Metabolism and mode of action of (R)-9-(3,4-dihydroxybutyl)guanine in herpes simplex virus-infected vero cells.

K Stenberg,A Larsson,R Datema

doi:10.1016/s0021-9258(17)35908-2

Abstract

The metabolism and mode of action of the anti-herpes compound buciclovir [R)-9-(3,4-dihydroxybutyl)-guanine, BCV) has been studied in herpes simplex virus-infected and uninfected Vero cells. In uninfected cells, a low and constant concentration of intracellular BCV was found, while in herpes simplex virus-infected cells, an increasing concentration of BCV phosphates was found due to metabolic trapping. The major phosphorylation product was BCV triphosphate (BCVTP) which was 92% of the total amount of BCV phosphates. BCV phosphates were accumulated to the same extent in cells infected with either a herpes simplex virus type 1 or a herpes simplex virus type 2 strain while thymidine kinase-deficient mutants of herpes simplex virus type 1 were 10 times less efficient in accumulating BCV phosphates. In uninfected Vero cells, the concentration of the phosphorylated forms of BCV was less than 1% of that found in herpes simplex virus-infected cells. The BCVTP formed in herpes simplex virus-infected cells was highly stable, as 80% of the amount of BCVTP was still present even 17 h after removal of extracellular BCV. BCV was a good substrate for herpes simplex virus type 1- and type 2-induced thymidine kinases but not for the cellular cytosol or mitochondrial thymidine kinases. BCV monophosphate could be phosphorylated by cellular guanylate kinase to BCV diphosphate. BCVTP was a selective and competitive inhibitor to deoxyguanosine triphosphate of the purified herpes simplex virus type 1- and type 2-induced DNA polymerases. BCVTP could neither act as an alternative substrate in the herpes simplex virus type 2 or cellular DNA polymerase reactions, nor could [3H]BCV monophosphate be detected in DNA formed by herpes simplex virus type 2 DNA polymerase, or be detected in nucleic acids extracted from herpes simplex virus type 1-infected cells. These data indicate that BCVTP may inhibit the herpes simplex virus-induced DNA polymerase without being incorporated into DNA.

Highlights

BCV phosphates were accumulated to the same extent of HSV-1 and HSV-2 multiplication in vitro and in vivo
The intracellular material consisted after 30 min incubation of 92% BCV, 5% BCVMP, and 3%BCV triphosphate (BCVTP)
In cells where HSV thymidine kinase was induced, BCVwas phosphorylated more rapidly and theintracellular concentration of BCVTP increased to 50 times the extracellular concentration ofBCV

Summary

Introduction

BCV phosphates were accumulated to the same extent of HSV-1 and HSV-2 multiplication in vitro and in vivo 8).BCV hasa high selectivity for HSV thymidine kinase compared to the cellular thymidine kinases which might be the basis for BCV’s selectivity as an antiviral agent ( 7 ) .In. BCV phosphates. In uninfected Vero cells, the concen- HSV-infected cells, BCV preferentially inhibits viral DNA tration of the phosphorylated forms ofBCV was less synthesis [7, 9] which is the cause of BCV’s antiviral effect. Than 1%of that found in herpes simplex virus-infected Since cell proliferation and DNA synthesis of uninfected cells cells. The BCVTP formed inherpes simplex virusinfected cells was highly stable, as 80% of the amount of BCVTP was still presenteven 17 h after removal of extracellular BCV

Methods

Results

Conclusion