Mechanism of Antiviral Action of (E)-5-(2-Bromovinyl)-2′-Deoxyuridine (BVDU): Direct Evidence with [14C]BVDU in Herpes Simplex Virus-Infected Cells

Abstract

The mechanism of action of (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) was investigated using a 14C-radiolabelled derivative. A remarkable accumulation of [14C]BVDU was detected in herpes simplex virus type 1 (HSV-1)-infected cells, in greater quantities (18-fold) than in uninfected cells. Furthermore, selective incorporation of [14C]BVDU was measured exclusively into HSV-1 DNA. Further studies on the cellular kinetic and metabolic fate of BVDU indicated that in HSV-1 -infected cells it was phosphorylated intracellularly to its mono-(BVDU-MP) and triphosphate (BVDU-TP) derivatives. The amount of BVDU-MP was 10 times higher than BVDU-TP. BVDU entered infected cells very quickly and was rapidly converted into BVDU-MP which, in turn, was further metabolized to BVDU-TP. After 5 h incubation, 13% of initial radioactivity was found in the SDS phase, representing mainly DNA and proteins, of HSV-1-infected cells and only 2% in uninfected cells, further confirming the selective incorporation of the compound into viral DNA. Following removal of BVDU from culture medium, the cellular decay of BVDU-phosphorylated derivatives was rather slow, with an estimated half-life of 10 h, and their disappearance from the cytosol was paralleled by a progressive accumulation of radioactivity in the SDS phase. These results give direct evidence for the selective cellular uptake, metabolism and viral DNA accumulation of BVDU in HSV-1-infected cells, representing the major factors for its powerful and specific anti-herpesvirus activity.