Molecular Mechanism Of Cellular Ubiquitin Conjugating Enzyme UBE2L6 Promoting Senecavirus A Replication

Seneca virus A(SVA),previously called Seneca Valley Virus(SVV).SVA infection can cause primary blisters in nasolabial mirror,tongue and hoof crown of finishing pigs,resulting in ulceration,loss of appetite and lameness.Moreover,piglets with acute infection can cause death.At present,there is no commercial vaccine and effective treatment.Since 2012,SVA infection and epidemic have occurred in pig farms in many provinces in China,causing great economic losses.In this study,SVV-CH-SD,a SVA strain isolated from Shandong Province,China,was used to complete the sequencing analysis of the whole gene sequence of this virus,and the molecular characteristics of the strain were identified.In addition,the reverse genetic platform of the strain was constructed.iTRAQ quantitative proteomics was used to determine the differentially expressed proteins in Swine Testicular cells(ST)infected with SVA.The results showed that E2 ubiquitin-conjugating enzyme UBE2L6 could significantly promote SVA replication.The contents of this research as following:1.Complete genomic sequencing of an SVA isolate SVV-CH-SD and construction of infectious clones of SVV-CH-SDIn this study,four pairs of specific primers were designed according to the sequence of SVA in Genbank.The whole genome of SVV-CH-SD strain was sequenced by race combined with RT-PCR.The results showed that the full genome was 7286 nucleotides(nt)in length and contained a single open reading frame(ORF)of 6546 nt,encoding a 2182 amino acid(aa).A phylogenetic analysis showed that the isolate shares highest sequence homology(98.52%)with SVA strain USA-GBI26-2015.A genetic recombination analysis revealed that strain SVV-CH-SD might be a major parent of SVA strain USA-IA40380-2015.According to the gene characteristics of the SVV-CH-SD strain,we divided it into four segments,and established Mlu Ⅰ,Afl Ⅱ and Asc Ⅰ restriction endonuclease sites through synonymous mutation of gene sites to achieve complete splicing of the whole genome,and obtained the recombinant plasmid pCMV-rSVA-SD containing the full length of the virus gene.Baby hamster kidney cells(BHK-21)were transfected with pCMV-rSVA-SD and a virus rSVA-SD was obtained.Rescued viruses were confirmed by plaque assay,growth characteristics,IFA and identification of genetic markers Afl Ⅱ,which indicated the successful establishment of SVA reverse genetic techniques.2.Proteomics of swine testicular cells infected with Senecavirus AIn order to further understand the changes in intracellular protein caused by swine testicular cells infected with SVA,ST cells were plated on a 6-well plate,and infected SVA strain SVV-CH-SD with 0.1 MOI.At 12 and 24h after infection,the differential proteins were analyzed by mass spectrometry combined with relative and absolute quantitative allele tagging(iTRAQ).The results showed that there were 24 up-regulated and 9 down regulated differentially expressed proteins at 12 h post infection(12 hpi).At 24 hpi,308 differentially expressed proteins were up-regulated and 132 were down-regulated.We randomly selected five up-regulated genes[Interferon-induced GTP-binding protein 2(Mx2),interferon-induced tetrapeptide repeat protein 1(IFIT1),interferon-stimulating gene 15(ISG15),Ubiquitin-conjugating enzyme UBE2L6(UBE2L6)and inflammatory response protein 6(RSAD2)]and three down regulated genes[Annexin A6(ANXA6),sperm perinuclear RNA binding protein(STRBP),transforming B cell complex subunit 70(SWAP70)]for qRT-PCR detection,and the expression changes were consistent with iTRAQ results.Subsequently,the functional enrichment analysis of differential proteins showed that these differential proteins are mainly involved in important biological processes such as cellular processes,metabolic processes,biological regulation,and the response of biological processes to stimulusbinding.In addition,the STRING database was used to analyze and draw a protein interaction network diagram.The results show that these proteins participate in the SVA infection process through mechanisms such as antiviral response and protein post-translational modification.This research provides a lot of molecular information for in-depth study of SVA infection and its interaction mechanism with the host.3.Ubiquitin-conjugating enzyme UBE2L6 promotes Senecavirus A proliferation by stabilizing the viral RNA polymeraseWe selected BST2,DTX3L,Mx2,GBP1,IFIT1,UBE2L6 and TRIM21 protein genes from the iTRAQ differential protein data at 24 hours after SVA infection of ST cells.And constructed the recombinant expression plasmid using the pCAGGS eukaryotic expression vector.Then,the above plasmids were transfected into BHK-21 cells.24 hours later,0.01 MOI SVA strain SVV-CH-SD was inoculated.It was found that ube216 significantly promoted SVA replication 16 hours after infection.On the contrary,we designed 3 pairs of sgRNAs for the second and third exon regions of UBE2L6 gene,cloned them into pX-459 vector,and successfully obtained a BHK-21 Knockout UBE2L6 cell line(BHK-UBE2L6-KO)through CRISPR/Cas9 technology and monoclonal screening.Subsequently,SVA was infected with the BHK-UBE2L6-KO cell line,and the level of SVA replication was detected 16 hours after infection.The results showed that knocking out endogenous UBE2L6 inhibited SVA replication.Furthermore,UBE2L6 and SVA viral proteins(L-eGFP,VP1,VP2,VP3,VP4-eGFP,2B,2C,3A,3C and 3D)were co-transfected into BHK-21 cells for co-immunoprecipitation(Co-IP).It was found that UBE2L6 only interacts with SVA’s RNA polymerase(3D protein).Considering that UBE2L6 is an E2 ubiquitin conjugating enzyme,we generated three UBE2L6 mutant-expressing plasmids(C86S,C98S and C102S).The results demonstrated that UBE2L6 ubiquitination modification 3D process depends on cysteine 86 of UBE2L6.Secondly,we generated a panel of ubiquitin mutants(pUb-K48,pUb-K63,pUb-K48R and pUb-K63R)and using the proteasome inhibitor MG132 to found that UBE2L6 mediates K48/K63 mixed ubiquitin chain modifies 3D and inhibits 3D protein targeted proteasome degradation.Finally,reverse genetic technology was used to rescue and obtain 3D mutant recombinant viruses[rK169R and rK321R]and their revertants[rK169R(R)and rK321R(R)].Compared with wild-type SVA,the replication ability of recombinant SVA[rK169R and rK321R]were significantly lower in BHK-21cells,and the levels of 3D ubiquitination in the mutants were nearly undetectable,whereas 3D ubiquitination in rSVA and the revertants were similar and robust,which indicated that the lysines at residues 169 and 321 of 3D are the required ubiquitination sites.These data indicate that UBE2L6 targets 3D in the process of SVA infection to promote virus replication,which is helpful to understand the molecular mechanism of virus infection and potential targets to control SVA infection.In summary,this study clarified the molecular characteristics of the SVV-CH-SD gene of the SVA isolated strain in Shandong Province with the highest homology to the American strain,and successfully constructed a reverse genetic platform for this strain.Quantitative proteomics(iTRAQ)was used to map the differentially expressed protein profiles of SVA infected Swine Testicular cells.It was found for the first time that host ubiquitin binding enzyme UBE2L6 could ubiquitinate SVA RNA polymerase 3D and promote SVA replication in BHK-21 cells.It enriches the theoretical basis of SVA infection and pathogenic mechanisms,and has important theoretical significance and application prospects for the prevention and control of the disease.