Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA
Cell proteins bind specifically to West Nile virus minus-strand 3' stem-loop RNA
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细胞蛋白与西尼罗河病毒负链 3 茎环 RNA 特异性结合
作者：
Pei Yong Shi;Weike Li;Margo A. Brinton
西尼罗病毒（West Nile virus，WNV）基因组RNA的5 '端非编码区（NCR）前96个核苷酸被发现形成了在黄病毒中保守的茎环（stem-loop，SL）结构。互补负链3' NCR RNA被认为是正链RNA合成的启动子，形成相应的预测SL结构。RNase探针检测证实了WNV 3'负链茎环RNA [WNV（-）3' SL RNA]的末端二级结构的存在。用BHK S100细胞质提取物和体外合成的WNV（-）3' SL RNA作为探针进行RNA-蛋白结合研究。通过凝胶迁移率变动试验检测了三种RNA-蛋白质复合物（复合物1、2和3），并通过凝胶迁移率变动和紫外诱导交联竞争试验证实了RNA-蛋白质相互作用的特异性。通过UV诱导的与WNV（-）3' SL RNA的交联，检测到分子量为108、60、50和42 kDa的4种BHK细胞蛋白。初步定位研究表明，所有四种蛋白质都与WNV 3'负链RNA的前75个核苷酸结合，该区域包含末端SL。黄病毒抗性表型先前显示在小鼠中作为单个常染色体显性等位基因遗传。抗性细胞和敏感细胞的感染效率相似，但抗性细胞（C3 H/RV）产生的基因组RNA比同源的敏感细胞（C3 H/He）少。在C_3H/RV和C_3H/He细胞提取物中发现了三种RNA-蛋白复合物和四种UV诱导的交联细胞蛋白，其迁移率与在BHK细胞提取物中检测到的WNV（-）3' SL RNA的迁移率相同。然而，C3 H/RV复合物1的半衰期比C3 H/He复合物1的半衰期长三倍。可能的是，其中一种抗性细胞蛋白对黄病毒负链RNA的结合活性增加可能导致正链RNA合成减少，如在黄病毒抗性表型中观察到的。
The first 96 nucleotides of the 5'noncoding region (NCR) of West Nile virus (WNV) genomic RNA were previously reported to form thermodynamically predicted stem-loop (SL) structures that are conserved among flaviviruses. The complementary minus-strand 3' NCR RNA, which is thought to function as a promoter for the synthesis of plus-strand RNA, forms a corresponding predicted SL structure. RNase probing of the WNV 3' minus-strand stem-loop RNA [WNV (-)3' SL RNA] confirmed the existence of a terminal secondary structure. RNA-protein binding studies were performed with BHK S100 cytoplasmic extracts and in vitro-synthesized WNV (-)3' SL RNA as the probe. Three RNA-protein complexes (complexes 1,2, and 3) were detected by a gel mobility shift assay, and the specificity of the RNA-protein interactions was confirmed by gel mobility shift and UV-induced cross-linking competition assays. Four BHK cell proteins with molecular masses of 108, 60, 50, and 42 kDa were detected by UV-induced cross-linking to the WNV (-)3' SL RNA. A preliminary mapping study indicated that all four proteins bound to the first 75 nucleotides of the WNV 3' minus-strand RNA, the region that contains the terminal SL. A flavivirus resistance phenotype was previously shown to be inherited in mice as a single, autosomal dominant allele. The efficiencies of infection of resistant cells and susceptible cells are similar, but resistant cells (C3H/RV) produce less genomic RNA than congenic, susceptible cells (C3H/He). Three RNA-protein complexes and four UV-induced cross-linked cell proteins with mobilities identical to those detected in BHK cell extracts with the WNV (-)3' SL RNA were found in both C3H/RV and C3H/He cell extracts. However, the half-life of the C3H/RV complex 1 was three times longer than that of the C3H/He complex 1. It is possible that the increased binding activity of one of the resistant cell proteins for the flavivirus minus-strand RNA could result in a reduced synthesis of plus-strand RNA as observed with the flavivirus resistance phenotype.
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影响因子：
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