Translating ribosomes inhibit poliovirus negative-strand RNA synthesis

Poliovirus has a single-stranded RNA genome of positive polarity that serve s two essential functions at the start of the viral replication cycle in in fected cells. First, it is translated to synthesize viral proteins and, sec ond, it is copied by the viral polymerase to synthesize negative-strand RNA . We investigated these two reactions by using HeLa S10 in vitro translatio n-RNA replication reactions. Preinitiation RNA replication complexes were i solated from these reactions and then used to measure the sequential synthe sis of negative- and positive-strand RNAs in the presence of different prot ein synthesis inhibitors. Puromycin was found to stimulate RNA replication overall. In contrast, RNA replication was inhibited by diphtheria toxin, cy cloheximide, anisomycin, and ricin A chain. Dose-response experiments showe d that precisely the same concentration of a specific drug was required to inhibit protein synthesis and to either stimulate or inhibit RNA replicatio n This suggested that the ability of these drugs to affect RNA replication was linked to their ability to alter the normal clearance of translating ri bosomes from the input viral RNA. Consistent with this idea was the finding that the protein synthesis inhibitors had no measurable effect on positive strand synthesis in normal RNA replication complex-es. In marked contrast, negative-strand synthesis was stimulated by puromycin and was inhibited by cycloheximide. Puromycin causes polypeptide chain termination and induces the dissociation of polyribosomes from mRNA, Cycloheximide and other inhibi tors of polypeptide chain elongation "freeze" ribosomes on mRNA and prevent the normal clearance of ribosomes from viral RNA templates. Therefore, it appears that the poliovirus polymerase a-as not able to dislodge translatin g ribosomes from viral RNA templates and mediate the switch from translatio n to negative-strand synthesis. Instead, the initiation of negative-strand synthesis appears to be coordinately regulated with the natural clearance o f translating ribosomes to avoid the dilemma of ribosome-polymerase collisi ons.