Effects Of P53 On FMDV Replication And Pathogenicity

?Objective? In this study,We knocked down Tp53 using shRNA technology,knocked out Tp53 by CRISPR/cas technology,and up-regulated p53 by exogenous overexpression plasmid,and then by constructing Tp53 knockout(Tp53-/-)cell lines and developing Tp53-/-C57BL/6 mice,we aimed to reveal the biological function of p53 in FMDV replication and pathogenicity in mice.?Methods?(1)PK-15 cells(porcine kidney 15 cell line)were treated with FMDV for 1 h,and then were analyzed by RNA-seq method and verified by RT-qPCR(real-time quantitative polymerase chain reaction)to reveal the host's early response expression profile against FMDV infection;(2)BHK-21 and PK-15 cells were used as materials,and cells were pretreated with MG132 and MDM2 inhibitors,and then were treated with FMDV,RT-qPCR and WB(western blotting)were performed to detect the expression of p53 at mRNA and protein levels in infected and uninfected cells,and then to reveal the effect of FMDV on p53 in BHK-21 and PK-15 cells and related corresponding pathways;(3)knockdown the expression of Tp53 in BHK-21 cells by pretreated with shRNA(short hairpin RNA)and then treated with FMDV to study the effect of down-regulation of p53 expression on FMDV replication.(4)BHK-21 cells were transfected with Tp53 gene overexpression plasmid HA-Tp53 and Flag-Tp53,and the up-regulation of p53 expression was identified with WB method,and then cells were treated with FMDV to detect the effect of p53 overexpression on FMDV replication.(5)The Tp53-/-BHK-21 and PK-15 cell lines were constructed by using the CRISPR/cas9 system and identified by WB and sequencing methods.Then,the knockout cell lines were used to study the effect of p53 knockout on FMDV replication in BHK-21 and PK-15 cells;(6)The lethality of FMDV against WT(wild type)and Tp53-/-mice was studied by using purchased and bred Tp53-/-C57BL/6 mices;(7)WT and Tp53-/-micee peritoneal macrophages(RPM?)were isolated with physiological saline and sputum shaking method,and then were treated with FMDV after adherence,and the expression of each cytokine was detected by RT-qPCR;(8)1-month-old WT and Tp53-/-mice were treated with FMDV(100LD50)for 72 h,then their hearts,spleens and muscles were fixed in 4% paraformaldehyde,and then HE staining was used to analyze the effect of Tp53 knockout on the pathogenicity of FMDV.?Results?(1)After RNA-seq analysis and RT-qPCR verification,PK-15 cells up-regulated cytokines such as TNF,CXCL2,CCL20,CCL4 and IL6 to stimulate inflammatory response against FMDV infection after treatment with FMDV for 1 h;(2)MG132 experiments showed that FMDV inhibited the expression of p53 mRNA and protein levels through ubiquitination pathway within 1 h after FMDV infection,and then treated with MDM2 inhibitor,it was found that FMDV inhibited expression of p53 in BHK-21 cells through MDM2-independent ubiquitination pathway at the early stage of infection;(3)Tp53 knockdown with shRNA can inhibit the replication of FMDV in BHK-21 cells;(4)Up-regulation of p53 expression thtough transfected with exogenous HA-Tp53 and Flag-Tp53 overexpression plasmids can not significantly affect the replication of FMDV in BHK-21 cells;(5)Tp53-/-BHK-21 and PK-15 cell lines were successfully obtained by using the CRISPR/cas9 system,and we found that FMDV replication was suppressed in Tp53-/-cells.While,MDA5,RIG-I,and TLR3 were up-regulated in the Tp53-/-cells;(6)Compared with WT C57BL/6 mice,Tp53-/-mice had higher survival rate against FMDV infection;By isolated mouse RPM? and infected with FMDV,we found that Tp53-/-can up-regulate the expression of cytokines that exert major antiviral functions,such as IFNB-1,TNF,and IL-6;(8)Results of HE staining shown that FMDV has higher pathogenicity to WT mice than Tp53-/-mice.?Conclusion? In the early stage of infection,FMDV inhibits the expression of p53 by a non-MDM2-specific ubiquitination-proteasome pathway,while the host mainly resists infection by inflammatory reaction;in long-term infection,knockdown and knockout of p53 inhibits FMDV replication and pathogenicity to mice thhrough upregulating the expression of IFNB-1,IRF-3,ISRE,TNF,IL6,RIG-I,MDA-5,etc.