Comparison of the capacity of different viral internal ribosome entry segments to direct translation initiation in poly(A)-dependent reticulocyte lysates

Polyadenylation stimulates translation of capped eukaryotic mRNAs and those carrying picornaviral internal ribosome entry segments (IRESes) in vivo. Rabbit reticulocyte lysates (RRL) reproduce poly(A)mediated translation stimulation in vitro after partial depletion of ribosomes and ribosome-associated factors. Here, we have evaluated the effects of varying different parameters (extent of extract depletion, cleavage of eIF4G, concentrations of KCl, MgCl2 and programming mRNA) on IRES-driven translation efficiency and poly(A)-dependency in ribosome-depleted RRL. For comparison, the study included a standard capped, polyadenylated mRNA. Dramatic differences were observed in the abilities of the different IRESes to direct translation in ribosome-depleted extracts. While the hepatitis A virus IRES was incapable of driving translation in physiological conditions in depleted RRL, mRNAs carrying the foot-and-mouth disease virus and hepatitis C virus IRESes were translated significantly better than a standard cellular mRNA in the same conditions. Indeed, the capacities of these IRESes to direct translation in ribosome-depleted RRL were similar to those reported previously in certain cell lines. Both the abilities of the IRESes to drive translation and their individual salt optima in ribosomedepleted extracts suggest that these elements have dramatically different affinities for some component(s) of the canonical translation machinery. Finally, using poliovirus as an example, we show that the ribosome-depleted system is well suited to the study of the translational capacity of naturally occurring IRES variants.