RNA binding specificity of Hepatitis C virus core protein

1. 我们开发了一种称为电泳法（ERA）的活性凝胶方法，通过凝胶中固定的RNA结合蛋白能够在电泳中捕获RNA。通过一些GST截断的HCV核心融合蛋白进行ERA分析发现，RNA结合区定位在4 -18和46-80氨基酸上，分别被命名为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。我们提出了一个共识序列PKPXRK(S/T) (R/K)R作为RNA/DNA结合基序。在此结果的基础上，HCV核心蛋白在1到90个氨基酸残基之间用于随后的电泳凝胶迁移量转移试验（EMSA）。核心蛋白对双链RNA （ds）具有高亲和力，而对大多数单链RNA （ss）具有低亲和力。干扰素诱导的dsRNA活化蛋白激酶（PKR）的激活在体外被分离的dsRNA核心蛋白强烈抑制。核心蛋白与HCV RNA区域的亲和力通过竞争EMSA与^<32> p标记的dsRNA进行测定。在5'-NCR中，环状3a （193-209 nt）和3d （288-321 nt）的sense (+) RNA链与其他片段相比表现出强烈的竞争。在3’-NCR中，核心蛋白对3’x尾部环1和环3的正义链和反义链的亲和力均高于环2等其他区域。当环1或环3与环2连接时，感觉链环1-2或环2-3与其反义rna相比，与dsRNA表现出强烈的竞争，这表明3' x尾部的环2在增强感觉链结合中起着重要作用。我们认为，核、5′-NCR和3′-NCR复合物在包衣起始过程中起重要作用。

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.