Regulation Mechanism Of The Ubiquitin-proteasome System During The Infection Of Classical Swine Fever Virus

The innate immune system of the creatures is formed along with the evolution in a long time,which eliminates the pathogenic microorganism or exogenous substances by identification and suppression.Meanwhile,the microorganism developed strategies to evade the recognition and suppress the function of the host immune system in the interplay with the host,contributing to its efficient replication within the host and leading to severe damage.While viruses could only replicate in the cell,study of the mechanism of virus infection and replication will be of great significance for virus prevention and control.Classical swine fever（CSF）is an acute thermal and high contact infectious disease caused by classical swine fever virus（CSFV）,the transmission of which caused severe damage to the swine industry and resulted in great economic losses.The infection of CSFV leads to disorder of the immune system and the great amount of virus in the body results in CSF.The ubiquitin-proteasome system（UPS）involves in the infection of many viruses and modulates the innate immune system,playing significantly modulating roles in the proliferation of many viruses.Studies about the relation between CSFV and the UPS are limited and the role of UPS on CSFV replication is unclear.In this study,interplay between the UPS and CSFV was analysed in aspects of both the virus and the host.To reveal the relationship of CSFV and the UPS,the ubiquitinatn levels of cellular proteins in PK-15 cells infected or uninfected with CSFV are analysed.The results showed that ubiquitination levels of total cellular proteins were upregulated at 12 hpi and 24 hpi,and downregulated at 48 hpi in CSFV infected cells,indicating that CSFV infection affects the ubiquitination of cellular proteins and the UPS may involve in the replication of CSFV.To explore the expression of viral proteins,plasmids encoding viral proteins N^pro,C,p7,E1,NS2,NS4A,NS4B and NS5A were transfected in PK-15 cells.Results showed that no bands were detected at the expected position.MG132,the inhibitor of UPS,was added to the medium after transfection to investigate the function of UPS on viral protein expressions.And the results showed that viral proteins,N^pro,C,p7,NS2,NS4A,NS4B and NS5A were detected by Western blot,indicating that MG132 upregulats the expression levels of them and they are possibly degraded by the 26S proteasome.Viral nonstructural protein NS4A acts as a cofactor of NS3 protein in the cleavage of viral polyprotein and studies about NS4A protein are limited.In order to further study the relations between NS4A protein and the UPS,plasmid encoding the NS4A protein was transiently transfected in PK-15 cells and expression of NS4A protein at different time points were examined.Results showed that NS4A protein was only detectable at 12 hpt.Further study with MG132 and 3-MA showed that it was degraded by the 26S proteasome after expression.While there are ubiquitin-dependent and-independent degradation by the proteasome,co-IP assay was conducted to detect whether NS4A protein was ubiquitinated.The result revealed that NS4A protein was polyubiquitinated.While ubiquitination most occurs on lysine residues（K）,the four K within NS4A protein were replaced separately or together by arginine（R）to examine their influences on the degradation of NS4A protein.Results showed that neither single mutation nor total mutation restrained its degradation.Truncated forms of NS4A protein,NS4AΔ1-4,were then constructed and expressed in cells.The results showed that expression level of NS4AΔ4 was nearly not influenced by MG132,indicating that residues responsible for NS4A protein degradation located within the first 22amino acids at the N-terminal.Considering that there is no overlap between NS4AΔ1 and NS4AΔ3 protein,and they were both degraded by the proteasome,there must be more than one residue responsible for the degradation of NS4A protein.Immunoprecipitation coupled with mass spectra analysis was performed to screen NS4A-interacting cellular proteins,which contributes to further investigation of the mechanism of the degradation of NS4A protein and its potential role in CSFV replication.The degradation mechanism of C protein was analysed in order to reveal the relationship between C protein and the UPS.The result showed that C protein was also degraded by the proteasome in PK-15 and 3D4/2 cells,which was partly inhibited by MG132and there might other ways for its degradation.While C protein was cleaved by cellular protein SPP and only the cleaved form of C protein was examined in this study.A series of truncated forms of C protein were expressed in cells to confirm the exact residue crucial for its degradation.The results showed that C_Δ5 protein was undetectable,C_Δ7 protein was detected