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Daniel J. Carr, Todd R. Wuest; Herpes Simplex Virus-1 Drives Corneal Lymphangiogenesis via Infected Cell Protein-4 Dependent Expression of VEGF-A. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5798.
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To determine the mechanism(s) by which HSV-1 infection drives VEGF-A up-regulation.
Male C57BL/6 (wild type, WT), MyD88 deficient (MyD88-/-), and TRIF deficient (TRIF-/-) mice or transfected human or mouse cells were infected with HSV-1. Mice were exsanguinated at times PI, and the corneas were evaluated for VEGF-A expression by ELISA. Real time RT-PCR was undertaken to evaluate expression of VEGF-A mRNA levels at times PI in the presence or absence of acyclovir, cycloheximide, or inhibitors of the MAP kinase pathway. siRNA knockdown experiments were conducted to evaluate EGR1 involvement of VEGF-A induction. Reporter constructs were engineered to characterize the VEGF-A promoter region in transfected cells which was further evaluated by electromobility shift assays (EMSA). Mutant HSV-1 were used to further evaluate those components of the pathogen required to up-regulate VEGF-A expression. Data was analyzed by ANOVA and Tukey’s post hoc T test for significance (p<.05).
The absence of TRIF or MyD88 did not impact on VEGF-A expression or lymphangiogenesis in the cornea of HSV-1 mice. In vitro, de novo protein synthesis but not viral replication or MEK1/2, JNK, or p38 was required for VEGF-A up-regulation based on corneal epithelial cells infected with HSV-1. In cells transfected with reporter constructs with sequential deletions of the human VEGF-A promoter, expression depended on a short stretch of DNA from -85 to -52 bp (relative to transcription start site). The -85 to -52 bp segment required for HSV-1 elicited VEGF-A transcription contains consensus sequences for the transcription for EGR-1 and three "GC boxes." HSV-1 infection up-regulates EGR-1 levels which promote transcription at EGR-1 consensus sequences within the HSV-1 genome. However, further promoter analysis revealed the lack of EGR-1 involvement in VEGF-A up-regulation. Instead, GC box 3 or any 2 GC box elements were found to be instrumental in VEGF-A up-regulation in response to HSV-1 infection. Surprisingly, the binding of nuclear proteins from uninfected and 6 hour PI HSV-1 nuclear extracts was apparently unaffected by GC box mutation in the probe spanning -88/+55bp as none of the mutants showed differential shifts by EMSA analysis. HSV-1 ICP4 but not ICP0 was found to bind the proximal VEGF-A promoter and contribute to VEGF-A transcriptional up-regulation.
Up-regulation of VEGF-A is due in part to the viral transactivator infected cell protein-4 (ICP4) through interaction with the proximal VEGF-A promoter.
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