The antiviral xanthate compound D609 inhibits herpes simplex virus type 1 replication and protein phosphorylation
Introduction
The antiviral xanthate compound D609 (tricyclodecan-9-yl-xanthogenate) inhibits a wide variety of enveloped and non-enveloped DNA and RNA viruses without inhibiting the growth of the cells (Sauer et al., 1984). In addition to its antiviral activity, D609 also inhibits the growth of certain tumors in animal models without toxicity (Amtmann et al., 1985Amtmann and Sauer, 1987Fürstenberger et al., 1989). Such a broad spectrum of activity suggests that D609 inhibits a fundamental metabolic process required for virus replication and tumor growth. The work of Müller-Decker (1989) showed that phospholipid turnover was inhibited by D609 suggesting that phospholipase C activity may be inhibited by D609.
Protein phosphorylation was suggested as a target for D609 by the demonstration that phosphorylation, but not synthesis, of the regulatory protein NS of vesicular stomatitis virus (VSV) was inhibited following treatment of VSV-infected cells with D609 (Müller-Decker et al., 1987). D609 treatment also inhibits the phosphorylation of the P protein of respiratory syncytial virus (Villanueva et al., 1991).
HSV proteins are phosphorylated by viral and cellular protein kinases and protein phosphorylation appears to be important for virus replication (Wilcox et al., 1980). HSV-1 encodes two protein kinase activities, US3 PK and UL13 PK (Purves et al., 1987b, Purves et al., 1991, Smith and Smith, 1989). The US3 PK is not essential for viral growth in tissue culture but is important for growth in neurons (Fink et al., 1992, Glorioso et al., 1994) and primary cell lines (unpublished data).
The replication of herpes simplex virus type 1 (HSV-1) is inhibited by D609, but by an unknown mechanism (Sauer et al., 1984). Thus, the primary aim of this study was to determine whether D609 inhibits viral and cellular protein kinases (PKs) thereby identifying the mechanism of antiviral action. Proteins are synthesized and phosphorylated at several times during the replication of HSV-1 (Wilcox et al., 1980), thus an additional aim was to determine when the drug-sensitive step occurs during the replication cycle of HSV-1. A model for D609 activity is presented.
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Cells and viruses
Vero cells were obtained from the American Type Culture Collection and were maintained in Modified autoclavable minimum essential medium with Earle's salts (EMEM) from GIBCO supplemented with 10% newborn calf serum, 20 U/ml penicillin and 20 μg/ml streptomycin and 29.2 μg/ml l-glutamine. HSV type 1 strain F (which contains and expresses the US3 gene) and strain R7041 (a US3 deletion mutant which does not express the US3 gene) (Longnecker and Roizman, 1987) was obtained from Dr B. Roizman,
D609 inhibits HSV-1 production
Vero cells infected at different multiplicities of infection (MOI) were treated with several concentrations of D609 at levels well below toxicity (Rosenthal et al., 1987, Sauer et al., 1984). The amount of virus produced after 24 h was determined by plaque assay on Vero cells.
The extent of anti-herpetic activity of D609 was dependent upon the MOI of virus (Table 1). At an MOI of 1 PFU/cell or less, concentrations of D609 as low as 3.8 μM reduced virus production by 50% and 75.2 μM D609
Discussion
The ability of D609 to inhibit the replication of RNA and DNA, enveloped and naked capsid viruses as well as certain tumors suggests a common target which is selectively important for their growth and replication. The inhibition of HSV-1 replication and protein phosphorylation with similar concentration dependence and the inhibitory activity of D609 on purified viral and cellular protein kinases indicates that protein phosphorylation is the target for D609 anti-HSV activity and that the target
Acknowledgements
This work was supported in part by grants from Merz and Co., Frankfurt, the Ohio Board of Regents Research Challenge Program and a Faculty Research Grant from The University of Akron.
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Current Address: Department of Natural Sciences, Walsh University, North Canton, OH 44720, USA.