Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins
Hepatitis C Virus Nonstructural Protein 5A: Biochemical Characterization of a Novel Structural Class of RNA-Binding Proteins
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DOI：
10.1128/jvi.01319-10
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发表时间：
2010-12-01
影响因子：
5.4
通讯作者：
Cameron, Craig E.
中科院分区：
文献类型：
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作者：
Hwang, Jungwook;Huang, Luyun;Cameron, Craig E.
丙型肝炎病毒（HCV）非结构蛋白5A（NS 5A）在体外表现出对富含G/U的RNA的偏好。NS 5A RNA结合活性及其在基因组中的靶位点的生物学分析将通过NS 5A-RNA复合物的描述来促进。我们证明，尿嘧啶碱基的C-4羰基，并通过推断，鸟嘌呤碱基的C-6羰基与NS 5A相互作用。5至6个核苷酸（nt）的富含U的RNA足以与NS 5A高亲和力结合。NS 5A的最小RNA结合结构域由残基2005至2221组成（称为结构域I-plus）。该蛋白质的该区域包括氨基末端结构域I以及随后的分隔结构域I和II的接头。该接头区是适应性突变的位点。对于仅含有残基2194至2419（结构域II和III）的NS 5A衍生物，未观察到富含U的RNA结合活性。质谱分析的NS 5A-聚（rU）复合物确定域I和II的网站与RNA的相互作用。通过戊二醛交联证明NS 5A的二聚化。这种二聚化可能是由结构域I-plus介导的，因为这种蛋白质的二聚体被交联捕获。未观察到结构域II-III蛋白的二聚体。当富含U的RNA存在时，NS 5A的单体-二聚体平衡偏向于二聚体，而当富含A的RNA存在时则不然，这与NS 5A二聚体是蛋白质的RNA结合活性形式一致。这些数据提供了NS 5A-RNA复合物的分子视角，并提出了HCV和细胞基因表达调控的可能机制。
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) exhibits a preference for G/U-rich RNA in vitro. Biological analysis of the NS5A RNA-binding activity and its target sites in the genome will be facilitated by a description of the NS5A-RNA complex. We demonstrate that the C-4 carbonyl of the uracil base and, by inference, the C-6 carbonyl of the guanine base interact with NS5A. U-rich RNA of 5 to 6 nucleotides (nt) is sufficient for high-affinity binding to NS5A. The minimal RNA-binding domain of NS5A consists of residues 2005 to 2221 (referred to as domain I-plus). This region of the protein includes the amino-terminal domain I as well as the subsequent linker that separates domains I and II. This linker region is the site of adaptive mutations. U-rich RNA-binding activity is not observed for an NS5A derivative containing only residues 2194 to 2419 (domains II and III). Mass spectrometric analysis of an NS5A-poly(rU) complex identified domains I and II as sites for interaction with RNA. Dimerization of NS5A was demonstrated by glutaraldehyde cross-linking. This dimerization is likely mediated by domain I-plus, as dimers of this protein are trapped by cross-linking. Dimers of the domain II-III protein are not observed. The monomer-dimer equilibrium of NS5A shifts in favor of dimer when U-rich RNA is present but not when A-rich RNA is present, consistent with an NS5A dimer being the RNA-binding-competent form of the protein. These data provide a molecular perspective of the NS5A-RNA complex and suggest possible mechanisms for regulation of HCV and cellular gene expression.