Regulation of PKR expression by HSV-1: Potential approach in cancer therapy

Introduction: Protein kinase R (PKR) is a serine-threonine kinase able to trigger different cellular processes in response to specific stress signals. In particular, the PKR activation during herpes simplex virus 1 (HSV-1) infection is an efficient way to inhibit virus replication. Additionally with its antiviral role, PKR is involved in inflammation and in the cancer and neurodegenerative diseases (1). Previous results have demonstrated that, in HSV-1 infected cells, the mitogen-activated extracellular signal-regulated kinase (MEK), a well-characterized kinase in the cancer pathway, blocks phospho-PKR accumulation. Otherwise, in ΔVHS HSV-1 mutant infected cells, MEK protein is not able to control phospho-PKR accumulation (2). This finding highlights the important involvement of VHS tegument protein of herpes simplex in the regulation of PKR phosphorilation and leads us to investigate the role of additional teguments protein Ser/Thr kinases US3 and UL13 on VHS/PKR control. The study of viral proteins and their contribution in PKR regulation could contribute to develop new HSV-1-based vector for cancer therapy (3). Materials and methods: HEp-2 cells were infected with 10 PFU/cell with HSV-1 (F) wildtype virus, R2621 (ΔVHS), R7356 (ΔUL13) or R7041 (ΔUS3) mutant viruses. 293T cells were transfected with the plasmids designed as follow pVHS, pUS3 and pUL13. Infected or transfected cells were subjected to proteins extraction for Western blot analysis and probed with polyclonal antibodies directed to p-PKR (thr446), total PKR and GAPDH. Infected or transfected cells were subjected to RNA extraction and Real-time PCR was used to detect the transcriptional levels of PKR. Results and Conclusions: The results demonstrate, for the first time, the involvement of viral proteins Us3 and UL13 in the regulation of PKR-phosphorylation. In particular, the cells transfected with US3, UL13 and VHS plasmids, separately, show a decreased accumulation in phosphorylated form of PKR. Otherwise, the failure of the viral protein expression, due to deleted virus infection, results in the phospho-PKR accumulation. The PKR control mediated by viral proteins of HSV-1 and the potential role of PKR as a target in cancer therapy, remarks the importance to develop HSV-1-based vectors, able to regulate positively or negatively PKR and modulate the cancerous cellular environmental.