RNA binding specificity of Hepatitis C virus core protein

1.我们建立了一种活性凝胶方法--电泳法(ERA)，利用凝胶中固定的RNA-bindg蛋白在凝胶中捕获RNA。与谷胱甘肽S转移酶截短的丙型肝炎病毒核心融合蛋白的EREA结果显示，核糖核酸结合区位于4-18和46-80位氨基酸，分别命名为RbRI和RbRⅡ。Rbr I与其他RNA/DNA结合蛋白有同源性，如核糖体蛋白S6。我们提出了一个共识序列PKPXRK(S/T)(R/K)R作为RNADNA结合基序。根据这一结果，将1到90个氨基酸残基的丙型肝炎病毒核心蛋白用于随后的凝胶迁移率改变分析(EMSA)。核心蛋白与双链(Ds)RNA亲和力高，与大多数单链(Ss)RNA亲和力低。在体外，干扰素诱导的dsRNA活化蛋白激酶(PKR)的激活被核心蛋白通过隔离的dsRNA强烈抑制。用竞争EMSA与~(32)&gt；P标记的dsRNA竞争测定核心蛋白与丙型肝炎病毒核糖核酸区域的亲和力。在5‘-NCR中，3a环(193-209nt)和3d环(288-321nt)的正义(Sense)RNA链与dsRNA显示出较强的竞争。在3‘-NCR中，核心蛋白与3’X尾部环1和3的正义链和反义链的亲和力高于包括环2在内的其余区域。当环1或3与环2连接时，正义链环1-2或环2-3与dsRNA表现出强烈的竞争，这表明3‘X尾部的环2在促进正义链结合方面发挥了重要作用。我们认为核心、5‘-NCR和3’-NCR的复合体在胶囊化的起始过程中起着重要的作用。

1. We developed an activity gel method named electrophoretic assay (ERA), by which immobilized RNA-bindig protein in the gel was able to trap RNA in electrophoresis. ERA by a number of Glutathione S-transferase (GST)-truncated HCV core fusion proteins revealed that RNA binding regions were mapped at amino acids 4 -18 and 46-80, which were named RBRI, and RBR II, respectively. RBR I shared homologies with other RNA/DNA binding proteins, such as ribosomal protein S6. We proposed a consensus sequence PKPXRK(S/T) (R/K)R as a RNA/DNA binding motif.2. On the basis of this result, HCV core protein between 1 and 90 amino acid residues was used for a subsequent electrophoretic gel mobility shift assay (EMSA). The core protein showed a high affinity to double-stranded (ds) RNA irrespective of its sequence and low affinity to most single-stranded (ss) RNA.3. Activation of interferon inducible dsRNA activated protein kinase (PKR) was strongly inhibited by core protein by sequestered dsRNA, in vitro.4. The affinities of the core protein to HCV RNA region was determined by competitive EMSA with ^<32>P-labeled dsRNA. In 5'-NCR, sense (+) RNA strands of loops 3a (193-209 nt) and 3d (288-321 nt) showed strong competition with dsRNA in contrast other fragments. In 3'-NCR, core protein showed higher affinity to both sense and antisense strands of loops 1 and 3 in 3'X tail than the rest regions including loop 2. When loops 1 or 3 was joined to loop 2, sense strand loop 1-2 or loop 2-3 showed strong competition with dsRNA in contrast to their antisense RNAs, indicating that the loop 2 in 3'X tail plays an important role in enhancement of sense strand-binding.5. We propose that the complex of core, 5'-NCR and 3'-NCR plays an important role in initiation of encapsidation.