CELL CYCLE PROGRESSION DURING HSV-1 REPLICATION REQUIRES ERK1 FUNCTIONS IN HEP-2 CELLS

Introduction: The herpes simplex virus 1 is able to readdress different cellular pathways, including cell cycle, to facilitate its replication and spread (1). Here, we have analyzed: i) HSV-1 control in the cell cycle progression and in the phosphorylation state of cyclin E and CDK2; ii) the recruitment and the different cellular compartmentalization of ERK1/2 proteins during viral replication; iii) the interaction between ERK, cell cycle progression and HSV-1 replication. Methods: HEp-2 and HEp-dnERK (dominant negative transfectans HEp-2) cell lines (2), were infected with HSV-1. Infected cells were subjected to citoplasmatic and nuclear proteins extraction and probed with antibodies directed to: phospho-ERK1/2, phopho and total cyclin E, CDK-2, ICP0, β-actin, GAPDH, Histone H3 and Us11. To analyse the cell cycle phases, infected and control HEp-2 cells were analysed by flow cytometer analysis. Phosphonoacetic acid was used as an inhibitor of DNA synthesis in HSV-infected cells. Relative and absolute quantification of Real-time PCR was performed in HEp-2 and in HEp-dnERK cells to analysed the fold change of ICPO and Cyclin E transcripts and the amount of viral DNA, respectively (3). Results: The results show that the cell cycle progression to S phase increases following HSV-1 infection and decreases when viral DNA accumulation is blocked by PAA treatment. We also demonstrate the activation and nuclear translocation of ERK in response to HSV-1 infection and the ERK1 control on cyclin E and CDK2 activation. The accumulation of total and phopho-cyclin E decreases when ERK1 activity is absent. Similarly, the accumulation of immediate early (ICP0) and late (Us11) viral proteins, the viral growth and the HSV-1 DNA replication, the levels of viral (ICP0) and cellular (Cyclin E) transcripts are reduced in HEp-dnERK compared to the HEp-2 cells. Discussion: Several studies have shown that HSV-1 is able to influence some key events occurring in G1/S phases and regulates the MAPK signaling pathways, positively or negatively to stimulate productive replication. We demonstrated that the lack of ERK1 activity affects the viral genes expression, viral yield and the Cyclin E transcripts accumulation, highlighting the capability of HSV-1 to drive ERK1/2 cellular compartmentalization and the phosphorylation state of cyclin E. Such regulation seems to be a crucial prerequisite for efficient replication and is essential for virus grown.