Ribosome sequencing (Ribo-seq) technology was employed to analyze key sequences regulating translation elongation in the Senecavirus A (SVA) genome and to evaluate their impact on viral replication, thereby providing a theoretical foundation for elucidating the molecular mechanisms of SVA. Which could lay a foundation for clarifying the role of RNA elements in SVA coding region in translation regulation.

Ribo-seq was performed using rSVA-eGFP as a model to generate a genome-wide translation profile of SVA and to identify ribosome pausing peaks. Synonymous mutations were then designed for the sequences corresponding to these pausing sites, and full-length cDNA recombinant plasmids were constructed. Replication-competent recombinant viruses were subsequently rescued through reverse genetics. The rescued viruses were subjected to serial blind passages, RT-PCR, sequencing, and growth-curve analyses to evaluate the effects of the mutations on viral replication. Representative recombinant viruses were selected for secondary Ribo-seq analysis to elucidate the effects of the modification site on viral replication and protein translation.

Ribo-seq data revealed significant ribosomal translation-stall peaks in VP1, 2C, and 3D genes of SVA, suggesting the presence of key motifs that impede ribosome translocation. To test this hypothesis, synonymous mutations were introduced into these three regions to construct recombinant viruses. The results indicated that only the mutant in the 3D region successfully rescued replication-competent virus, and the introduced mutation remained stable after 20 consecutive passages, with no significant impact on viral growth kinetics. These findings indicated that the motifs in the 3D region were nonessential for viral replication. In contrast, mutations in the VP1 and 2C regions failed to produce viable viruses, demonstrating that the motifs in these regions were essential for viral replication. Further Ribo-seq analysis of the rescued virus revealed that the pausing peaks in both the 2C and 3D regions disappeared after mutation. This suggested that the key motifs in the 2C region, if any, were not the sole determinant of the translation rate of viral proteins, whereas the motif in the 3D region, although not required for viral replication, could modulate the translation efficiency of viral proteins.

Through genome-wide Ribo-seq profiling of SVA, this study identified critical sequence motifs that regulate translational elongation. The motifs in the VP1 and 2C regions were essential for viral replication, while the motif in the 3D region, though non-essential for viral replication, could affect protein translation rates.