猪流行性腹泻病毒经其附属蛋白抑制细胞凋亡

Porcine epidemic diarrhea virus inhibits cellular apoptosis via their accessory protein

摘要:

【背景】orf3位于猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV) s基因与e基因之间，是目前发现的PEDV唯一一个附属基因，编码附属蛋白(ORF3蛋白)。我们前期研究初步发现ORF3蛋白对PEDV诱导的细胞凋亡有影响。【目的】研究ORF3蛋白在PEDV侵染复制过程中的毒力作用机制。【方法】实验用3种PEDV：rDR13att-?ORF3 (orf3基因全部敲除)、DR13-ORF3att (携带有C端截短orf3)、rDR13att-ORF3wt (携带全长orf3基因)感染Vero细胞，观察病变情况，再用活细胞成像仪、流式细胞仪、DNA断裂的原位末端标记法[terminal deoxynucleotidyl transferase (TDT)-mediated dUTP nick end labeling，TUNEL]等方法检测不同感染时间点的细胞凋亡情况，然后用蛋白质印迹方法分析PEDV感染宿主细胞中主要凋亡相关蛋白(如Caspase-3)的活化或裂解，最后进行转录组测序研究病毒感染细胞中差异基因的表达情况，再用荧光定量PCR验证转录组结果。【结果】rDR13att-?ORF3引起较多的细胞病变，活细胞成像仪的动态观察结果显示，3种病毒侵染的细胞凋亡水平随着时间的延长均高于正常阴性细胞，但敲除orf3的病毒感染细胞后细胞凋亡率比其他两种病毒更高；敲除orf3病毒感染细胞凋亡率显著高于其他两种病毒；病毒rDR13att-?ORF3感染细胞后TUNEL阳性细胞数比DR13-ORF3att和rDR13att-ORF3wt更多；表达ORF3蛋白的重组PEDV可以抑制Caspase-3的活化；ORF3蛋白对受感染细胞Heat shock 70 kD protein 1B (HSP70)基因转录有促进作用，荧光定量PCR结果表明rDR13att-ORF3wt感染细胞的HSP70表达量高于rDR13att-?ORF3感染细胞。【结论】PEDV通过ORF3蛋白抑制细胞凋亡，而且这种作用可能是通过抑制Caspase-3的活化或增加HSP70的产生来完成的。

Abstract:

[Background] orf3 is located between the s and e genes of porcine epidemic diarrhea virus (PEDV) genome. It is the only accessory gene found in PEDV and encodes the ORF3 protein. Our earlier study showed that the ORF3 protein might has an effect on cell apoptosis induced by PEDV infection. [Objective] To study the virulence mechanism of ORF3 protein in the process of PEDV infection and replication. [Methods] Vero cells were infected with 3 different PEDVs, namely rDR13att-?ORF3 (with orf3 deleted from the genome), DR13-ORF3att (with a N-terminal 92 aa truncated orf3) and rDR13att-ORF3wt (with full length orf3). We observed cell pathogenic effect (CPE). Then different methods such as live cell imager, flow cytometry, TUNEL assay were used for apoptosis analysis of the cells at different time points of infection. Analysis of major apoptosis-related protein such as the cleaved Caspase-3 was also carried out by Western blotting in PEDV-infected cells. Finally, transcriptome sequencing was used to study the differential expression genes within the cells infected by the different viruses. Transcriptome results were further verified by real-time PCR. [Results] rDR13att-?ORF3 induced more CPEs in the infected cells than the other two viruses. Dynamic observation results of live cell imager showed all the three viruses induced obvious apoptosis in the infected cells. However, higher apoptotic level was detected in the cells infected with rDR13att-?ORF3 than the cells infected by the other two viruses (P<0.05). The flow cytometry also checked the apoptotic difference with the cells infected with the viruses: higher proportion of apoptotic cells among the cells infected with rDR13att-?ORF3. The result was further confirmed by terminal deoxynucleotidyl transferase (TDT)-mediated dUTP nick end labeling (TUNEL) staining. There are more TUNEL staining cells among the cells infected with rDR13att-?ORF3 than the cells infected by the other two viruses. The result of Western blotting showed rDR13att-ORF3wt can inhibit Caspase-3 activation in the infected cells. Transcriptome analysis found there was a significantly higher-level heat shock 70 kD protein 1B (HSP70) transcription in cells infected with rDR13att-ORF3wt than in the cells infected with rDR13att-?ORF3. Real-time PCR showed the expression of HSP70 in cells infected by rDR13att-ORF3wt was higher than that of rDR13att-?ORF3. [Conclusion] ORF3 protein inhibited cellular apoptosis induced by porcine epidemic diarrhea virus infection. The effect may be through inhibition of Caspase-3 activation (cleavage) or promotion of HSP70 expression.