RNA binding specificity of Hepatitis C virus core protein

1.我们发展了一种活性凝胶电泳分析法（ERA），将RNA结合蛋白固定在凝胶中，使其在电泳过程中捕获RNA。用谷胱甘肽S-转移酶（GST）截短的HCV核心融合蛋白进行ERA分析，发现其RNA结合区位于4 - 18和46-80位氨基酸，分别命名为RBRI和RBR II。RBR I与其他RNA/DNA结合蛋白（如核糖体蛋白S6）具有同源性。我们提出了一个共有序列PKPXRK（S/T）（R/K）R作为RNA/DNA结合基序.在此结果的基础上，将1至90个氨基酸残基之间的HCV核心蛋白用于随后的电泳凝胶迁移率变动测定（EMSA）。核心蛋白与双链RNA（ds RNA）的亲和力较高，与大多数单链RNA（ss RNA）的亲和力较低.在体外实验中，核心蛋白通过隔离的dsRNA强烈抑制干扰素诱导的dsRNA活化蛋白激酶（PKR）的激活.用竞争性EMSA法测定核心蛋白与HCV RNA区域的亲和力<32>。在5 '-NCR中，环3a（193-209 nt）和环3d（288-321 nt）的正义（+）RNA链与dsRNA的竞争性较强，而其它片段则相反。在3 '-NCR中，核心蛋白对3' X尾中的环1和3的正义链和反义链的亲和力均高于包括环2的其余区域。当环1或环3与环2连接时，正义链环1-2或环2-3与dsRNA的竞争性强于反义RNA，表明环2在增强正义链结合中起重要作用。我们认为，核心、5 '-NCR和3'-NCR的复合物在引发腺苷酸化中起重要作用。

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.