High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA
High affinity interaction between nucleocapsid protein and leader/intergenic sequence of mouse hepatitis virus RNA
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DOI：
10.1099/0022-1317-81-1-181
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发表时间：
2000-01-01
影响因子：
3.8
通讯作者：
Tahara, SM
中科院分区：
文献类型：
--
作者：
Nelson, GW;Stohlman, SA;Tahara, SM
小鼠肝炎病毒(MHV)核衣壳蛋白(N)是病毒粒子的主要结构蛋白。它既与病毒基因组RNA结合，又与亚基因组mRNAs结合，在病毒RNA依赖的RNA转录、基因组复制、包装和翻译等复制过程中具有结构和非结构作用。这些过程都涉及N蛋白和病毒RNA之间的RNA-蛋白质相互作用。为了更好地了解这种多功能蛋白的核糖核酸结合特性，我们在大肠杆菌中表达了N蛋白作为与谷胱甘肽-S-转移酶融合的蛋白。对全长和部分N蛋白片段进行RNA结合特性的生化分析，以确定RNA结合域。以MHV 5‘-前导序列为配体时，N蛋白与GST-N蛋白融合产物具有相似的结合活性，其解离常数(K-d)为14 nM，与病毒RNA结合的最小N蛋白片段为含有177-231残基的55个氨基酸片段，K-d为32 nM。从这些研究中推断出一个被N蛋白识别的一致的病毒序列；AAUCYAAAC被确定为N蛋白的潜在最小配体。虽然核心的UCYAA序列经常在病毒基因组中随机重复，但含有一个或多个UCYAA重复序列的配体在与N蛋白的结合方面没有区别。总之，这些数据表明N蛋白与MHV mRNA 5‘端的保守RNA序列之间存在高亲和力的特异性相互作用。
The nucleocapsid (N) protein of mouse hepatitis virus (MHV) is the major virion structural protein. It associates with both viral genomic RNA and subgenomic mRNAs and has structural and nonstructural roles in replication including viral RNA-dependent RNA transcription, genome replication, encapsidation and translation. These processes all involve RNA-protein interactions between the N protein and viral RNAs. To better understand the RNA-binding properties of this multifunctional protein, the N protein was expressed in Escherichia coli as a chimeric protein fused to glutathione-S-transferase (GST). Biochemical analyses of RNA-binding properties were performed on full-length and partial N protein segments to define the RNA-binding domain. The full-length N protein and the GST-N protein fusion product had similar binding activities with a dissociation constant (K-d) of 14 nM when the MHV 5'-leader sequence was used as ligand, The smallest N protein fragment which retained RNA-binding activity was a 55 aa segment containing residues 177-231 which bound viral RNA with a K-d of 32 nM. A consensus viral sequence recognized by the N protein was inferred from these studies; AAUCYAAAC was identified to be the potential minimum ligand for the N protein. Although the core UCYAA sequence is often tandemly repeated in viral genomes, ligands containing one or more repeats of UCYAA showed no difference in binding to the N protein. Together these data demonstrate a high-affinity, specific interaction between the N protein and a conserved RNA sequence present at the 5'-ends of MHV mRNA.