新城疫病毒对宿主核糖体蛋白RPL11降解机制的初步研究

Preliminary study on degradation mechanism of host ribosomal protein RPL11 by Newcastle disease virus

本研究使用蛋白标记药物Puromycin标记NDV感染后DF-1细胞新合成的蛋白,发现NDV感染后新蛋白合成量显著下调,而病毒NP蛋白的含量却随感染时间的延长呈增加趋势。为探究NDV的感染对DF-1细胞核糖体大亚基L11蛋白(RPL 11)表达水平的影响,分别收获NDV(MOI 1)感染后不同时间(8 h、16 h、24 h和32 h)和不同剂量NDV(MOI 1、0.1)感染后相同时间的细胞样品,采用western blot、激光共聚焦、荧光定量PCR分别检测内源性RPL 11的蛋白表达和mRNA转录水平。Western blot结果显示NDV感染后核糖体蛋白RPL 11的表达量显著降低,并且与病毒感染时间和感染剂量呈正相关。激光共聚焦结果显示,NDV感染后细胞核与细胞质中的RPL 11荧光信号均明显减弱。但荧光定量PCR结果显示,该基因的转录水平却无明显变化,表明其蛋白表达量的减少发生在翻译后修饰环节。构建重组质粒pFlag-RPL 11转染DF-1细胞24 h后再感染不同剂量NDV(MOI 1、0.1),通过western blot检测外源性RPL 11的表达水平。结果显示外源性RPL 11蛋白的表达量在病毒感染后也呈明显下降趋势,并也与病毒感染剂量呈正相关。在NDV感染(MOI 1)的DF-1细胞培养基中添加泛素蛋白酶体抑制剂MG132,病毒滴度测定结果表明该药物对病毒增殖无明显影响,却能够显著抑制NDV感染后RPL 11蛋白的降解,表明RPL 11的降解依赖泛素蛋白酶体途径。上述结果证实NDV感染细胞后能够引起RPL 11蛋白的泛素化降解,且该降解过程能够被MG132所抑制。本研究为解析NDV感染后宿主细胞内蛋白翻译的变化提供了新的着眼点。

In this study,puromycin was used to label the newly synthesized proteins in NDV infected DF-1 cells.It was found that the amount of new synthesis of proteins was significantly decreased after NDV infection,while the content of viral NP protein showed an increasing trend with the persistence of infection time.In order to explore the effect of NDV infection on the expression level of ribosomal large subunit L11 protein(RPL11)of DF-1 cells,the cell samples were collected at different time(8 hours,16 hours,24 hours and 32 hours)after infection with different doses of NDV(MOI 1 and 0.1).Western blot,confocal laser scanning and fluorescence quantitative PCR were used to detect the protein expression and m RNA transcription level of endogenous RPL11 in the cell samples.Western blot results showed that the expression of RPL11 was significantly reduced after NDV infection,and it was positively correlated with virus infection time and dose.The results of confocal laser scanning showed that the fluorescence signals of RPL11 both in nucleus and cytoplasm were significantly decreased after NDV infection.However,the results of fluorescence quantitative PCR showed no significant change in the transcription level of the RPL11 gene,indicating that the reduction of protein expression occurred in the post-translational modification.The recombinant plasmid p Flag-RPL11 was constructed and transfected into DF-1 cells for 24 hours and then infected with different doses of NDV(MOI 1,0.1).The expression level of exogenous RPL11 was detected by western blot.The results showed that the expression of exogenous RPL11 protein was significantly decreased after virus infection,and it was also positively correlated with virus infection dose.The ubiquitin proteasome inhibitor MG132 was added to the culture medium of DF-1 cells infected with NDV(MOI 1).The results of viral titer test showed that MG132 had no significant effect on virus proliferation,but it could significantly inhibit the degradation of RPL11 protein after NDV infection,indicating that the degradation of RPL11 depended on ubiquitin proteasome pathway.These results confirmed that NDV could induce ubiquitination degradation of RPL11 protein,and the degradation process could be inhibited by MG132.This study provides a new insight into the changes of protein translation in host cells after NDV infection.