竹花叶病毒卫星RNA编码P20蛋白的同型和异型相互作用

竹花叶病毒卫星RNA(satBaMV)是一个长度为836个核苷酸(不包括polyA)的单链正义RNA分子,可编码一20ku的卫星蛋白(P20).satBaMV的复制和包被需依赖竹花叶病毒(BaMV).P20是核酸结合蛋白,能促进satBaMV在寄主植物的长距离移动.利用细菌双杂交系统(BTH)和pull-downassays研究了P20自身、P20与BaMV蛋白以及BaMV蛋白之间的相互作用.研究表明:P20自身的相互作用是最强的;P20与甲基转移酶(MET)和衣壳蛋白(CP)之间有明显的相互作用;三基因连锁蛋白之间亦存在强的相互作用;CP与三基因连锁蛋白之间有明显的相互作用.删减分析表明,位于P20N端包括RNA结合位点在内的15个氨基酸是P20自身相互作用所必需的.N端缺失可导致P20间相互作用消失.P20的β折叠结构也是P20间相互作用所必需.此外,P20与烟草细胞色素C还原酶和β微管蛋白之间有较强的相互作用.BaMV蛋白与P20之间的同型和异型相互作用对BaMV及其卫星RNA在寄主植物中的移动起重要作用.

Homotypic and Heterotypic Interactions of SatBaMV-encoded P20 Protein

Satellite RNA ofBaMV (satBaMV), a single-stranded positive-sense RNA of 836 nucleotide [excluding 3′ poly(A) tail]containing a single open reading frame which encodes a nonstructural protein of 20 ku (P20), depends on BaMV for its replication and encapsidation. P20 is a nucleic-acid-binding protein, which facilitates the long distance movement of satBaMV in plants.Protein-protein interactions were analyzed by a bacterial two-hybrid system and pull-down assays to investigate whether P20 could self-associate and/or interact with the helper virus proteins. Self-interaction of P20 was the strongest among the viral protein-protein interactions detected in vivo and in vitro. Significant interactions of P20 with methyltransferase (MET) and capsid protein (CP) of BaMV were evidenced. Interactions among triple gene block protein 1, 2 and 3 (TGBp1, TGBp2 and TGBp3) of BaMV were also significant. BaMV CP also exhibited a strong self-interaction and strong affinity to TGBp1, TGBp2 and TGBp3. By deletion analysis,the minimal region delineated for the self-interaction of P20 was the N-terminal 15 amino acids which include the RNA binding motif of P20. N-terminal deletion resulted in the loss of self-interaction of P20. Deletion analysis also confirmed the importance of β-sheet structure of P20 in the P20-P20 interaction. P20 showed significant interactions with two host (Nicotiana benthamiana) proteins,cytochrome-C reductase and β-tubulins. The homotypic and heterotypic interactions of BaMV proteins and P20 in vivo thus suggest that the protein-protein interactions may directly exert an effect on the BaMV and satBaMV RNA movement as a protein complex in the infected host plants.