Abstract
Purpose: :
Herpes Zoster causes serious ocular infections that can result in blindness. Our studies address the VZV regulatory proteins, with the goal that these are strong possible targets for antiviral development. We have addressed the role of a viral protein kinase (from VZV ORF66), a multifunctional protein that affects both viral and host cell targets. To further investigate the role of the kinase in the context of virus, recombinant VZV expressing EGFP–ORF66 proteins were developed to probe growth, induction of cytopathic effect and MHC–I downergulation in ocular and non ocular cell types.
Methods: :
The Recombinant VZV were generated using the overlapping cosmid system in the wild type VZV background. These expressed (1) functional EGFP–ORF66 (2) point inactivated kinase dead EGFP–ORF66 (3) a "rescued" recombinant VZV expressing active EGFP–ORF 66 (4) A translational stop EGFP–ORF66 at codon 80. Each was evaluated for growth rates and MHC–I surface downregulation by Flow analyses: and for induced cellular cytopathic effects and on MeWos, human foreskin and corneal fibroblasts, and rpe cells.
Results: :
VZV with functional EGFP ORF66 kinase or "rescued" kinase demonstrated growth rates identical to wild type VZV, induced actin reorganization and generated strong cytopathic effect identical to wild type VZV on all cells. However, VZV lacking ORF66 or expressing dead kinase demonstrated severely impaired growth rates specific to corneal fibroblasts and rpe cells, did not induce actin stress fiber breakdown and demonstrated little cpe in both ocular cell types. Growth on permissive cells for 66– and 66+ VZV revealed that, while MHC–I was still downregulated in the absence of kinase, it was consistently more modest as compared to cells infected with VZV that had functional 66.
Conclusions: :
These studies indicate two roles for the kinase on the host cell environment: One in which the VZV ORF66 kinase affects actin reorganization and induces cpe that may be important for spread in corenal fibroblast cells, and the second is that the kinase influences surface MHC–I in the context of viral infection.
Keywords: varicella zoster virus • cytoskeleton • protein structure/function