FIGURE 5.

mRNAs partition to free RNPs or membrane-bound polysomes in response to CBV infection. HeLa cells were mock-infected (A,B) or infected with CBV (C,D) for 6 h and processed to yield cytosol and ER fractions. Polyribosome profiles of cytosolic (free) (A,C) and ER (membrane-bound) (B,D) ribosomes were determined by velocity sedimentation on 15%–50% linear sucrose gradients. Gradient fractions were collected and analyzed by UV spectroscopy. Total RNA was isolated from individual gradient fractions and the precise sedimentation of ribosomal subunits and ribosomes was determined through analysis of ribosomal RNA by agarose denaturing gels. Gels were transferred to nylon membranes and processed for Northern analysis using probes directed against BiP, GRP94, GAPDH, actin, c-myc mRNAs, and CBV RNAs.