The RNA Encompassing the Internal Ribosome Entry Site in the Poliovirus 5′ Nontranslated Region Enhances the Encapsidation of Genomic RNA

Abstract

Poliovirus replicons were constructed which contain the internal ribosome entry site (IRES) of encephalomyocarditis virus (EMCV) substituted for the poliovirus IRES. To monitor gene expression and encapsidation, the gene encoding firefly luciferase was substituted for the P1 gene. Replicons can be encapsidated following serial passage in the presence of a recombinant vaccinia virus, VV-P1, which expresses the poliovirus P1 protein following infection. Encapsidation of the wild-type replicon (PV-Luc) was accomplished at either 33 or 37°C; the lower temperature actually resulted in greater amounts of encapsidated replicon. In contrast, the replicon with the EMCV IRES element (EMCV-Luc) was not efficiently encapsidated at 37°C and, following serial passage with VV-P1 at 37°C, was not amplified. EMCV-Luc was efficiently encapsidated, however, following serial passage with VV-P1 at 33°C. Using the encapsidated EMCV-Luc obtained at 33°C, we found that cells infected with EMCV-Luc at 33 or 37°C produced similar amounts of luciferase. Encapsidated EMCV-Luc and PV-Luc had similar thermal stability at 33 and 37°C. A single-round encapsidation analysis revealed that less EMCV-Luc was encapsidated at 37 than at 33°C; less EMCV-Luc was encapsidated at 33°C compared to PV-Luc at either 37 or 33°C. The results of our studies suggest that in addition to influencing translation/replication, the IRES region of poliovirus can function to enhance encapsidation.