| Porcine transmissible gastroenteritis virus(TGEV)can cause porcine transmissible gastroenteritis(TGE),which is characterized by diarrhea,vomiting and severe dehydration.It is easy to co-infect with other pathogens.Therefore,the healthy development of pig industry is greatly threatened.Infection of pig testicular cells(ST)in vitro by TGEV not only causes a typical cytopathic effect(CPE),but also induces the production of type I interferon.The mechanism of TGEV leading to cell CPE and the mechanism of inducing interferon production have been explored by the predecessors.Since its discovery,p53 has been a hot topic of research.Research on its anti-tumor,stress,cell cycle regulation and innate immunity has been very deep.It is known that a variety of viral infections can activate p53 and induce apoptosis and interferon production to exert the innate defense of the body.Previous studies have shown that TGEV infection of pig kidney cells(PK-15)can cause significant up-regulation of p53 and induce apoptosis.The fact that phosphorylation of p53 causes activation of the interferon-activated pathway,which in turn induces the production of interferon,has also been reported.So what role does p53 play in the early infection of TGEV? After p53 activation,what mechanism is used to regulate the production of interferon? What is the impact of the presence of p53 on viral replication? The answers to these questions are rarely reported so far.In this study,porcine kidney cells(PK-15cells)of wild type(WT)and knockout type(p53-/-)of TGEV and p53 were used as research materials.The effect of p53 signal on IFN-beta production after virus infection was clarified,and then the molecular regulation mechanism of p53 on IFN-beta induction in this process was attempted to explain.In this study,the cells were infected with CRISPR-Cas9 lentiviral system targeting pig p53 gene,and positive clones were screened by puromycin.Western blotting and cell genome sequencing were used to identify stable p53 knockout PK-15 cell line.Then,the real-time quantitative PCR method was used to detect the transcriptional levels of various genes and the factors involved in p53-regulated interferon-producing pathways to explain the effect of p53 on viral replication in TGEV-infected host cells the induction of TGEV-induced type I interferon via the p53 signaling pathway.In this way,the important role of p53 in host cell anti-TGEV infection was clarified,and the molecular mechanism of p53 signaling pathway affecting TGEV-induced type I interferon was further revealed.The main results of this study are as follows:1.The results of gene sequencing showed that the p53 gene deleted a base T at the 454 base position,resulting in the arginine of the site 102 p53 protein becoming serine,and the frameshift was terminated at 113 site;Western blot results showed that the constructed p53-/-PK-15 cells failed to detect the expression of p53 protein.2.At the 0.1 MOI of TGEV infection,the mRNA level of IFN-? was significantly increased in the early stage of infection(6 hpi),peaked at 24 h,and the mRNA level of IFN-? in WT PK-15 cells decreased after 36h;The mRNA level of IFN-? in p53-/-PK-15 cells was basically synchronous with that of WT PK-15 cells,but the peak value was delayed at 36 h,and the IFN-? mRNA level was lower than that of WT PK-15 cells at each time.The IFN-?assay showed that the IFN-? content increased in a time-dependent manner,and the concentration in p53-/-PK-15 cells was significantly inhibited compared with the concentration in WT PK-15 cells.3.Detection of key factors in the TLR pathway induced by IFN-? production showed that TGEV infection resulted in up-regulation of TRIF and TRAM mRNA levels in WT and p53-/-PK-15 cells.And mRNA expression in TGEV-infected p53-/-PK-15 cells was significantly blocked before 36 h after infection compared to levels in WT cells.Resveratrol treatment,a pathway inhibitor,significantly reduced TRIF expression in WT PK-15 cells infected with TGEV,and the expression of IFN-? induced by TGEV in p53-/-PK-15 cells and WT PK-15 cells.Quantitative results of key factors in the RLR pathway showed that MDA5,RIG-I and IPS-1 mRNA levels were also up-regulated in infected WT and p53-/-PK-15 cells.Before 36 hours after infection,the levels of these factors in p53-/-PK-15 cells infected with TGEV were significantly lower than those in WT cells.The results of the pathway inhibitor BX795 showed that BX795 not only significantly down-regulated the mRNA level of RIG-I,but also inhibited TGEV-induced IFN-? mRNA expression in p53-/-PK-15 cells and WT PK-15 cells.4.Quantification of the viral genome and subgenomic protein factors showed that the transcriptional levels of viral genomes and subgenomics in p53-/-PK-15 cells were significantly higher than those of WT PK-15 cells at 24 and 36 hpi.5.Quantitative analysis of viral genomic RNA(gRNA)and subgenomic RNA(ORF,N)showed that the transcription levels of viral gRNA,ORF and N genes in p53-/-PK-15 cells were significantly higher than those in WT-PK-15 cells at 24 and 36 hours after infection.Interferon-stimulated gene examination showed that IRF3,IRF9,ISG15,and ISG20 in T PK-15 cells had a higher increase than p53-/-PK-15 cells in a time-dependent manner before36 h infection.In addition,poly(I:C)treatment significantly inhibited the expression of TGEV gRNA,N and ORF7 in WT PK-15 cells and p53-/-PK-15 cells,and the viral gene levelin WT PK-15 cells was significantly lower than that in p53-/-PK-15 cells.The same results were shown in the exogenous interferon-? assay,except that exogenous IFN-? was less potent than WT PK-15 cells in inhibiting TGEV replication in p53-/-PK-15 cells.This study clarified the interaction between PK-15 cells and TGEV after TGEV infection.The molecular mechanism of TGEV-induced type I interferon was first revealed by p53 signaling pathway,and the antiviral effect mechanism of p53 in early TGEV infection was elucidated.It provides certain ideas and methods for further research on new antiviral drugs and treatment methods,and provides a theoretical basis for controlling the harm of TGEV in the breeding industry. |
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