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Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.

ABSTRACT: In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the packaging of the DNA into capsids, indicative of a defect in the encapsidation process. To uncover the molecular target of the inhibition, resistant HSV-1 isolates were generated, and the mutation responsible for the resistance was mapped using marker transfer techniques. Each of three independent isolates had point mutations in the UL6 gene which resulted in independent single-amino-acid changes. One mutation was located in the N terminus of the protein (E121D), while two were located close together in the C terminus (A618V and Q621R). Each of these point mutations was sufficient to confer drug resistance when introduced into wild-type virus. The UL6 gene is one of the seven HSV-1 genes known to play a role in DNA packaging. This novel class of inhibitors has provided a new tool for dissection of HSV-1 encapsidation mechanisms and has uncovered a new viable target for the treatment of herpesviral diseases.

SUBMITTER: van Zeijl M

PROVIDER: S-EPMC102102 | biostudies-literature | 2000 Oct

REPOSITORIES: biostudies-literature

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Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.

van Zeijl M M Fairhurst J J Jones T R TR Vernon S K SK Morin J J LaRocque J J Feld B B O'Hara B B Bloom J D JD Johann S V SV

Journal of virology 20001001 19

In our search for novel inhibitors of herpes simplex virus type 1 (HSV-1), a new class of thiourea inhibitors was discovered. N-(4-[3-(5-Chloro-2,4-dimethoxyphenyl)-thioureido]-phenyl)-acetamide and its 2-fluoro-benzamide derivative inhibited HSV-1 replication. HSV-2, human cytomegalovirus, and varicella-zoster virus were inhibited to a lesser extent. The compounds acted late in the replication cycle by impairing both the cleavage of concatameric viral DNA into progeny genome length and the pack ...[more]

PMID: 10982350

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Project description:Cleavage and encapsidation of newly replicated herpes simplex virus type 1 (HSV-1) DNA requires several essential viral gene products that are conserved in sequence within the Herpesviridae. However, conservation of function has not been analyzed in greater detail. For functional characterization of the UL6, UL15, UL28, UL32, and UL33 gene products of pseudorabies virus (PrV), the respective deletion mutants were generated by mutagenesis of the virus genome cloned as a bacterial artificial chromosome (BAC) in Escherichia coli and propagated in transgenic rabbit kidney cells lines expressing the deleted genes. Neither of the PrV mutants was able to produce plaques or infectious progeny in noncomplementing cells. DNA analyses revealed that the viral genomes were replicated but not cleaved into monomers. By electron microscopy, only scaffold-containing immature but not DNA-containing mature capsids were detected in the nuclei of noncomplementing cells infected with either of the mutants. Remarkably, primary envelopment of empty capsids at the nuclear membrane occasionally occurred, and enveloped tegument-containing light particles were formed in the cytoplasm and released into the extracellular space. Immunofluorescence analyses with monospecific antisera of cells transfected with the respective expression plasmids indicated that pUL6, pUL15, and pUL32 were able to enter the nucleus. In contrast, pUL28 and pUL33 were predominantly found in the cytoplasm. Only pUL6 could be unequivocally identified and localized in PrV-infected cells and in purified virions, whereas the low abundance or immunogenicity of the other proteins hampered similar studies. Yeast two-hybrid analyses revealed physical interactions between the PrV pUL15, pUL28, and pUL33 proteins, indicating that, as in HSV-1, a tripartite protein complex might catalyze cleavage and encapsidation of viral DNA. Whereas the pUL6 protein is supposed to form the portal for DNA entry into the capsid, the precise role of the UL32 gene product during this process remains to be elucidated. Interestingly, the defect of UL32-negative PrV could be completely corrected in trans by the homologous protein of HSV-1, demonstrating similar functions. However, trans-complementation of UL32-negative HSV-1 by the PrV protein was not observed.

Project description:PurposeInfection of the human cornea by herpes simplex virus type-1 (HSV-1) can cause significant vision loss. The purpose of this study was to develop an ex vivo model to visualize viral growth and spread in the cornea. The model was also used to analyze cytokine production and study the antiviral effects of zinc oxide tetrapods.MethodsA β-galactosidase-expressing recombinant virus, HSV-1(KOS)tk12, was used to demonstrate the ability of the virus to enter and develop blue plaques on human corneal epithelial (HCE) cells and corneal tissues. Freshly obtained porcine corneas were cultured and then scratched before infection with HSV-1(KOS)tk12. The blue plaques on the corneas were imaged using a stereomicroscope. Western blot analysis for HSV-1 proteins was performed to verify HSV-1 infection of the cornea. Using the ex vivo model, zinc oxide tetrapods were tested for their anti-HSV-1 potential, and a cytokine profile was developed to assess the effects of the treatment.ResultsCultured corneas and the use of β-galactosidase-expressing HSV-1(KOS)tk12 virus can provide an attractive ex vivo model to visualize and study HSV-1 entry and spread of the infection in tissues. We found that unlike cultured HCE cells, which demonstrated nearly 100% infectivity, HSV-1 infection of the cultured cornea was more restrictive and took longer to develop. We also found that the zinc oxide tetrapod-shaped nano- and microstructures inhibited HSV infection of the cultured cells, as well as the cultured corneas. The cytokine profile of the infected samples was consistent with previous studies of HSV-1 corneal infection.ConclusionsThe ability to visualize HSV-1 growth and spread in corneal tissues can provide new details about HSV-1 infection of the cornea and the efficacy of new cornea-specific antiviral drug candidates. The ex vivo model also demonstrates antiviral effects of zinc oxide tetrapods and adequately portrays the drug delivery issues that cornea-specific treatments face.

Dataset Information

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.

Publications

Novel class of thiourea compounds that inhibit herpes simplex virus type 1 DNA cleavage and encapsidation: resistance maps to the UL6 gene.

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