Laboratory of Virology and Immunology, GIGA-R, University of Liege (ULg), Liège, Belgium.
PLoS One. 2009 Nov 18;4(11):e7882. doi: 10.1371/journal.pone.0007882.
Available data suggest that the Varicella-Zoster virus (VZV) IE4 protein acts as an important regulator on VZV and cellular genes expression and could exert its functions at post-transcriptional level. However, the molecular mechanisms supported by this protein are not yet fully characterized. In the present study, we have attempted to clarify this IE4-mediated gene regulation and identify some cellular partners of IE4. By yeast two-hybrid and immunoprecipitation analysis, we showed that IE4 interacts with three shuttling SR proteins, namely ASF/SF2, 9G8 and SRp20. We positioned the binding domain in the IE4 RbRc region and we showed that these interactions are not bridged by RNA. We demonstrated also that IE4 strongly interacts with the main SR protein kinase, SRPK1, and is phosphorylated in in vitro kinase assay on residue Ser-136 contained in the Rb domain. By Northwestern analysis, we showed that IE4 is able to bind RNA through its arginine-rich region and in immunoprecipitation experiments the presence of RNA stabilizes complexes containing IE4 and the cellular export factors TAP/NXF1 and Aly/REF since the interactions are RNase-sensitive. Finally, we determined that IE4 influences the export of reporter mRNAs and clearly showed, by TAP/NXF1 knockdown, that VZV infection requires the TAP/NXF1 export pathway to express some viral transcripts. We thus highlighted a new example of viral mRNA export factor and proposed a model of IE4-mediated viral mRNAs export.
现有数据表明，水痘带状疱疹病毒（VZV）IE4 蛋白是 VZV 和细胞基因表达的重要调节剂，可在转录后水平发挥作用。然而，该蛋白支持的分子机制尚未完全阐明。在本研究中，我们试图阐明 IE4 介导的基因调控，并鉴定一些 IE4 的细胞伴侣。通过酵母双杂交和免疫沉淀分析，我们表明 IE4 与三种穿梭 SR 蛋白 ASF/SF2、9G8 和 SRp20 相互作用。我们将结合域定位在 IE4 RbRc 区域，并表明这些相互作用不是由 RNA 桥接的。我们还证明 IE4 与主要的 SR 蛋白激酶 SRPK1 强烈相互作用，并在 Rb 结构域内包含的残基 Ser-136 上的体外激酶测定中被磷酸化。通过 Northwestern 分析，我们表明 IE4 能够通过其富含精氨酸的区域结合 RNA，并且在免疫沉淀实验中，RNA 的存在稳定包含 IE4 和细胞输出因子 TAP/NXF1 和 Aly/REF 的复合物，因为这些相互作用对核糖核酸酶敏感。最后，我们确定 IE4 影响报告 mRNA 的输出，并通过 TAP/NXF1 敲低清楚地表明，VZV 感染需要 TAP/NXF1 输出途径来表达一些病毒转录本。因此，我们强调了一种新的病毒 mRNA 输出因子，并提出了一种 IE4 介导的病毒 mRNAs 输出模型。
