FIGURE 7.
Protein synthesis is compartmentalized to the ER during CBV infection. At 5.5 h post-infection, HeLa cells infected with CBV were methionine-starved for 30 min, then pulsed for 2 min with [35S]methionine/cysteine. To inhibit further incorporation of radiolabeled amino acid, cells were treated with 200 μM cycloheximide and subsequently fractionated to yield cytosol and ER. Polyribosome profiles of cytosolic (free) (A) and ER (membrane-bound) (B) ribosomes were determined by velocity sedimentation on 15%–50% linear sucrose gradients. Gradient fractions were collected and analyzed by UV spectroscopy. Total protein was isolated from individual gradient fractions, separated by SDS-PAGE, and analyzed by [35S]methionine incorporation determined by PhosphorImager analysis.