| Objective:In this study, we focus on the potential functions of ERK signaling pathway involved in the replication of herpes simplex virus type II(HSV2) and the distinct effects for the two subtypes, MEK1 and MEK2, during the early- and late- phase of ERK activation induced by HSV2 infection.Thus,we attempt to further disclose the complexity of MKE kinase, and explore the molecular mechanism of ERK pathway activation involved in HSV2 replication.Methods:1. HEK293 cells (human embryonic kidney 293 cells) were infected with HSV2 333 strain (kindly provided by Dr. WX Jia), then we observed the cytopathetic effect (CPE) induced by HSV2 and established a cell model to elucidate the interaction between HSV2 replication and ERK pathway activation.2. HEK293 cells were infected with HSV2 at a multiplicity of infection (MOI) of 1.5. Cells and supematants were collected at the different time points indicated in figure 2-1. Cells were lysed to detect the level of phosphorylated ERK 1/2 (p-ERKl/2) by Western blot analysis and collected supematants were assayed for virus titers.3. HEK293 cells were preincubated with U0126 (50uM) for 1 hour, and then infected with HSV2 at an MOI of 1.5. Cells and supematants were collected separately at same time points as described in method 2 to determine the effects of U0126 on the activation of ERK pathway induced by HSV2 replication.4. HEK293 cells were infected with HSV2 (live virus) or Ultraviolet-irradiated HSV2(UV-HSV2, dead virus) at an MOI of 10. At the indicated time points, cells were collected and lysed for Western blot to detect the level of p-ERK1/2 to investigate the impact of HSV2 and UV-HSV2 infection on the early and late phase of ERK pathway activation. 5. HEK293 cells were transfected with siRNAs specific for MEK1 or MEK2 (siMEKl or siMEK2), respectively, and a siRNA control (siMutlor siMut2) at the indicated doses. The cells were then infected with HSV2 and UV-HSV2 (MOI=10) separately at 48 hours p.L Cell lysates collected at 20 minutes p.i. or 4 hours p.i.were prepared to detect MEK1(or MEK2) and p-ERK1/2 levels by Western blot, then analyzed the distinct effects of knockdown of MEK1 or MEK2 on the early and late phase of ERK activation induced by HSV2 or UV-HSV2.Results:1. A typical cytopathetic effect was observed in HSV2 infected HEK293 cells.2. A biphasic activation of ERK pathway was observed(a transient early-phase and a sustained late-phase activation) in HEK293 cells following HSV2 infection at an MOI of 1.5.3. The intensity of ERK pathway activation was severely impaired by U0126, a specific inhibitor of MEK1/2, concurrently with a significantly reduction of the virus production which was represented by viral liters (50% tissue culture infectious dose,TCID50) of HSV2.4. HSV2 infection could tigger the biphasic activation of ERK,but just the early-but not late-phase activation of ERK pathway was shown in the cells infected with UV-irradiated HSV2 (dead virus).5. The knockdown of MEK1 inhibited both early-and late-phase activation of ERK pathway induced by live virus, whereas no significant effect was achieved by the knockdown MEK2.Conclusions:1. Our results reveal that the activaton of ERK pathway plays an essential role in the replication of HSV2 in HEK293 cells.2. Our data suggests that the mechanism involved in the early-phase and late-phase activation of ERK pathway induced by HSV2 infection might be different:the early phase ERK activation may relate to viral adsorbing, penetration and uncoating, while the late phase activation may result from viral biosynthesis through an unknown mechanism.3. Our findings demonstrate that the distinct functions of MEK subtypes for the biphasic activation of ERK pathway during virus infection and replication. The MEK1-ERKs model might be essential for activation of ERK pathway in the course of HSV2 replication. |
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