The Role Of Ubiquitination In PEDV Proliferation

Ubiquitination is an important protein post-translational modification,which plays a key role in regulating cell cycle,signal transduction,DNA repair,inflammation,antigen presentation and other cell physiological processes.Porcine epidemic diarrhea virus（PEDV）is a single-stranded positive-sense RNA virus with envelope,which mainly causes severe watery diarrhea in infected piglets and brings serious economic losses to the global swine industry.At present,studies have shown that various viruses infection depend on ubiquitination,but there are few reports of its effect on PEDV proliferation.In this thesis,PEDV variant strain AJ1102 as a representative,the role and mechanism of ubiquitination in PEDV proliferation were studied.The specific research contents are as follows:1.PEDV infection changes intracellular protein ubiquitination levelsIn order to analyze the effect of PEDV infection on intracellular protein ubiquitination levels,PEDV AJ1102 strain was inoculated into porcine intestinal epithelial cells（IPI-FX）,and samples were collected at different time points after inoculation.Intracellular protein ubiquitination levels were detected by Western Blot assay.The results showed that compared with uninfected control cells,intracellular protein ubiquitination levels were obviously reduced within 6-18 h after virus infection,while there was no significant difference at 24 h,indicating that PEDV infection can change intracellular protein ubiquitination levels,and the effect is more obviously at the early and middle stages of virus infection.2.Overexpression of ubiquitin promotes PEDV proliferationPEDV infection can change intracellular protein ubiquitination levels,suggesting that intracellular protein ubiquitination may be involved in regulating PEDV proliferation.In order to verify this hypothesis,and considering that intracellular protein ubiquitination are most common in K48 and K63 types,wild-type ubiquitin and K48 and K63 types ubiquitin eukaryotic expression plasmids were transfected into IPI-FX cells,and then inoculated with PEDV AJ1102 strain.The virus proliferation was detected by Western Blot and TCID₅₀assays.The results showed that overexpression of three types of ubiquitin could promote PEDV proliferation,and the effect of K63 type ubiquitin was most obviously,indicating that intracellular protein K63 type ubiquitination is most critical in PEDV proliferation.3.The ubiquitin-proteasome system is mainly involved in PEDV genome replicationThe ubiquitin-proteasome system（UPS）is the mainly way to degrade intracellular ubiquitinated protein,it is not clear whether it will participate in PEDV proliferation.Therefore,IPI-FX cells were treated with UPS inhibitor MG132,and then infected with PEDV AJ1102 strain.The virus proliferation was detected by Western Blot,RT-q PCR and TCID₅₀assays.The results showed that MG132 inhibited PEDV proliferation in a dose-dependent manner.Further analysis showed that MG132 inhibited proliferation PEDV genome replication,but had no significant effect on virus adsorption,invasion and release,indicating that UPS is mainly involved in PEDV genome replication.4.Overexpression of ubiquitin affects part of PEDV protein expressionOverexpression of ubiquitin can promote PEDV proliferation,in order to explore its effect on virus protein,PEDV protein eukaryotic expression plasmids were co-transfected with ubiquitin eukaryotic expression plasmid into HEK-293T cells,and PEDV protein expression levels were detected by Western Blot assay.The results showed that PEDV M protein,nsp2,nsp7,nsp8,nsp9,nsp13 and nsp16 expression levels were increased,while nsp16 expression level was increased most obviously,indicating that overexpression of ubiquitin can affect part of PEDV protein expression.5.Determination of ubiquitination types and ubiquitination sites of PEDV nsp16Since overexpression of ubiquitin had the most significantly effect on PEDV nsp16expression,the protein was selected for further study.Firstly,the interaction between PEDV nsp16 and ubiquitin was detected by Co-IP assay.It was found that PEDV nsp16interacted with wild-type ubiquitin and ubiquitin mutants K6-Ub,K11-Ub,K29-Ub,K48-Ub and K63-Ub,indicating that PEDV nsp16 has multiple ubiquitination types.In order to determine the specific ubiquitination sites of PEDV nsp16,eukaryotic expression plasmids with 20 lysine single point mutations in PEDV nsp16 were constructed and co-transfected with wild-type ubiquitin eukaryotic expression plasmid into HEK-293T cells.Co-IP assay showed that compared with wild-type nsp16,K185R mutant ubiquitination level was obviously decreased,while K280R mutant ubiquitination level was significantly increased,indicating that K185 and K280 are important ubiquitination sites of PEDV nsp16.6.PEDV nsp16 ubiquitination is beneficial to virus proliferationIn order to analyze the role of PEDV nsp16 ubiquitination in virus proliferation,PEDV AJ1102 strain infectious clone was used to construct recombinant viruses r AJ1102-K185R and r AJ1102-K280R with K185 and K280 mutations in nsp16,and wild-type virus r AJ1102 was simultaneously rescued.The proliferation curve showed that r AJ1102-K185R and r AJ1102-K280R had similar proliferation kinetics with r AJ1102 in Vero cells,but r AJ1102-K185R titer was obviously lower than that of r AJ1102,while r AJ1102-K280R titer was significantly higher than that of r AJ1102,indicating that PEDV nsp16 ubiquitination is beneficial to virus proliferation.In summary,this study reveals the mutual regulation between PEDV infection and intracellular protein ubiquitination,certifies that PEDV nsp16 ubiquitination can promote virus proliferation,improves understanding of the specific mechanisms of PEDV infection of cells.