Fig 6.

Site-specific PABP cleavage stimulates EMCV RNA synthesis and infectious virus production. (A) HeLa cells transfected with plasmids expressing WT PABP, the Q437N cleavage-resistant variant (as in the Fig. 4D legend), or the plasmid vector-only control were infected with EMCV (MOI = 10⁻³). After 48 h, lysates were prepared by freeze-thawing, and the amount of infectious virus produced was quantified by plaque assay in Vero cells. (B) HeLa cells were transiently transfected as for panel A, transfected with an EGFP-expressing plasmid, or left untransfected. After 48 h, cultures were metabolically labeled for 1 h as for panel A. Total protein was isolated and the amount of ³⁵S incorporated determined by trichloroacetic acid (TCA) precipitation. Acid-insoluble material was collected onto fiberglass filters and washed, and the radioactivity was quantified by counting in liquid scintillant. The relative amount of ³⁵S incorporation in untransfected HeLa cells was normalized to 100%. Error bars indicate the standard errors of the means. (C) Cell-free lysates prepared from HeLa cells ectopically expressing cleavage-resistant Flag-tagged PABP variant (Q437N) were loaded onto sucrose gradients, which were subsequently fractionated while absorbance at 254 nm was monitored. TCA-precipitated fractions were analyzed by immunoblotting using antibodies specific for Flag or ribosomal protein s6 (rpS6). Fraction 1, free ribosome subunits; fraction 2, disomes; fractions 3 to 5, polyribosomes. (D) HeLa cells transiently transfected with empty plasmid vector or a plasmid expressing Flag-tagged PABP (WT or Q437N) were infected with EMCV (MOI = 10) for 5 h. (Top) Infections were performed in the presence of [³H]uridine and actinomycin D. Total RNA was isolated, samples were precipitated with TCA, and the amount of acid-insoluble radioactivity quantified by counting in liquid scintillant. Errors bars indicate standard errors of the means. (Bottom) The amount of infectious virus produced after a single replication cycle was quantified by plaque assay. Error bars indicate standard errors of the means.