Identification of small molecule compounds that selectively inhibit varicella-zoster virus replication

Abstract

A series of nonnucleoside, N-alpha-methylbenzyl-N'-arylthiourea analogs were identified which demonstrated selective activity against varicella-zoster virus (VZV) but were inactive against other human herpesviruses, including herpes simplex virus. Representative compounds had potent activity against VZV early-passage clinical isolates and an acyclovir-resistant isolate. Resistant viruses generated against one inhibitor were also resistant to other compounds in the series, suggesting that this group of related small molecules was acting on the same virus-specific target. Sequencing of the VZV ORF54 gene from two independently derived resistant viruses revealed mutations in ORF54 compared to the parental VZV strain Ellen sequence. Recombinant VZV in which the wild-type ORF54 sequence was replaced with the ORF54 gene from either of the resistant viruses became resistant to the series of inhibitor compounds. Treatment of VZV-infected cells with the inhibitor impaired morphogenesis of capsids. Inhibitor-treated cells lacked DNA-containing dense-core capsids in the nucleus, and only incomplete virions were present on the cell surface. These data suggest that the VZV-specific thiourea inhibitor series block virus replication by interfering with the function of the ORF54 protein and/or other proteins that interact with the ORF54 protein.

FIG. 1.

Structures of Comp I, Comp II, and Comp III.

FIG. 2.

Indirect immunofluorescence analysis of VZV IE62 protein (A) or gB (B) expression in the presence or absence of Comp I. Panels on the left are stained with monoclonal antibodies specific for VZV proteins. Panels on the right show the adjacent field of cells stained with 4′,6-diamidino-2-phenylindole (DAPI).

FIG. 3.

Activity of selected compounds against the VZV strain Ellen Comp I-resistant isolates. (A) IC₅₀ (micrograms per milliliter) were derived from automated ELISA results of two separate experiments comparing the parental strain Ellen and the two individually isolated Ellen^r mutants. Comp II and Comp III, structurally related compounds within the VZV thiourea inhibitor series, were tested along with Comp I. (B) Plaque reduction assays show the percent reduction in plaque number for various concentrations of the three compounds against parental Ellen and the Ellen^r isolates. The data represent the average number of plaques observed in triplicate samples for all viruses at each indicated drug concentration. The dotted line indicates a 50% reduction in the number of plaques. Data were normalized so that the average number of plaques observed in the no-drug control samples for any one virus was 100.

FIG. 4.

In vitro growth kinetics of VZV Ellen^rA. HFF were inoculated with cell-associated or cell-free virus, and at various times postinfection the cells were harvested and virus titers were determined on fresh HFF monolayers. A comparison of the parental strain Ellen and the Ellen^rA isolate is shown. Data points represent the average of three independent assays.

FIG. 5.

ORF54 mutations associated with resistance to Comp I. The amino acid sequences of the ORF54 protein from parental VZV Ellen, recombinant Oka (ROka), and the ACV-resistant isolate (Ellen-ACV^r) were identical. The regions corresponding to amino acids 320 to 329 and 400 to 409 of ORF54 are shown. Changes identified for the various resistant isolates are boxed.

FIG. 6.

Strategy for construction of recombinant VZV with specific mutations in ORF54. (A) Cosmid clones used for generation of VZV ROka. (B) A 1.4-kb fragment derived from ORF54 of Ellen^rA was inserted into a 9.7-kb NheI subclone from cosmid MstIIA in place of the wild-type sequence. One of the two nucleotide changes observed for the 1.4-kb Ellen^rA ORF54 fragment resulted in the elimination of an NsiI restriction site and was used to identify NheI Ellen^rA clones. The altered NheI fragment was inserted into cosmid MstIIA, in place of the wild-type ROka sequence, to generate cosmid MstIIA-54A. The ORF54 region within MstIIA-54A was sequenced to confirm that the expected changes were present: two nucleotide changes resulting in Y-to-C and N-to-D substitutions at amino acids 324 and 408, respectively. (C) A 1.4-kb fragment derived from ORF54 Ellen^rB was inserted into a 9.7-kb NheI subclone derived from cosmid MstIIA-54A. The 1.4-kb Ellen^rB fragment regenerated an NsiI restriction site in ORF54 that was used to identify NheI Ellen^rB clones. The altered NheI fragment was inserted into MstIIA to generate cosmid MstIIA-54B. The ORF54 region within MstIIA-54B was sequenced to confirm that the expected change was present: a single nucleotide change resulting in a G-to-D substitution at amino acid 407.

FIG. 7.

In vitro growth kinetics of ROka54A3 and A4, containing ORF54 mutations derived from Ellen^rA. HFF were inoculated with VZV-infected cells, and at various times postinfection, cells were harvested and virus titers were determined on fresh HFF monolayers. Data points represent the average of three independent assays.

FIG. 8.

Activity of Comp I against parental and ORF54 mutant viruses. Plaque reduction assay data show the percent reduction in plaque number for VZV strains Ellen, Ellen^rA, ROka, and the ROka ORF54 mutants (ROka54A3, ROka54A4, and ROka54B) in the presence of increasing concentrations of Comp I. The data represent the average number of plaques observed in triplicate samples of all viruses at each drug concentration. Data were normalized so that the average number of plaques observed in the absence of Comp I was defined as 100.

FIG. 9.

Electron micrographs of nuclei from VZV ROka-infected (A, C, and D) or ROka54B-infected (B) melanoma cells in the absence (A) or presence (B to D) of 2 μg of Comp I per ml. The insets in panels A and D provide a higher-power image of selected capsid particles from their representative panels. Open arrow, empty capsid; striped arrow, aberrant capsid; solid arrow, full DNA-containing, dense-core capsid.

FIG. 10.

Electron micrographs of the cell membrane from VZV ROka-infected (A and B) or ROka54B-infected (C) melanoma cells in the absence (A) or presence (B and C) of 2 μg of Comp I per ml. The insets provide a more detailed view of selected viral particles (arrow) from their representative panels. N, nucleus; C, cytoplasm.