| Classical swine fever(CSF)is an infectious disease caused by classical swine fever virus(CSFV)that seriously threatens the domestic and foreign pig industry.Viruses,as organisms that parasitize host cells,rely heavily on various biological mechanisms of host cells for replication and propagation.Therefore,it is of great significance to study the interaction and mechanism between viruses and cells to further understand the pathogenic mechanism of viruses and study antiviral therapy.CSFV can induce innate immune responses after infecting pigs,and regulate the host cell cycle and apoptosis for completing self-proliferation.Ribosome protein S3(RPS3),a component of the 40 S ribosomal subunit,which plays an important role in various intracellular pathways.This article focuses on the effects and mechanisms of RPS3 on CSFV replication.The results are as follows:1.RPS3 inhibits the replication of CSFV,and CSFV down-regulates the expression of RPS3(1)Using recombinant lentiviral system,porcine macrophages(PAM)cell lines of overexpression or knockdown RPS3 were constructed.The results showed that the CSFV replication was significantly inhibited in RPS3-overexpressed PAM cells(P<0.01 or P<0.001),and significantly promoted in RPS3-knockdown PAM cells(P<0.01 or P<0.001),indicating that RPS3 inhibits CSFV replication.Addition of the supernatant from RPS3 over-expressing cells in CSFV infected PAM cells revealed that the supernatant from RPS3 over-expressing cells significantly inhibits CSFV replication(P<0.01 or P<0.001).(2)The expression of RPS3 was detected in PAM cells infected with CSFV.It was found that CSFV infection downregulates the expression of RPS3 in cells.2.CSFV inhibits the expression of innate immune factors induced by RPS3.(1)In PAM cells,overexpression of RPS3 significantly increased the m RNA levels of TLR3(P<0.001),TLR2,TLR4,TLR7,IL-8 and IFN-β(P<0.001),and the secretion of IL-8and IFN-β(P<0.001).However,interfering with RPS3 significantly reduced the m RNA levels of TLR3(P<0.01),TLR2,TLR4,TLR7,IL-8 and IFN-β(P<0.001),and the secretion of IL-8and IFN-β(P<0.001).This indicates that RPS3 up-regulates the expression of innate immune factors.(2)The m RNA levels of TLR2,TLR4,TLR7,IL-8 and IFN-β,and the secretion of IL-8and IFN-β were significantly increased in PAM cells infected with CSFV(P<0.001).It shows that CSFV infection promotes the expression of innate immune factors.(3)In CSFV infected cells,overexpression of RPS3 significantly increased the m RNA levels of TLR3,TLR4,TLR7,IFN-β(P<0.001),TLR2 and IL-8(P<0.01),and the secretion of IL-8 and IFN-β(P<0.001).Interfering with RPS3 significantly reduced the m RNA levels of TLR2,TLR3,TLR4,TLR7,IL-8 and IFN-β(P<0.001),and the secretion of IL-8 and IFN-β(P<0.001).In RPS3 overexpressing and interfering cells,CSFV infection could significantly down-regulate the m RNA expression levels of TLR2,TLR3,TLR4,TLR7,IL-8 and IFN-β(P<0.001)and significantly reduce IL-8 and IFN-β secretion(P<0.001).This indicates that CSFV inhibits the production of innate immune factors mediated by RPS3.3.The change of the expression levels of RPS3 leads to apoptosis,and CSFV inhibits apoptosis by down-regulating RPS3,thereby favoring CSFV replication.(1)In PAM cells,overexpression of RPS3 significantly increased the apoptosis rate compared with the control group(P<0.001),which significantly enhanced the activity of proapoptotic molecules caspase-3(P<0.001)and caspase-8(P<0.01),and significantly reduced cell viability(P<0.001);Knockdown of RPS3 significantly increased the apoptosis rate compared with the control group(P<0.001),which significantly enhanced the activity of proapoptotic molecules caspase-3(P<0.001)and caspase-8(P<0.01),and the cell viability was significantly reduced(P<0.001).This indicates that the stable expression of RPS3 is a necessary condition for maintaining cell viability.(2)In CSFV infected cells,overexpression of RPS3 significantly increased apoptosis(P<0.001),and significantly enhanced the activity of the pro-apoptotic molecules caspase-3(P<0.001)and caspase-8(P<0.01),and also significantly reduced cell viability(P<0.001).Comparing the apoptosis rates of RPS3-overexpressing cells in the presence or absence of CSFV,we found that CSFV infection significantly down-regulated RPS3-mediated apoptosis(P<0.001),while significantly attenuated the activity of apoptotic molecules caspase 3 and caspase 8(P<0.001),and significantly increased the cell activity(P<0.01).(3)In CSFV infected cells,interference with RPS3 had no effect on cell apoptosis,the activity of apoptosis molecules caspase-3 and caspase-8,and cell viability(P>0.05).CSFV infection of cells with low expression of RPS3 could significantly down-regulate cell apoptosis(P<0.001),at the same time significantly attenuate the activities of pro-apoptotic molecules caspase-3 and caspase-8(P<0.001),and significantly enhance cell viability(P<0.001).4.RPS3 can shorten the S phase of cells,and CSFV can arrest cells in S phase by downregulating RPS3,which is beneficial to CSFV replication.(1)In PAM cells,overexpression of RPS3 significantly reduced the proportion of S-phase cells(P<0.001),and significantly reduced the protein levels of Cyclin A and Cyclin D(P<0.001).Interference with RPS3 significantly increased the proportion of S phase cells(P<0.001)and significantly increased the protein levels of Cyclin A and Cyclin D(P<0.001).(2)CSFV infection significantly increased the proportion of S-phase cells(P<0.001),and significantly increased the protein levels of Cyclin A and Cyclin D(P<0.001).(3)In CSFV infected cells,overexpression of RPS3 significantly reduced the proportion of S-phase cells(P<0.001),and significantly reduced the protein levels of Cyclin A and Cyclin D(P<0.01);Interfering with RPS3 significantly increased the proportion of S-phase cells(P<0.001),and significantly increased the protein levels of Cyclin A and Cyclin D(P<0.001).CSFV infection of RPS3 overexpressing cells and RPS3 interference cells could significantly increase the proportion of cells in S phase(P<0.001),and significantly upregulate the protein levels of Cyclin A and Cyclin D(P<0.001).This indicates that CSFV can arrest cells in S phase by down-regulating RPS3.In summary,this study shows that RPS3 has the effect of inhibiting CSFV replication,and CSFV down-regulates the expression of RPS3.CSFV suppresses the expression of innate immune factors and apoptosis by down-regulating RPS3,and arrests the cells in S phase,which is beneficial to the replication of CSFV.The findings provide new scientific data for clarifying the effect and mechanism of RPS3 on CSFV replication. |
