Study of Conserved Motifs in Senecavirus A Genome
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.First, the translation rate of the whole genome of SVA was analyzed using rSVA-GFP as a model via Ribo-seq technology. Based on the significant ribosome enrichment peaks detected by Ribo-seq, synonymous mutations were introduced into the corresponding sequences. After chemical synthesis, these sequences were substituted into the full-length cDNA backbone to obtain recombinant plasmids. Subsequently, reverse genetics was employed to transfect the plasmid into BHK-21 cells to rescue the replication-competent recombinant virus. A series of experiments, including blind passage, RT-PCR, Sanger sequencing, and growth curve analysis, were conducted on the recombinant virus to characterize its biological properties. 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, suggesting that key motifs impeding ribosomal translocation may be present in these regions. It was hypothesized that these regions may contain key motifs that impede ribosomal translocation. To test this hypothesis, these regions were further synonymously mutated to construct recombinant viruses. The results indicated that only the mutant in the 3D region successfully rescued replication-competent viruse that remained genetically stable through 20 passages and had no significant impact on viral growth kinetics. It suggested that the motifs in the 3D region were nonessential for viral replication. In contrast, the motifs, corresponding to the Ribo-seq-specific peaks in the VP1 and 2C regions, were essential for viral replication. The further Ribo-seq analysis of the rescued virus revealed the absence of ribosomal stalling peaks in both the 2C and 3D regions. This suggested that the key motifs in the 2C region, if any, were not the sole determinant of the translation rate of viral proteins. In contrast, disrupting any such structures in the 3D region did not impact viral replication but could affect the rates of viral protein translation. Overall design: Ribo-seq profiling of Senecavirus A expressing green fluorescent protein (rSVA-GFP) and genetically modified rSVA-GFP at 24h post-infection in BHK-21 cells