Molecular dissection of the multifunctional 2b protein of Cucumber mosaic virus

为了研究黄瓜花叶病毒（CMV）2b蛋白在病毒致病中的作用，我们将CMV 2b基因进行了突变，并利用马铃薯X病毒载体和CMV 2b缺失RNA 2进行了表达，分析了突变体的毒力。在假定的磷酸化位点（在位置40处的塞纳）及其邻近位置的突变对2b蛋白的毒力具有强烈的影响。特别是39位氨基酸对2b的毒力有影响。当氨基酸由赖氨酸变为谷氨酸、由碱性氨基酸变为酸性氨基酸时，2b蛋白的毒力大大降低。通过将绿色荧光蛋白基因与突变的2b蛋白基因共渗入本氏烟草（Nicotianabenthamiana）叶片中，分析了突变的2b蛋白对RNA沉默的抑制作用。在这些实验中，39K到39E的突变大大降低了2b蛋白的RNA沉默抑制剂能力。综上所述，2b蛋白的毒力似乎与CMV 2b蛋白的RNA沉默抑制剂能力相一致。CMV 2b蛋白的毒力、RNA沉默抑制能力和核质穿梭之间的关系有待进一步研究，但本研究为全面了解2b蛋白在CMV致病中的功能提供了一定的基础。

To investigate the function(s) of the 2b protein of Cucumber mosaic virus (CMV) in the viral pathogenesis, we have made various mutant forms of the 2b gene and analyzed their virulence by expressing using Potato virus X vector or the 2b-deleted RNA2 of CMV. Mutations in the putative phosphorylation site (seine at a position of 40) and its neighboring positions have strong effects on the virulence of the 2b protein. Especially, the amino acid position at 39 affected the virulence of 2b. When the amino acid at the position was changed from lysine to glutaxnic acid, bacic amino acid to acidic amino acid, the virulence of the 2b protein was greatly reduced. By co-agroinfiltration of the green fluorescent protein gene with the mutated 2b protein genes into the leaves of Nicotiana benthamiana, we have analyzed RNA silencing suppression ability of the mutated 2b protein. In these experiments, the mutation of 39K to 39E greatly reduced RNA silencing suppressor capability of the 2b protein. Taken together, it seems probable that the virulence of the 2b protein coincide with the RNA silencing suppressor capability of the 2b protein of CMV. Further studies will be required to figure out the relationship among virulence, RNA silencing suppressor ability and nuclear-cytoplasm shuttle of the 2b protein of CMV ; however, this research provided some steps toward the complete understandings of the function(s) of the 2b protein of CMV pathogenesis.