Compounds that target host cell proteins prevent varicella-zoster virus replication in culture, ex vivo, and in SCID-Hu mice

Abstract

Varicella-zoster virus (VZV) replicates in quiescent T cells, neurons, and skin cells. In cultured fibroblasts (HFFs), VZV induces host cyclin expression and cyclin-dependent kinase (CDK) activity without causing cell cycle progression. CDK1/cyclin B1 phosphorylates the major viral transactivator, and the CDK inhibitor roscovitine prevents VZV mRNA transcription. We investigated the antiviral effects of additional compounds that target CDKs or other cell cycle enzymes in culture, ex vivo, and in vivo. Cytotoxicity and cell growth arrest doses were determined by Neutral Red assay. Antiviral effects were evaluated in HFFs by plaque assay, genome copy number, and bioluminescence. Positive controls were acyclovir (400 μM) and phosphonoacetic acid (PAA, 1 mM). Test compounds were roscovitine, aloisine A, and purvalanol A (CDK inhibitors), aphidicolin (inhibits human and herpesvirus DNA polymerase), L-mimosine (indirectly inhibits human DNA polymerase), and DRB (inhibits casein kinase 2). All had antiviral effects below the concentrations required for cell growth arrest. Compounds were tested in skin organ culture at EC99 doses; all prevented VZV replication in skin, except for aloisine A and purvalanol A. In SCID mice with skin xenografts, roscovitine (0.7 mg/kg/day) was as effective as PAA (36 mg/kg/day). The screening systems described here are useful models for evaluating novel antiviral drugs for VZV.

Varicella-Zoster Virus ORF12 Protein Activates the Phosphatidylinositol 3-Kinase/Akt Pathway To Regulate Cell Cycle Progression

“…We found that regulation of the cell cycle by VZV ORF12 protein was dependent on PI3K, which supports this hypothesis. This is further bolstered by observations that the inhibition of intracellular signaling pathways (34,35) or cell cycle regulators (51,52) by small molecules reduces VZV replication.…”

β-l-1-[5-(E-2-bromovinyl)-2-(hydroxymethyl)-1,3-(dioxolan-4-yl)] uracil (l-BHDU) prevents varicella-zoster virus replication in a SCID-Hu mouse model and does not interfere with 5-fluorouracil catabolism

“…The neutral red (NR) cytotoxicity assay was performed as described previously (Rowe et al, 2010). Cellular proliferation was evaluated by colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay following the method developed by Mosmann (Mosmann, T.1983).…”

“…Skin was divided into approximately 1-cm 2 pieces, cultured on NetWells (Corning, NY), and inoculated with VZV-BAC-Luc by scarification with a 27-gauge needle (Rowe et al, 2010; Taylor and Moffat, 2005). For bioluminescence imaging, skin explants were submerged in D-luciferin (300 μg/mL in PBS) for 1 hour before scanning in the IVIS® 50.…”

“…To address these questions, we evaluated L-BHDU ( 1 ), its 5′- O -L- valyl ester prodrug ( 2 ), and two phosphoramidate derivatives ( 3 & 4 ) in a range of models that address cytotoxicity and efficacy in culture and in vivo . We have developed systems for screening potential antiviral compounds against VZV that employ fully differentiated, intact human tissues and live animals in an attempt to more closely mimic what occurs during a natural infection (Rowe et al, 2010). The cytotoxic and antiviral effects of L-BHDU were first examined in a primary cell line, human foreskin fibroblasts (HFFs), and then ex vivo in a skin organ culture (SOC) model (Taylor and Moffat, 2005).…”

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

“…Study of the binding kinetics of VZV MCP and its mutants with 35B2 and related compounds identified in the SAR analysis will help us to see whether 35B2 interacts with a tertiary structural element conserved among herpesviruses. Further studies are also required to see whether such anti-MCP compounds are effective in assays that represent various aspects of VZV infections in vivo, such as T lymphocyte cultures, skin raft/organ cultures, and SCID-hu mouse models (25,28,35,46,51).…”