Project description:Phosphorylation is a widespread post-translational modification that regulates numerous biological processes. Viruses can alter the physiological activities of host cells to promote virus particle replication, and manipulating phosphorylation is one of the mechanisms. Senecavirus A (SVA) the causative agent of porcine idiopathic vesicular disease. Although numerous studies on SVA have been performed, comprehensive phosphoproteomics analysis of SVA infection is lacking. In the present study, we performed a quantitative mass spectrometry-based phosphoproteomics survey of SVA infection in instituto biologico-rim suino-2 (IBRS-2) cells. Three parallel experiments were performed and 4520 phosphosites were identified on 2084 proteins. Gene Ontology functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses showed that many of the phosphorylated proteins were involved in apoptosis and spliceosome pathways, and subcellular structure localisation analysis revealed that more than half were located in the nucleus. Motif analysis of proteins with differentially regulated phosphosites showed that proline, aspartic acid and glutamic acid were the most abundant residues in the serine motif, while proline and arginine were the most abundant in the threonine motif. Forty phosphosites on 27 proteins were validated by parallel reaction monitoring phosphoproteomics, and 30 phosphosites in 21 proteins were verified. Nine proteins with significantly altered phosphosites were further explored, and eight (SRRM2, CDK13, DDX20, DDX21, BAD, ELAVL1, PBK, and STAT3) may play a role in SVA infection. Finally, identified eight predicted kinases, and the kinase activity of inhibitor of nuclear factor kappa-B kinase subunit β and CDK9 was reversed following SVA infection. This is the first phosphoproteomics analysis of SVA infection of IBRS-2 cells, and the results greatly expand our knowledge of SVA infection. The findings provide a basis for studying the interactions of other picornaviruses and their mammalian host cells.

Project description: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.

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Senecavirus A isolate:SVA-715

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