宿主蛋白CCT5与狂犬病病毒M蛋白互作鉴定及其对病毒复制的影响

Identification of interaction between the host protein CCT5 and rabies virus M protein and the effects on viral replication

  • 摘要: 【目的】 明确宿主蛋白CCT5与狂犬病病毒M蛋白的互作关系及过表达CCT5基因对RABV复制的影响,为揭示CCT5蛋白在RABV复制过程中的作用机制提供理论依据。【方法】 采用实时荧光定量PCR和Western blotting分别检测RABV感染BHK-21细胞和N2a细胞后CCT5、M、N、P和G基因及其编码蛋白的相对表达量。PCR扩增CCT5基因并克隆至载体pcDNA3.0上构建真核表达载体,转染HEK-293T细胞后检测CCT5蛋白表达情况并筛选最佳转染时间。通过GST-pull down、Co-IP和细胞共定位试验验证CCT5蛋白与RABV M蛋白的互作关系,并探究CCT5基因过表达对RABV复制的影响。【结果】 RABV感染BHK-21细胞后,N、P、M和G基因相对表达量整体上有所升高,而CCT5基因相对表达量变化不明显。RABV感染N2a细胞后,CCT5蛋白相对表达量变化不明显。以真核表达载体pcDNA3.0-CCT5-Flag转染HEK-293T细胞,在转染36 h的CCT5蛋白相对表达量最高。GST pull-down验证结果显示,CCT5蛋白与RABV M蛋白在体外存在互作关系。Co-IP验证结果显示,CCT5蛋白与RABV M蛋白在细胞内存在互作关系。细胞内共定位验证结果显示,CCT5蛋白与RABV M蛋白在细胞质内存在广泛的共定位。CCT5基因过表达能从转录、翻译及子代病毒产生等多个层面促进RABV的复制。【结论】 宿主分子伴侣蛋白CCT5与RABV M蛋白在体内外均存在互作关系,且二者共定位于细胞质。RABV在不同细胞系中并不调控CCT5基因及其蛋白的表达,但过表达CCT5基因可促进RABV基因的转录和翻译水平及子代病毒产生,对RABV复制具有正向调控作用。

     

    Abstract: 【Objective】 This study aimed to clarify the effects of the interaction between the host protein CCT5 and rabies virus (RABV) M protein and overexpressed CCT5 gene on RABV replication, so as to provide a theoretical basis for the function and mechanism of CCT5 protein in RABV replication.【Method】 The relative expressions of CCT5, M, N, P, and G genes and their proteins after RABV infection of BHK-21 and N2a cells were detected by real-time fluorescence quantitative PCR and Western blotting, respectively. The CCT5 gene was amplified by PCR and cloned into the pcDNA3.0 vector to construct a eukaryotic expression vector. After transfection into HEK-293T cells, the expression of CCT5 protein was detected, and the optimal transfection time was selected. The interaction between CCT5 protein and RABV M protein was verified by GST-pull down, Co-IP, and cellular colocalization. The effect of CCT5 gene over-expression on RABV replication.【Result】 After RABV infection of BHK-21 cells, the relative expressions of N, P, M, and G genes generally increased, while the relative expression of CCT5 gene showed an inobvious change. RABV infection of N2a cells did not result in an obvious change in the relative expression of CCT5 protein. Transfection of HEK-293T cells with eukaryotic expression vector pcDNA3.0-CCT5-Flag resulted in the highest CCT5 relative protein expression at 36 h post-transfection. GST pull-down validation showed that CCT5 protein interacted with RABV M protein in vitro. Co-IP validation showed that CCT5 protein and RABV M protein interact intracellularly. Intracellular colocalization validation showed that CCT5 protein and RABV M protein had extensive colocalization in the cytoplasm. Overexpression of the CCT5 gene could promote RABV replication at multiple levels, including transcription, translation, and progeny virus production.【Conclusion】 The host chaperone protein CCT5 interacts with the RABV M protein both in vivo and in vitro, and the two are colocalized in the cytoplasm. RABV does not regulate the expression of the CCT5 gene and its protein in different cell lines, but the overexpressed CCT5 gene can promote the transcription and translation of the RABV gene and the production of progeny viruses, thus having a positive regulatory effect on RABV replication.

     

/

返回文章
返回