Figure 4: RACK1 aids PV translation prior and post host cell translational shut-off.

(A) ³⁵S metabolic pulse labeling of uninfected (mock) and PV-infected cells. Cells were mock-infected or infected with PV Mahoney at MOI of 1 (MOI of 10 for positive control) and ³⁵S pulse-labeled for 10 min at 185 minutes post infection. Total protein lysates were separated in 10% SDS-PAGE and visualized by exposure to a phosphor-screen. Poliovirus-specific protein products P1, 3CD, and 2C are indicated (Florez et al., 2005). (B) Same experiment as in (A), but at MOI of 10 and labeled at 160 minutes post infection. (C) Immunoblot analysis for expression of poliovirus proteins 3CD and 3D following infection at MOI of 1 and MOI of 10. (D) Expression of a PV-Luc replicon is inefficient in cells lacking RACK1. HAP1, RACK1 KO #1, and RACK-1 FLAG cells were transfected with in vitro transcribed PV-Luc replicon RNA. An aliquot of cells was removed 3, 5, 7, and 9 hours post RNA transfection and Firefly luminescence was measured. Error bars represent the standard error of the mean of at least three independent experiments. Left panel: PV-Luc translation is decreased in cells lacking RACK1 during the replication phase. Middle panel: To limit the observation to the early translation phase, PV-Luc replication was inhibited with 2 mM guanidine hydrochloride immediately after RNA transfection. Right panel: Translation of all PV-Luc reporters is completely inhibited upon treatment with 25 μg/ml cycloheximide. Cells were treated with cycloheximide immediately after RNA transfection.