Walter Brandon L, Parsley Todd B, Ehrenfeld Ellie, Semler Bert L
Department of Microbiology and Molecular Genetics, College of Medicine, University of California, Irvine, California 92697, USA.
J Virol. 2002 Dec;76(23):12008-22. doi: 10.1128/jvi.76.23.12008-12022.2002.
The limited coding capacity of picornavirus genomic RNAs necessitates utilization of host cell factors in the completion of an infectious cycle. One host protein that plays a role in both translation initiation and viral RNA synthesis is poly(rC) binding protein 2 (PCBP2). For picornavirus RNAs containing type I internal ribosome entry site (IRES) elements, PCBP2 binds the major stem-loop structure (stem-loop IV) in the IRES and is essential for translation initiation. Additionally, the binding of PCBP2 to the 5'-terminal stem-loop structure (stem-loop I or cloverleaf) in concert with viral protein 3CD is required for initiation of RNA synthesis directed by poliovirus replication complexes. PCBP1, a highly homologous isoform of PCBP2, binds to poliovirus stem-loop I with an affinity similar to that of PCBP2; however, PCBP1 has reduced affinity for stem-loop IV. Using a dicistronic poliovirus RNA, we were able to functionally uncouple translation and RNA replication in PCBP-depleted extracts. Our results demonstrate that PCBP1 rescues RNA replication but is not able to rescue translation initiation. We have also generated mutated versions of PCBP2 containing site-directed lesions in each of the three RNA-binding domains. Specific defects in RNA binding to either stem-loop I and/or stem-loop IV suggest that these domains may have differential functions in translation and RNA replication. These predictions were confirmed in functional assays that allow separation of RNA replication activities from translation. Our data have implications for differential picornavirus template utilization during viral translation and RNA replication and suggest that specific PCBP2 domains may have distinct roles in these activities.
微小核糖核酸病毒基因组RNA有限的编码能力使得在感染周期的完成过程中必须利用宿主细胞因子。一种在翻译起始和病毒RNA合成中都起作用的宿主蛋白是聚（rC）结合蛋白2（PCBP2）。对于含有I型内部核糖体进入位点（IRES）元件的微小核糖核酸病毒RNA，PCBP2结合IRES中的主要茎环结构（茎环IV），对翻译起始至关重要。此外，PCBP2与病毒蛋白3CD协同结合到5'-末端茎环结构（茎环I或苜蓿叶形结构）对于脊髓灰质炎病毒复制复合物指导的RNA合成起始是必需的。PCBP1是PCBP2的高度同源异构体，以与PCBP2相似的亲和力结合脊髓灰质炎病毒茎环I；然而，PCBP1对茎环IV的亲和力降低。使用双顺反子脊髓灰质炎病毒RNA，我们能够在PCBP缺失的提取物中在功能上解偶联翻译和RNA复制。我们的结果表明，PCBP1拯救RNA复制，但不能拯救翻译起始。我们还生成了PCBP2的突变版本，在三个RNA结合结构域中的每一个中都含有定点损伤。与茎环I和/或茎环IV的RNA结合的特定缺陷表明这些结构域在翻译和RNA复制中可能具有不同的功能。这些预测在允许将RNA复制活性与翻译分离的功能测定中得到证实。我们的数据对病毒翻译和RNA复制过程中微小核糖核酸病毒模板的差异利用具有启示意义，并表明特定的PCBP2结构域在这些活动中可能具有不同的作用。
