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P. R. Kinchington, A. Erazo, M. Walters; Cellular Targets of a Viral Protein Kinase Required for Growth of Varicella Zoster Virus in Primary Corneal Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3872.
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Varicella zoster virus (VZV) causes blinding corneal disease when it reactivates from latency to cause herpes zoster. Infections of the corneal layers trigger complex immune mediated stromal disease, neurotrophic keratopathy with corneal nerve damage, and may lead to long term ocular pain that can be difficult to treat. We have recently shown that a virally encoded protein kinase, 66PK, which is not required for VZV growth on most cell types, is nevertheless essential for VZV growth in primary corneal fibroblasts. The object of this work was to identify cellular targets of the viral protein kinase with the goal to determining roles that mediate VZV growth in corneal cells.
A protein kinase A (PKA)-substrate phosphoserine-specific antibody that recognizes some 66PK targets was used to probe and then precipitate protein substrates from cells infected with VZV or adenoviruses expressing 66PK. Based on mass spectrometry ID, specific antibodies were used to demonstrate 66PK-mediated phosphorylation of the cellular proteins in corneal cells expressing the kinase by VZV infection, adenovirus transduction or after plasmid DNA transfection, in the absence and presence of inhibitors of PKA.
The predominant protein efficiently phosphorylated by 66PK and recognized by the PKA substrate-specific antibody was an abundant nuclear matrix protein that has roles in nuclear retention of hyperedited RNAs, which undergoes rapid degradation upon phosphorylation by PKA to free up the nuclear matrix. This correlates with reduced efficiency of capsid formation in corneal cells infected by VZV lacking the kinase. We also found HDAC2 was phosphorylated when the 66PK was expressed, and its role in the control of chromatin remodeling correlates with an observed increase in transcription from transfected promoters in 66PK expressing cells. Phosphorylation of either protein occurred in cells inhibited with PKA inhibitors, suggesting they were direct substrates for the viral kinase.
The VZV 66PK protein kinase has a profound influence on the nucleus to influence transcription and nuclear integrity. While HDACs are known to be targets of the related US3 kinase of HSV-1, the ID of matrin as a substrate is unprecedented.
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