IRES Mutation Confers a Replicative Advantage to SVA by Enhancing Translation Efficiency

The internal ribosome entry site (IRES) is a cis-acting element found in certain RNA viruses. In virus-infected cells, the Senecavirus A (SVA) IRES can directly recruit the small ribosomal subunit to an internal initiation codon on the mRNA, enabling translation initiation independently of both the 5^' cap structure of the viral genome and the host cell eukaryotic initiation factor 4F (eIF4F). As a small RNA virus, SVA frequently accumulates genetic mutations during transmission. This study aimed to characterize the mutational and evolutionary patterns of the SVA genome during its transmission within host cells exhibiting enhanced innate immunity. SVA was serially passaged for 80 generations in preactivated 3D4/21 cells that had been established in an antiviral innate immune state. During serial passaging, two stable single-nucleotide mutations were consistently identified in the IRES region of the viral genomic RNA from passages 60-80: a uridine (U) insertion at genomic position 109, and a guanine (G)-to-adenine (A) substitution at position 265. Furthermore, the rescued SVA mutants harboring these mutations exhibited significantly higher replication titers than the parental strain. Notably, our results indicated that the mutant virus was unable to evade host antiviral innate immunity, whereas it significantly enhanced the translational activity of the SVA IRES. Collectively, these findings provide a foundation for understanding how single nucleotide polymorphisms (SNPs) in the 5^' untranslated region (UTR) region influence viral IRES activity and the replication capacity of recombinant viruses.