Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes
Functional interaction of heterogeneous nuclear ribonucleoprotein C with poliovirus RNA synthesis initiation complexes
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DOI：
10.1128/jvi.79.6.3254-3266.2005
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发表时间：
2005-03-01
影响因子：
5.4
通讯作者：
Semler, BL
中科院分区：
文献类型：
--
作者：
Brunner, JE;Nguyen, JHC;Semler, BL
我们先前已经证明，细胞蛋白质特异性地与脊髓灰质炎病毒负链RNA的3'端相互作用。我们现在报告的身份，这种蛋白质作为异质核核糖核蛋白（hnRNP）C1/C2。与脊髓灰质炎病毒RNA的RNP复合物的形成严重受损的赖氨酸，高度保守的脊椎动物，与重组hnRNP C1的RNA识别基序（RRM）中的谷氨酰胺的取代，这表明该结合是由蛋白质中的RRM介导的。我们还表明，在谷胱甘肽S-转移酶（GST）下拉试验中，GST/hnRNP C1与脊髓灰质炎病毒多肽3CD（病毒RNA依赖性RNA聚合酶3D（pol）的前体）以及P2和P3（非结构蛋白的前体）结合。在hnRNP C1（C1 DeltaC）的辅助结构域的截断减少了这些蛋白质-蛋白质的相互作用。当GST/hnRNP C1 DeltaC加入到体外复制反应中时，观察到RNA合成的显着减少，与补充野生型融合蛋白的反应相反。使用脊髓灰质炎病毒负链三叶草RNA，从体外复制反应中间接功能性耗尽hnRNP C，导致RNA合成减少。GST/hnRNP C1的加入反应拯救RNA合成到接近模拟耗尽的水平。此外，我们证明了脊髓灰质炎病毒的正链和负链RNA存在于从感染的HeLa细胞制备的细胞质提取物中，与hnRNP C1/C2共免疫沉淀。我们的研究结果表明，hnRNP C1在脊髓灰质炎病毒感染细胞的正链RNA合成中发挥作用，可能在起始水平。
We had previously demonstrated that a cellular protein specifically interacts with the 3' end of poliovirus negative-strand RNA. We now report the identity of this protein as heterogeneous nuclear ribonucleoprotein (hnRNP) C1/C2. Formation of an RNP complex with poliovirus RNA was severely impaired by substitution of a lysine, highly conserved among vertebrates, with glutamine in the RNA recognition motif (RRM) of recombinant hnRNP C1, suggesting that the binding is mediated by the RRM in the protein. We have also shown that in a glutathione S-transferase (GST) pull-down assay, GST/hnRNP C1 binds to poliovirus polypeptide 3CD, a precursor to the viral RNA-dependent RNA polymerase, 3D(pol), as well as to P2 and P3, precursors to the nonstructural proteins. Truncation of the auxiliary domain in hnRNP C1 (C1DeltaC) diminished these protein-protein interactions. When GST/hnRNP C1DeltaC was added to in vitro replication reactions, a significant reduction in RNA synthesis was observed in contrast to reactions supplemented with wild-type fusion protein. Indirect functional depletion of hnRNP C from in vitro replication reactions, using poliovirus negative-strand cloverleaf RNA, led to a decrease in RNA synthesis. The addition of GST/hnRNP C1 to the reactions rescued RNA synthesis to near mock-depleted levels. Furthermore, we demonstrated that poliovirus positive-strand and negative-strand RNA present in cytoplasmic extracts prepared from infected HeLa cells coimmunoprecipitated with hnRNP C1/C2. Our findings suggest that hnRNP C1 has a role in positive-strand RNA synthesis in poliovirus-infected cells, possibly at the level of initiation.