FIG. 6.

Translational initiation from downstream AUG codons requires phosphorylation of eIF2α. (a) SFV induces reduction in phospho-eIF4E levels. BHK-21 cells were infected with SFV-NP, SFVC-NP, or IAV (flu) as indicated. As controls, cells were either incubated in medium lacking bovine serum (0%) or incubated in medium containing elevated levels (20%). Lysates harvested at 12 h p.i. were subjected to SDS-PAGE and processed for Western blotting using antiserum specific for phospho-eIF4E or actin. (b) SFV induces phosphorylation of eIF2α. MEF obtained either from WT mice or from mice expressing a mutant variant of eIF2α unable to become phosphorylated (Ser₅₁A) were infected with SFV-NP or SFVC-NP as indicated. Lysates harvested at 12 h p.i. were subjected to SDS-PAGE and processed for Western blotting using antiserum specific for phospho-Ser51-eIF2α. (c) Both virus-induced protein synthesis inhibition and the ability of the SFV capsid gene to serve as a translation enhancer require phosphorylation of eIF2α. Lysates from WT cells (MEF) and mutant cells unable to phosphorylatable eIF2α (MEF Ser₅₁A) were infected with SFV-NP or SFVC-NP and analyzed by Western blotting for the expression of NP. The expression from the SFV vector does not suffer from host cell translational shutdown in mutant cells (rightmost lane). (d) Mitochondrial targeting of SFV-expressed NP requires phosphorylation of eIF2α. WT (MEF) and mutant cells unable to phosphorylate eIF2α (MEF Ser₅₁A) were infected with SFV-NP and SFVC-NP as indicated and processed for immunofluorescence using Abs to the carboxy-terminal NP epitope (α-C), fully folded NP (α-F), or mitochondria (α-mt). Confocal images were obtained using identical settings for exposure and laser intensity between the samples illustrating the various expression levels. Mitochondrial NP was detectable only in WT cells infected with SFV-NP (upper left image). (e) Mitochondrial targeting of SFV1-NP requires phosphorylation of eIF2α. Immunolabeled samples of WT (MEF) and mutant cells (MEF Ser₅₁A) from the same immunolabeled SFV1-NP specimens shown in Fig. 6d, with the exception that the settings during the confocal imaging were adjusted in order to acquire next-to-saturated images, thus illustrating the mitochondrial localization of SFV1-NP in WT cells stained with the α-C pAb.