Figure 7.
Silencing of eIF2A expression inhibits translation of SV 26S mRNA in PKR+/+, but not in PKR0/0 cells. The indicated cell type was transfected with eIF2A-specific or control siRNAs as described in Materials and Methods. (A) Fifty hours later, poly(A)+ mRNAs were isolated from cells and subjected to Northern blot analysis using a murine eIF2A probe. The ∼2-kb eIF2A transcript is indicated. The blot was also probed with a β-actin probe as a loading control. The level of eIF2A mRNA was quantified by densitometry and corrected by the amount of β-actin mRNA detected in each sample. The amount of eIF2A mRNA with respect to β-actin mRNA found in PKR+/+ and PKR0/0 cells was arbitrarily assigned as 1 a.u., arbitrary units. (B) Effect of eIF2A silencing on SV 26S mRNA translation. Cells transfected with the indicated siRNA were infected with SV or VSV at an MOI of 25 PFU/cell. (Upper panel) Six hours later, cells were pulsed with [35S]Met-Cys for 30 min and analyzed by SDS-PAGE followed by autoradiography. (Lower panel) Analysis of eIF2α phosphorylation in these samples is also shown. (C) Silencing of eIF2A does not affect translation of genomic SV mRNA. Transfected cells were infected with recombinant SV-Luc, and the luciferase activity of cell extracts was assayed 6 hpi.