Sean Polen, Nguyen Joseph H C, Semler Bert L
Department of Microbiology and Molecular Genetics, School of Medicine, University of California, Irvine, CA 92697, USA.
Virology. 2008 Sep 1;378(2):243-53. doi: 10.1016/j.virol.2008.05.007. Epub 2008 Jul 25.
Poliovirus, a member of the enterovirus genus in the family Picornaviridae, is the causative agent of poliomyelitis. Translation of the viral genome is mediated through an internal ribosomal entry site (IRES) encoded within the 5' noncoding region (5' NCR). IRES elements are highly structured RNA sequences that facilitate the recruitment of ribosomes for translation. Previous studies have shown that binding of a cellular protein, poly(rC) binding protein 2 (PCBP2), to a major stem-loop structure in the genomic 5' NCR is necessary for the translation of picornaviruses containing type I IRES elements, including poliovirus, coxsackievirus, and human rhinovirus. PCBP1, an isoform that shares approximately 90% amino acid identity to PCBP2, cannot efficiently stimulate poliovirus IRES-mediated translation, most likely due to its reduced binding affinity to stem-loop IV within the poliovirus IRES. The primary differences between PCBP1 and PCBP2 are found in the so-called linker domain between the second and third K-homology (KH) domains of these proteins. We hypothesize that the linker region of PCBP2 augments binding to poliovirus stem-loop IV RNA. To test this hypothesis, we generated six PCBP1/PCBP2 chimeric proteins. The recombinant PCBP1/PCBP2 chimeric proteins were able to interact with poliovirus stem-loop I RNA and participate in protein-protein interactions. We demonstrated that the PCBP1/PCBP2 chimeric proteins with the PCBP2 linker, but not with the PCBP1 linker, were able to interact with poliovirus stem-loop IV RNA, and could subsequently stimulate poliovirus IRES-mediated translation. In addition, using a monoclonal anti-PCBP2 antibody (directed against the PCBP2 linker domain) in mobility shift assays, we showed that the PCBP2 linker domain modulates binding to poliovirus stem-loop IV RNA via a mechanism that is not inhibited by the antibody.
脊髓灰质炎病毒是小核糖核酸病毒科肠道病毒属的成员，是脊髓灰质炎的病原体。病毒基因组的翻译是通过5'非编码区（5'NCR）内编码的内部核糖体进入位点（IRES）介导的。IRES元件是高度结构化的RNA序列，有助于募集核糖体进行翻译。先前的研究表明，细胞蛋白多聚（rC）结合蛋白2（PCBP2）与基因组5'NCR中的一个主要茎环结构结合，对于含有I型IRES元件的小核糖核酸病毒（包括脊髓灰质炎病毒、柯萨奇病毒和人鼻病毒）的翻译是必需的。PCBP1是一种与PCBP2具有约90%氨基酸同一性的异构体，不能有效刺激脊髓灰质炎病毒IRES介导的翻译，最可能的原因是其与脊髓灰质炎病毒IRES内茎环IV的结合亲和力降低。PCBP1和PCBP2之间的主要差异存在于这些蛋白质的第二个和第三个K-同源（KH）结构域之间的所谓连接域中。我们假设PCBP2的连接区域增强了与脊髓灰质炎病毒茎环IV RNA的结合。为了验证这一假设，我们生成了六种PCBP1/PCBP2嵌合蛋白。重组PCBP1/PCBP2嵌合蛋白能够与脊髓灰质炎病毒茎环I RNA相互作用并参与蛋白质-蛋白质相互作用。我们证明，具有PCBP2连接子而非PCBP1连接子的PCBP1/PCBP2嵌合蛋白能够与脊髓灰质炎病毒茎环IV RNA相互作用，并随后刺激脊髓灰质炎病毒IRES介导的翻译。此外，在迁移率变动分析中使用单克隆抗PCBP2抗体（针对PCBP2连接域），我们表明PCBP2连接域通过一种不受该抗体抑制的机制调节与脊髓灰质炎病毒茎环IV RNA的结合。
