Activation of Host Translational Control Pathways by a Viral Developmental Switch
Figure 3
Phosphorylation of the translational repressor 4E-BP1 in response to KSHV lytic reactivation.
A. Activation of the PI3-Kinase (PI3K) /AKT/TSC pathway by diverse stimuli results in phosphorylation and subsequent inactivation of 4E-BP1 by mTORC1. Hyperphosphorylated 4E-BP1 releases the cap-binding protein eIF4E (shown bound to the mRNA 5′ cap), making it available to bind eIF4G and assemble a functional eIF4F complex composed of eIF4E, eIF4G, and eIF4A. The macrolide rapamycin inhibits mTORC1 and prevents 4E-BP1 phosphorylation. B. Hyperphosphorylated 4E-BP1 accumulates following KSHV reactivation. TREx BCBL1 or TREx BCBL1-RTA cells were untreated (−) or treated (+) with TPA+DOX in the presence or absence of rapamycin (Rap). At 48 h post-induction, total protein was harvested, fractioned by SDS-PAGE in a 17.5% gel and analyzed by immunoblotting using anti-4E-BP1. Arrowheads indicate the differential electrophoretic migration of the hypo- and hyper-phosphorylated 4E-BP1 forms. C. Inhibition of 4E-BP1 phosphorylation does not affect KSHV reactivation. Top panel: TREx BCBL-1 cells were induced with TPA+DOX in the presence and absence of rapamycin (rap). The fraction of ORF59 positive cells was quantified as described in the legend to Fig. 1. Bottom panel: Total protein was isolated, fractionated by SDS-PAGE and analyzed by immunoblotting using the indicated antisera. RhoGDI: loading control.