Study On Porcine Circovirus 2 Infection Mechanism Mediated By Src And Abl Tyrosine Kinase Family

Porcine circovirus 2(PCV2)causes huge economic losses in the global swine industry,however its interaction with host is complicated and remained unclear.Therefore,it is urgent to research on the mechanism of PCV2 infection and develop a new control strategy.As we previously reported,the end of Loop CT in the Cap of all PCV2 isolates had a strictly conserved PXXP motif that supposed to interact with the tyrosine kinase containing Src homology 3(SH3)domain / Src homology 2(SH2)domain.Studies showed that this interaction play a critical role in molecular ‘cross-talk’ between some viruses and host cells.However,the relationship between PCV2 and tyrosine kinase has not been well understood.This study aims to explore the invasion mechanism of PCV2 into PK15 cells from the perspective of tyrosine kinase.1.PCV2 infection increase the genes expression of Abl and Src family kinasesIn this study,we first detected mRNA expression of 11 tyrosine kinase genes at various time points after PCV2 infection into Porcine kidney epithelial cells(PK15),results indicated that Hck,Fyn,Fgr,Abl,Frk,Lck,and Src genes were up-regulated during PCV2 infection.We inferred that these tyrosine kinase genes may be involved in the life cycle of PCV2.2.Proliferation of PCV2 in PK15 cells requires Src family kinasesIn order to verify the hypothesis,we used the inhibitors PP2(Src kinase family inhibitor)and STI571(Abl kinase family inhibitor)against the above genes,together with PCV2 and PCV2 virus-like particles(VLPs)to study the role of these tyrosines in PCV2 infection.Indirect immunofluorescence(IFA)and cell ELISA showed that the inhibitors(STI571 and PP2)did not affect PCV2 VLP invasion.Subsequently,we used PCV2 rescued virus to study the effect of inhibitors(STI571 and PP2)on the proliferation of PCV2.The results showed that STI571 and PP2 decreased PCV2 replication,synthesis of viral DNA,expression of Cap protein and virions release.It is noteworthy that PP2 has ability to inhibit the proliferation of PCV2 more than STI571,and TCID50 showed that the inhibitory effect of PP2 on PCV2 proliferation was 2-fold more than that of STI571.Taken together,these results suggest that the Src and Abl tyrosine family kinase genes are involved in the proliferation of PCV2,and the proliferation of PCV2 is particularly dependent on Src family kinases.3.Src family kinases act cooperate with platelet-derived growth factor receptor(PDGFR)on the proliferation of PCV2Exploring the ways in which Src family kinases participate in PCV2 proliferation,and whether the Src family kinases acts independently or corelate with other proteins in the proliferation of PCV2,will help clarify the mechanism of PCV2 infection.Based on previous studies,members of the Src family of protein tyrosine kinases combine with the activated PDGFR through their SH2 domain,and causes activation of the intrinsic activity of the Src family kinases.Moreover,PDGFR has been shown to be related to the life cycle of many viruses.Hence,we hypothesized that Src family kinases may act cooperate with PDGFR on the proliferation of PCV2.We firstly detected the expression of PDGFR mRNA at different time points in PCV2 infected PK15 cells,and then used PDGFR-specific inhibitor(Crenolanib)to further confirm the role of PDGFR in PCV2 infection.IFA results indicated that the Crenolanib inhibits PCV2 proliferation in a drug-dependent manner.Based on the our results,it can be concluded that src family kinases cooperate with platelet-derived growth factor receptor(PDGFR)act on the proliferation of PCV2.This study laid a theoretical foundation for revealing the molecular mechanism of PCV2 infection and provided a reference for the development of potential therapeutic inhibitors against PCV2 infection,however the synergy mechanism between the Src tyrosine family kinase and PDGFR remains to be furthered study.