Structural determinants of insert retention of poliovirus expression vectors with recombinant IRES elements

Although picornaviruses provide attractive vectors for expression of foreign genes, poor genetic stability restricts their use for immunization purposes. A new prototype vector was generated to increase foreign insert retention, by shifting of the initiation codon to a cryptic AUG within the internal ribosomal entry site (IRES) and replacement of IRES domain VI with foreign ORFs. Using our strategy to replace regulatory noncoding sequences with unrelated foreign genetic material, we generated stable poliovirus-based expression vectors with robust long-term expression of foreign ORFs. Our studies revealed that size and predicted secondary structure formed by the heterologous sequences govern long-term retention and efficiency of expression of foreign inserts replacing IRES structures. These observations indicate that, with certain limitations imposed by structural preferences, foreign sequences can functionally replace IRES substructures in stable picornavirus immunization vectors.