Identification of a Varicella-Zoster Virus Replication Inhibitor That Blocks Capsid Assembly by Interacting with the Floor Domain of the

A novel anti-varicella-zoster virus compound, a derivative of pyrazolo[1,5-c]1,3,5-triazin-4-one (coded as 35B2), was identified from a library of 9,600 random compounds. This compound inhibited both acyclovir (ACV)-resistant and-sensitive strains. In a plaque reduction assay under conditions in which the 50 % effective concentration of ACV against the vaccine Oka strain (V-Oka) in human fibroblasts was 4.25M, the 50 % effective concentration of 35B2 was 0.75M. The selective index of the com-pound was more than 200. Treatment with 35B2 inhibited neither immediate-early gene expression nor viral DNA synthesis. Twenty-four virus clones resistant to 35B2 were isolated, all of which had a mutation(s) in the amino acid sequence of open read-ing frame 40 (ORF40), which encodes the major capsid protein (MCP). Most of the mutations were located in the regions corre-sponding to the “floor ” domain of the MCP of herpes simplex virus 1. Treatment with 35B2 changed the localization of MCP in the fibroblasts infected with V-Oka but not in the fibroblasts infected with the resistant clones, although it did not affect steady-state levels of MCP. Overexpression of the scaffold proteins restored the normal MCP localization in the 35B2-treated infected cells. The compound did not inhibit the scaffold protein-mediated translocation of MCP from the cytoplasm to the nucleus. Electronmicroscopic analysis demonstrated the lack of capsid formation in the 35B2-treated infected cells. These data indicate the feasibility of developing a new class of antivirals that target the herpesvirus MCPs and inhibit normal capsid formation by a mechanism that differs from those of the known protease and encapsidation inhibitors. Further biochemical studies are re-quired to clarify the precise antiviral mechanism