Effect Of Prrsv Infection On Cell Cycle And Its Mechanism

Porcine reproductive and respiratory syndrome virus(PRRSV)is a pathogen of porcine reproductive and respiratory syndrome(PRRS).The main features of the disease are sow abortion and piglet respiratory diseases.The disease spreads widely in the herd and has caused a serious impact on the development of the global swine industry and is also a major problem in the prevention and control of swine disease.At present,there is no effective treatment for the disease.To increase farming benefits,comprehensive prevention and control measures should be adopted to reduce or block the occurrence and spread of the disease.In order to reduce the adverse effects of PRRS and better control of the disease,more in-depth understanding and research of pathogenesis of PRRSV should be proceeded.In eukaryotic cells,cell growth and division is carried out in the form of cell cycle.The cell cycle checkpoint acts as a negative feedback adjustment mechanism to ensure proper and reasonable cell cycle events.When DNA is damaged,these checkpoints are activated to play their roles and interrupt the cell cycle.The cell cycle resumes after DNA damage repair.If the damage can be repaired in time,it will lead to cell apoptosis.The relative signaling pathways are activated by cell cycle arrest induced by damage,which in turn affects the expression of the cell cycle regulatory proteins or their phosphorylation.Numerous studies have shown that many viruses and their associated proteins can disrupt cell cycle and induce cell apoptosis during infection.Although there are many studies about the pathogenesis of PRRSV,the effect of PRRSV infection on cell cycle and its mechanism are rarely reported.In this study,MARC-145 cell were infected with PRRSV strain SD16 and its infection on cell cycle and the mechanism were explored.The main contents are as follows:1.Significant cytopathic effects(CPE)were observed in MARC-145 cells infected with PRRSV strain SD16 under inverted microscope.A large number of PRRSV N proteins were detected using IFA and Western blot.2.According to the instruction of Cell Counting Kit-8(CCK-8),the standard curve of the number of MARC-145 cells and 450 nm absorbance(OD450)was plotted and the regression equation “ y = 0.3098 ln(x)-1.7214,R2 = 0.998 ” was obtained.MARC-145 cells were infected with different multiplicity of infection(MOI)PRRSV strains SD16.The absorbance values at different time after infection were measured.According to the regression equation,the number of cells at different time after infection was calculated.The results showed that the proliferation of MARC-145 cells was inhibited by infection with different doses of PRRSV strain SD16.1 MOI infection dose was chosed for following study with comprehensive consideration.3.PRRSV infection could induce apoptosis of MARC-145 cells with Annexin V-PI double staining and the number of apoptotic cells increased with the prolongation of infection time.PRRSV infection induced MARC-145 cell cycle arrest in G2/M phase by PI staining and flow cytometry detection.4.Using IFA and Western blot analysis,it was observed that CyclinB1 was highly expressed and prominently localized in the nucleus of PRRSV infected cells,the level of phosphorylated Cdc2(p-Cdc2)(Tyr15)in PRRSV infected cells was obviously higher and the nuclear distribution was increased than that in mock cells,and the expression of p-p53(Ser15)was significantly increased in PRRSV infected cells.The expression of 14-3-3σ was significantly increased in PRRSV infected cells and p21 expression was significantly increased with Western blot analysis.Then the interactions between p21 and Cdc2-CyclinB1 complex in MARC-145 cells induced by PRRSV infection was further confirmed with co-immunoprecipitation experiments.These results indicated that cell cycle arrest in G2/M phase of PRRSV infected MARC-145 cells was associated with activation of p53/p21 signaling pathway.5.The difference of Wee1,Myt1 transcription level was not statistically significant and protein expression of Wee1 and Myt1 were increased in MARC-145 cells infected with PRRSV using real-time quantitative PCR(qPCR)and Western blot.The expression of Cdc25 C was not obvious,but the expression of p-Cdc25C(Ser345)was significantly increased and mainly localized in tcytoplasm.The increase of Chk1 and p-Chk1(Ser345)expression is very obvious.These results showed that cell cycle arrest in G2/M phase induced by PRRSV infection was associated with activation of Chk/Cdc25 C signaling pathway.In summary,PRRSV infection activated p53/p21 signaling pathway and Chk/Cdc25 signaling pathway and induced MARC-145 cell cycle arrest in G2/M phase.