDV L was replaced by the same amount of a plasmid encoding green fluorescent protein. The positive control (+) consisted of untreated cells and was set at 100% in each experiment. For an internal control for transfection efficiency, a plasmid encoding Renilla luciferase under the control of a polymerase II promoter was cotransfected. Seventy-two hours after transfection, the cells were lysed and analyzed for CAT protein and luciferase levels. CAT values were normalized for transfection efficiency and are expressed for each sample as the percentage of the value with the untreated control. Data shown are means (± standard deviations) for three independent experiments.