首页> 美国卫生研究院文献>Journal of Virology >Replication of Poliovirus Requires Binding of the Poly(rC) Binding Protein to the Cloverleaf as Well as to the Adjacent C-Rich Spacer Sequence between the Cloverleaf and the Internal Ribosomal Entry Site
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Replication of Poliovirus Requires Binding of the Poly(rC) Binding Protein to the Cloverleaf as Well as to the Adjacent C-Rich Spacer Sequence between the Cloverleaf and the Internal Ribosomal Entry Site

机译:脊髓灰质炎病毒的复制需要将poly(rC)结合蛋白结合到苜蓿叶苜蓿和苜蓿叶苜蓿与内部核糖体进入位点之间的邻近C-富集间隔序列

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摘要

The 5′ nontranslated region of poliovirus RNA contains two highly structured regions, the cloverleaf (CL) and the internal ribosomal entry site (IRES). A cellular protein, the poly(rC) binding protein (PCBP), has been reported to interact with the CL either alone or in combination with viral protein 3CDpro. The formation of the ternary complex is essential for RNA replication and, hence, viral proliferation. PCBP also interacts with stem-loop IV of the IRES, an event critical for the initiation of cap-independent translation. Until recently, no special function was assigned to a spacer region (nucleotides [nt] 89 to 123) located between the CL and the IRES. However, on the basis of our discovery that this region strongly affects the neurovirulent phenotype of poliovirus, we have embarked upon genetic and biochemical analyses of the spacer region, focusing on two clusters of C residues (C93-95 and C98-100) that are highly conserved among entero- and rhinoviruses. Replacement of all six C residues with A residues had no effect on translation in vitro but abolished RNA replication, leading to a lethal growth phenotype of the virus in HeLa cells. Mutation of the first group of C residues (C93-95) resulted in slower viral growth, whereas the C98-100A change had no significant effect on viability. Genetic analyses of the C-rich region by extensive mutagenesis and analyses of revertants revealed that two consecutive C residues (C94-95) were sufficient to promote normal growth of the virus. However, there was a distinct position effect of the preferred C residues. A 142-nt-long 5′-terminal RNA fragment including the CL and spacer sequences efficiently bound PCBP, whereas no PCBP binding was observed with the CL (nt 1 to 88) alone. Binding of PCBP to the 142-nt fragment was completely ablated after the two C clusters in the spacer were mutated to A clusters. In contrast, the same mutations had no effect on the binding of 3CDpro to the 142-nt RNA fragment. Stepwise replacement of the C residues with A residues resulted in impaired replication that covaried with weaker binding of PCBP in vitro. We conclude that PCBP has little, if any, binding affinity for the CL itself (nt 1 to 88) but requires additional nucleotides downstream of the CL for its function as an essential cofactor in poliovirus RNA replication. These data reveal a new essential function of the spacer between the CL and the IRES in poliovirus proliferation.
机译:脊髓灰质炎病毒RNA的5'非翻译区包含两个高度结构化的区域,苜蓿叶(CL)和内部核糖体进入位点(IRES)。据报道,一种细胞蛋白即poly(rC)结合蛋白(PCBP)可以单独或与病毒蛋白3CD pro 组合与CL相互作用。三元复合物的形成对于RNA复制以及病毒增殖至关重要。 PCBP还与IRES的茎环IV相互作用,这对启动不依赖帽的翻译至关重要。直到最近,还没有为位于CL和IRES之间的间隔区(核苷酸[nt] 89至123)分配特殊功能。但是,基于我们发现该区域强烈影响脊髓灰质炎病毒神经毒性表型的发现,我们着手对间隔区进行了遗传和生化分析,重点研究了两个C残基簇(C93-95和C98-100)。在肠道病毒和鼻病毒中高度保守。用A残基替换所有六个C残基对体外翻译没有影响,但废除了RNA复制,导致HeLa细胞中病毒的致死生长表型。第一组C残基(C93-95)的突变导致病毒生长较慢,而C98-100A的变化对生存力没有明显影响。通过广泛的诱变和回复株分析对富C区进行遗传分析,发现两个连续的C残基(C94-95)足以促进病毒的正常生长。但是,优选的C残基具有明显的位置效应。包含CL和间隔序列的142nt长的5'-末端RNA片段有效结合PCBP,而单独使用CL(nt 1至88)未观察到PCBP结合。间隔物中的两个C簇突变为A簇后,PCBP与142-nt片段的结合被完全消除。相反,相同的突变对3CD pro 与142-nt RNA片段的结合没有影响。用A残基逐步替换C残基会导致复制受损,这与体外PCBP的结合较弱有关。我们得出的结论是,PCBP对CL本身(核苷酸1至88)的结合亲和力很小(如果有的话),但在CL下游需要附加的核苷酸,因为它作为脊髓灰质炎病毒RNA复制中的必需辅助因子起作用。这些数据揭示了CL和IRES之间的间隔子在脊髓灰质炎病毒增殖中的新的重要功能。

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