to express at a low level and C_Δ8 protein was expressed at a relatively high level without MG132,indicating that residues 260-262 at the C-terminal may mediate the degradation of C protein.Expression of mutant C proteins,C_M260-261 and C_M260-263 with several amino acid mutations,were nearly not affected by MG132,indicating that residues M260 and L260 mostly affected the degradation of C protein.IP was performed with anti-EGFP antibody in PK-15 cells expressing C_Δ2,C_M260-261 or EGFP protein to confirm whether turnover of C protein was due to posttranslational modification.And results showed that C protein was not ubiquitinated compared with the EGFP protein,indicating that C protein might be degraded via Ub–independent pathway.Besides,expression of a series of truncated C proteins showed that residues 262-267 at the C-terminal were important for the cleavage of C protein by SPP.Besides,there may be interaction between the degradation and cleavage of C protein.Considering the modulating role of 26S proteasome in the degradation of CSFV viral proteins N^pro,C,p7,NS2,NS4A,NS4B and NS5A,the impact of proteasome inhibitor MG132 on CSFV replication was further investigated.Firstly,viability of PK-15 cells was examined under different concentrations of MG132 by CCK-8 assay.And the appropriate concentration of MG132 which had no effect on cell viability was 0.1μM.Thus,PK-15cells infected with CSFV were treated with 0.1μM MG132 for 24 h or 48 h.The results showed that MG132 decreased CSFV RNA level by 1.7 fold at 48 hpi,and virus titers by3.5 fold at 24 hpi and 6.9 fold at 48 hpi,indicating that MG132 could strongly inhibited the replication of CSFV.To reveal the underlying molecular mechanism,critical immune molecules were examined in CSFV infected cells with or without MG132 by q RT-PCR.The result showed that IFN-αwas slightly affected due to CSFV infection and MG132 did not alter the levels of IFN-αcompared with DMSO or untreated groups,indicating that the inhibitory role of MG132 in CSFV replication is not caused by IFN-α.Previous studies have revealed that IFN stimulated genes（ISGs）encoded proteins play important inhibitory roles in the replication of CSFV,so we next examined the transcription levels of three ISGs（Mx1,OAS1 and PKR）by q RT-PCR.The result showed that CSFV infection upregulated the transcription level of Mx1,OAS1 and PKR.MG132 upregulated the transcription level of the Mx1 and OAS1 compared with DMSO and untreated groups,indicating that the ISGs may play critical roles in the inhibition of CSFV replication by MG132.High doses of MG132 were further used to test the role of MG132 in the replication of CSFV.And the result showed that 0.5μM MG132 decreased CSFV RNA level by 2.1 fold,and 1.0μM MG132 decreased CSFV RNA level by 5.7 fold.The levels of Mx1,OAS1 and PKR were all increased by 0.5μM and 1.0μM MG132 compared with the DMSO group.While transcription level of ISGs was controlled by the JAK-STAT pathway,critical protein STAT1 in this pathway was examined by q RT-PCR and Western blot.Results showed that CSFV infection had no obvious effect on the m RNA level of STAT1,but decreased the level of STAT1 protein and increased the level of p STAT1 compared with the uninfected goup,showing that JAK-STAT pathway was activated due to CSFV infection.MG132 had no obvious effect on the level of p STAT1 compared with DMSO or untreated groups.The exact viral protein responsible for the degradation of STAT1 protein was further analysed by Western blot.The result showed that NS4A protein and its truncated NS4AΔ4 protein could decrease the level of STAT1 compared with EGFP control,indicating that NS4A protein may involve in the regulation of STAT1 as well as the JAK-STAT pathway during CSFV infection.Since the translocation of STAT1 from cytoplasm to nucleus is critical for the activation of JAK-STAT pathway,we further analysed the effect of MG132 on the localization of STAT1.Resutls showed that MG132 induced the accumulation of STAT1 in the nucleus of adjacent cells of the CSFV infected cells compared with DMSO or untreated groups,indicating that MG132 may play an antiviral role by inducing the translocation of STAT1 and the activation of JAK-STAT pathway in the uninfected cells.In conclusion,a close relationship between the UPS and CSFV was identified by the examination of ubiqitination level of cellular proteins in CSFV infected cells and the detection of the role of 26S proteasome in viral proteins N^pro,C,p7,NS2,NS4A,NS4B and NS5A.Examination of the JAK-STAT pathway under different conditions showed that viral protein NS4A mediated the degradation of STAT1 protein.The upregulation of expression of ISGs may play important roles in the inhibition of CSFV by MG132.Our study contributes to further investigation of the mechanism of the immune escape caused by CSFV and the prevention and control methods of CSF.