Effect And Mechanism Of Interferon Stimulated Gene 15 On The Replication Of Classical Swine Fever Virus

Classical swine fever(CSF)is a highly infectious disease caused by classical swine fever virus(CSFV),which causes serious economic damage to the pig industry in China.Interferons(IFN)establish an antiviral line of defense by inducing transcription and expression of interferon-stimulated genes(ISGs).As an important antiviral factor,ISGs play an important role in the host defense and elimination of viruses.Therefore,study of the antiviral mechanisms of ISGs is important.ISG15 is one of the potent antiviral molecules in the IFN signaling pathway.ISG15 exerts an antiviral effect by binding to a target protein to regulate cell processes or acting as a monomer to effect on viruses.Our research focuses on the impact of ISG15 on CSFV replication and antiviral mechanisms.The results were shown as follows:1.The effect of ISG15 on CSFV replication(1)Three shRNA sequences targeting ISG15 were designed and inserted into the lentivirus interference vector.ISG15 overexpression or ISG15 knockdown cell lines were established by package and infection of recombinant lentiviruses in Porcine alveolar macrophage(PAM).Cell lines were infected with CSFV Shimen strain and the level of CSFV replication were examined.The results showed that CSFV replication was significantly inhibited in cell line stably overexpressing ISG15(P<0.01 or P<0.001)whereas was significantly promoted in ISG15 knockdown cell line(P<0.01 or P<0.001),suggesting that ISG15 is an anti-CSFV effector.(2)IFN induced ISG15 expression in a dose-dependent manner,suggesting that ISG15 can be induced by IFN in PAM cells.At the same time,it was also found that IFN also caused an increase in the level of ISGylation expression.Under the conditions of IFN pretreatment,knockdown of ISG15 expression promoted CSFV replication more significantly than those in PAMs in absence of IFN(P<0.01),demonstrating that ISG15 exerts the antiviral effect of IFN.(3)PAM cells were infected with CSFV.The expression of ISG15 and ISGylation levels were detected.It was found that CSFV promoted the expression of ISG15,but did not affect the level of ISGylation.2.ISG15 inhibits CSFV replication depending on the ISGylation pathway(1)The binding region to target protein of the ISG15 was mutated by a point mutation method to construct a mutant of ISG15 which could not bind the target protein.Mutant identification results showed that the ISGylation product was significantly reduced in PAM cells transfected with ISG15 mutant.ISG15 mutants with CSFV infection did not significantly changed CSFV replication(P>0.05),suggesting that ISG15 inhibits CSFV replication in an ISGylation pathway.In shISG15-1 cell line,supplementation with mutant ISG15 did not inhibit CSFV replication,indicating that ISG15 inhibits CSFV depending on ISGylation pathway.(2)Three shRNA sequences designed for USP18 were inserted into the lentiviral interference vector,and a stable knockdown USP18 expression PAM cell line was established by lentiviral package and infection.In IFN-pretreated shUSP18 cells,CSFV replication was significantly down-regulated,and the level of ISGylation was significantly induced(P<0.05 or P<0.001),indicating that ISGylation activation plays an important role in the inhibition of CSFV during IFN treatment.(3)The downstream of the IFN pathway were detected in ISG15 knockdown and USP18 knockdown cell lines,respectively,and it was found that knockdown of ISG15 and knockdown of USP18 showed the same up-regulation on ISGs(P<0.001).However,knockdown of ISG15 and knockdown of USP18,showed opposite effect on CSFV replication.The results showed that in terms of inhibition of CSFV,the effect on ISGylation was more pronounced than the effect on ISGs.3.ISGylation affects CSFV replication by regulating the process of autophagy(1)CSFV was infected with cells overexpressing ISG15 and knocking down ISG15,respectively.It was found that overexpression of ISG15 significantly inhibited autophagy(P<0.01),whereas knocking down ISG15 promoted autophagy,indicating that ISG15 can inhibit autophagy(P<0.05).Autophagy inhibitor treatment of shISG15-1 cells,upregulation of knockdown of ISG15 on CSFV replication was significantly eliminated,and the upregulation of autophagy was also inhibited(P<0.05 or P<0.01),indicating that ISG15 affects CSFV by regulating autophagy.In the cells pretreated with rapamycin,overexpression of ISG15 inhibited autophagy(P<0.001),whereas knockdown of ISG15 levels promoted autophagy(P<0.05),indicating that ISG15 inhibits rapamycin-induced autophagy.(2)Overexpression of the autophagy protein BECN1(Beclin1)rescued the inhibitory effect of ISG15 on autophagy levels and CSFV replication,suggesting that ISG15 may affect autophagy by regulating the expression of BECN1,thereby inhibiting CSFV replication.The BECN1 sequence was directly bind to the binding domain of ISG15 to construct a mimetic of ISGylated BECN1.CSFV infection revealed that CSFV replication and autophagy levels were not up-regulated in cells with overexpressing mimics,suggesting that ISGylated BECN1 may fail to function as wild BECN1 in autophagy.The interaction of HERC5,the key enzyme in the ISGylation system,with BECN1 was verified by Co-IP,GST pull-down and laser confocal techniques,further demonstrating that ISG15 can regulate autophagy protein BECN1 though ISGylation.Taken together,this study demonstrates that ISG15 effectively inhibits CSFV replication;ISG15 inhibits CSFV replication depending on ISGylation;ISG15 regulates autophagy by ISGylating BECN1,thereby affecting CSFV replication.This study provides a novel theoretical basis for further understanding of the anti-CSFV mechanisms of IFN.