翻译后修饰可能影响黄瓜花叶病毒2b 蛋白抑制子活性及稳定性

Implication of post-translational modifications in suppressor activity and stability of the Cucumber mosaic virus 2b protein

摘要:

黄瓜花叶病毒 (Cucumber mosaic virus，CMV) 编码的2b 蛋白具有RNA 沉默抑制子功能，为了研究翻译后修饰对2b 功能的影响，利用反向PCR 定点突变方法对CMV-Q 株系2b 蛋白的1 个预测的磷酸化位点 (S40) 和2 个预测的泛素化/SUMO 化位点 (K22，K39) 进行了点突变，同时将点突变体插入植物表达载体。通过农杆菌共注射法对3 个2b 突变体的抑制子活性进行了分析，结果证明，当S40 突变为A (2b^S40A) 后，2b

关键词:黄瓜花叶病毒; 2b 蛋白; 翻译后修饰; 抑制子活性; 蛋白稳定性

Abstract:

To gain insights into the function of potential post-translational modifications on the activity of the Cucumber mosaic virus (CMV)-encoded silencing suppressor protein 2b, one predicted phosphorylation site (S40) and two predicted ubiquitination/sumoylation sites (K22 and K39) in CMV-Q2b protein were individually or simultaneously mutated by site-directed mutagenesis methods. These Q2b mutants were inserted into plant expression vectors, expressed in plant leaves, and then analyzed for their silencing suppressor activities. The results showed that S40A mutation greatly impaired both the local and systemic silencing suppressor activity, and the K22R mutation has no significant effect on the suppressor activity, while the K22R/K39R double mutation reduced the systemic silencing suppressor activity. To test if the decrease of suppressor activity were due to protein accumulation changes, western blot were performed to moniter the protein level of Q2b mutants. The results indicated that mutations of both K22 and K39 to R or S40 to A all significantly reduced the accumulation of the Q2b protein in plants, while the single mutation of K22 to R did not alter the accumulation of Q2b protein, suggesting that two potential post-translational modification sites, K39 and S40, contribute to the suppressor activity and stability of 2b protein in plant cells.

Key words:Cucumber mosaic virus; 2b protein; post-translational modifications; suppressor activity; protein stability