Study On The Role Of Host Protein Cydophilin A In Replicaiton And Proliefration Of Classical Swine Fever Virus

Classical swine fever virus (CSFV) is a small, enveloped virus with anonsegmented positive-stranded RNA genome, belonging to the genus Pestiviruswithin the family Flaviviridae. CSFV is the pathogen of classical swine fever (CSF), ahighly contagious swine disease with high morbidity and mortality, featuringsymptoms of hemorrhagic fever and immune suppression. The disease usually leadsto substantial economic losses to the pig industry worldwide. The viral genome isapproximately12.3kb in size and contains a single large open reading frame (ORF).This ORF is translated into a polyprotein which is further processed into11~12mature proteins by viral and cellular proteases. Although CSFV in vitro does notcause significant cytopathologic effect (CPE), the infection in vivo was able to causeserious cell damage and disease, this illustrates the very complicated interactionbetween viral proteins and host proteins. However, CSFV-host interaction and howCSFV infection regulate the expression of cellular genes and signal transductionpathways remain unknown. Previous study in our lab has identified21differentiallyexpressed protein in CSFV-infected PK-15cells by two-dimensional gelelectrophoresis (2-DE), including up-regulated cyclophilin A (CyPA) with2.7-foldincrease. Recent reports have suggested that cyclophilin A plays an important role inthe infection process of some viruses, such as HIV, HCV, and so on. Until now, theeffect of cyclophilin A on CSFV infection is not clear.To elucidate the role of cyclophilin A in CSFV replication, Cyclosporin A (CsA),the specific inhibitor of CyPA peptidyl-prolyl cis-trans isomerase activity, was used totreat PK-15cells prior to CSFV infection. CSFV-infected cells were collected at48hand72h post infection (p.i.) and subsequently subjected to detect the copy number ofviral genome by Realtime PCR and to measure the virus titer as TCID50. Resultsshowed that at48h and72h p.i., various concentrations of CsA were all found to significantly inhibit CSFV replication in PK-15cells, the efficiency of CSFVreplication was reduceded to44.6%~78.7%(p<0.01or p<0.05). Furthermore, theproduction of infectious CSFV was also prevented after treatment of PK-15cells withhigh concentrations of CsA, at48h and72h p.i. TCID50was decreased by2.8~83.2fold (p<0.05or p<0.01), which is consistent with the results of viral genomereplication in the presence of CsA. These results suggest that the peptidyl-prolylcis-trans isomerase function of CyPA is critical for CSFV replication.In order to further validate that CyPA is critical for CSFV replication, CyPA wasdown-regulated in PK-15cells by RNAi. After treatment with60nM siRNA, PK-15cells were infected with CSFV, and then the viral genome copies and virus titers weremeasured. Western blot results showed that CyPA expression was down-regulated inPK-15cells, in which CSFV genome copies at48h and72h p.i. were significantlylower than the control group (p<0.05) with1.9-and1.2-fold decrease, respectively. Inaddition, the infectious virus titer analyzed at48h and72h p.i decreased by7.1and11.0times, respectively, which was significantly lower than the negative control(p<0.05). These results indicated that down-regulated expression of CyPA induced bysiRNA also inhibited the replication and proliferation of CSFV in PK-15cells. Toup-regulate the expression of CyPA, PK-15cells were transfected withp3xFLAG-CMV-10-CyPA containing the CyPA gene and then infected with CSFV at6h after transfection. As expected, up-regulate expression of CyPA promotes thereplication and proliferation of CSFV in PK-15cells, but CSFV genome copies andvirus titers were not significantly different from that of the control group (p>0.05),indicating up-regulate expression of CyPA did not play an important role in the lifecycle of CSFV in PK-15cells.In order to study the interaction between CyPA and viral and host proteins,co-immunoprecipitation (Co-IP) was used to screen proteins interacted with CyPA inCSFV-infected PK-15cells, and19host proteins were identified, including translationelongation factor1α (EF1α), heat shock protein60(HSP60), T-complex protein1.Several studies have been shown that EF1α and HSP60are involved in the virusinfection and replication in target cells, thus they play a significant role inpathogenesis of related viruses. To illustrate the interaction between CyPA and these host factors, works are under progress to unravel the mechanism of CyPA in CSFVinfection and pathogenesis.In all, the present study provides the first report about the role of host proteinCyPA in replication and proliferation of CSFV. CyPA was identified as a critical factorinvolved in virus replication in CSFV-infected PK-15cells, and this providesimportant insights into elucidating the pathogenesis of CSFV.