The Role And Mechanism Of US10 And TRAF6 During Classical Swine Fever Virus Infection

Classical swine fever?CSF?,caused by classical swine fever virus?CSFV?,is a severe,high mortality rate and highly contagious viral disease of domestic pigs and wild boars,which has resulted in huge economic losses in the pig industry worldwide.CSF is classified as a notifiable and former List A disease by OIE.CSFV N^pro has been actively studied but remains incompletely understood.Few studies have investigated the cellular proteins that interact with N^pro and their participation in viral replication.CSFV inhibits host cell immune response for itself continuous replication during CSFV infection.At present,interactions between CSFV and host become a research focus.The research of cellular proteins interactive with CSFV and their roles in CSFV replication mechanism can further explain the immune escape mechanism of CSFV and provide an effective strategies in response to CSF to inhibit CSFV replication.This article mainly investigated the roles of ribosomal protein?uS10?,interactive with CSFV N^pro,and tumor necrosis factor receptor-associated factor 6?TRAF6?,interactive with CSFV NS3,in CSFV replication and their mechanisms.The results were shown as follows:?1?Identification of cellular proteins that interact with CSFV N^pro and NS3 protein.Here,the yeast two-hybrid?Y2H?system was employed to screen N^pro-interacting and NS3-interacting proteins from a porcine alveolar macrophage?PAM?cDNA library,and a BLAST search of the NCBI database revealed that 15 cellular proteins interacted with N^pro,and 26 cellular proteins interacted with NS3.The identified proteins were analyzed by enrichment of GO categories and singaling pathway.These GO enrichments included DNA binding,RNA binding,protein binding,ion binding,transcription factor activity,gene expression,translation,biosynthetic and metabolic processes.The pathway analysis of these identified proteins included the cytosolic DNA-sensing pathway,RIG-I-like receptor signaling pathway,Toll-like receptor signaling pathway,ubiquitin-mediated proteolysis,ribosome and endocytosis,starch and sucrose metabolism and the insulin-signaling pathway.Based on these findings,the N^pro-cellular protein interaction network and NS3-cellular protein interaction network were constructed.?2?uS10 interacts with N^pro and inhibits CSFV replication.We choose ribosomal protein S20,also known as universal S10?uS10?from intracellular proteins interactive with N^pro for further research.Co-immunoprecipitation and glutathione S-transferase pull-down technologies were used to verify the N^pro-uS10 interactions in cells and in vitro.Furthermore,we determined CSFV replication in stably overexpressing or knockdown uS10 PAM constructed by lentivirus infection.uS10 overexpression inhibited CSFV replication,whereas the knockdown of uS10 promoted CSFV replication in PAM,revealing the antiviral role of uS10.In addition,N^pro or CSFV reduced u S10 expression in PAM in a proteasome-dependent manner,indicating that the N^pro-uS10 interaction might contribute to persistent CSFV replication.Further research showed that uS10 knockdown reduced toll-like receptor 3?TLR3?expression and that uS10 overexpression increased TLR3 expression.Notably,uS10knockdown did not promote CSFV replication following TLR3 overexpression.Conversely,uS10 overexpression did not inhibit CSFV replication following TLR3 knockdown.These results revealed that uS10 inhibits CSFV replication by modulating TLR3 expression.?3?TRAF6 interacts with NS3 and inhibits CSFV replication.We choose tumour necrosis factor receptor-associated factor 6?TRAF6?from intracellular proteins interactive with NS3 for further research.Co-immunoprecipitation and glutathione S-transferase pull-down technologies were used to verify the NS3-TRAF6 interaction in cells and in vitro.Furthermore,we determined CSFV replication in stably overexpressing or knockdown TRAF6 PAM constructed by lentivirus infection.We observed that TRAF6 overexpression significantly inhibited CSFV replication,and TRAF6 knockdown promoted CSFV replication in PAM.Additionally,TRAF6 was degraded during CSFV infection or NS3 expression exclusively,indicating that CSFV and TRAF6 were mutually antagonistic and that TRAF6degradation might contribute to persistent CSFV replication.Moreover,nuclear factor-kappa B?NF-?B?activity and interferon?IFN?-?and interleukin?IL?-6 expression were increased in TRAF6-overexpressing cells,whereas TRAF6-knockdown cells exhibited decreased NF-?B activity and IFN-?and IL-6 levels.Notably,TRAF6 overexpression did not reduce CSFV replication following inhibition of NF-?B activation by p65 knockdown.Our findings revealed that TRAF6 inhibits CSFV replication via activation of NF-?B-signaling pathways along with increases in the expression of its targets IFN-?and IL-6.In conclusion,CSFV N^pro interacted with uS10 and CSFV NS3 interacted with TARF6.This study first reported the inhibitory effect of uS10 and TRAF6 on CSFV replication,and the downregulation of uS10 and TRAF6 during CSFV infection.This work addresses novel aspects concerning the mechanisms associated with CSFV evasion of innate immune responses and continuous infection.These data also provides a novel theoretical basis for further understanding the physiological functions of CSFV N^pro and NS3 protein,and the relationship between virus and host.