E2 ubiquitin-conjugating enzyme UBE2L6 promotes Senecavirus A proliferation by stabilizing the viral RNA polymerase

Author summary Senecavirus A (SVA) is a newly emerging pathogen causing swine idiopathic vesicular disease and epidemic transient neonatal losses. Infections have been reported in many pork producing countries, yet the mechanism of SVA replication remains poorly understood. In this study, we found that UBE2L6, an E2 ubiquitin-conjugating enzyme, is up-regulated in SVA-infected BHK-21 cells. The viral RNA dependent RNA polymerase (RdRp) 3D is ubiquitinated by UBE2L6, and the lysines at residues 169 and 321 of 3D are the required ubiquitination sites. The level of replication of recombinant viruses harboring ubiquitination-deficient 3D was significantly decreased compared to parental SVA. Our data demonstrate that UBE2L6 ubiquitinates SVA 3D, thereby facilitating SVA infection. These results may make it possible to identify novel targets for disease treatment. Senecavirus A (SVA), discovered in 2002, is an emerging pathogen of swine that has since been reported in numerous pork producing countries. To date, the mechanism of SVA replication remains poorly understood. In this study, utilizing iTRAQ analysis we found that UBE2L6, an E2 ubiquitin-conjugating enzyme, is up-regulated in SVA-infected BHK-21 cells, and that its overexpression promotes SVA replication. We determined that UBE2L6 interacts with, and ubiquitinates the RNA-dependent RNA polymerase of SVA, (the 3D protein) and this ubiquitination serves to inhibit the degradation of 3D. UBE2L6-mediated ubiquitination of 3D requires a cystine at residue 86 in UBE2L6, and lysines at residues 169 and 321 in 3D. Virus with mutations in 3D (rK169R and rK321R) exhibited significantly decreased replication compared to wild type SVA and the repaired viruses, rK169R(R) and rK321R(R). These data indicate that UBE2L6, the enzyme, targets the 3D polymerase, the substrate, during SVA infection to facilitate replication.