Interactions and Nuclear Import of the N and P Proteins of Sonchus Yellow Net Virus, a Plant Nucleorhabdovirus

We have characterized the interaction and nuclear localization of the nucleocapsid (N) protein and phosphoprotein (P) of sonchus yellow net nucleorhabdovirus. Expression studies with plant and yeast cells revealed that both N and P are capable of independent nuclear import. Site-specific mutagenesis and deletion analyses demonstrated that N contains a carboxy-terminal bipartite nuclear localization signal (NLS) located between amino acids 465 and 481 and that P contains a karyophillic region between amino acids 40 and 124. The N NLS was fully capable of functioning outside of the context of the N protein and was able to direct the nuclear import of a synthetic protein fusion consisting of green fluorescent protein fused to glutathione S-transferase (GST). Expression and mapping studies suggested that the karyophillic domain in P is located within the N-binding domain. Coexpression of N and P drastically affected their localization patterns relative to those of individually expressed proteins and resulted in a shift of both proteins to a subnuclear region. Yeast two-hybrid and GST pulldown experiments verified the N-P and P-P interactions, and deletion analyses have identified the N and P interacting domains. N NLS mutants were not transported to the nucleus by import-competent P, presumably because N binding masks the P NLS. Taken together, our results support a model for independent entry of N and P into the nucleus followed by associations that mediate subnuclear localization.

我们对苦苣菜黄网核弹状病毒（sonchus yellow net nucleorhabdovirus）的核衣壳（nucleocapsid, N）蛋白与磷蛋白（phosphoprotein, P）的相互作用及核定位特征进行了系统性表征。通过植物与酵母细胞的表达实验证实，N蛋白与P蛋白均可独立完成核输入过程。定点诱变与缺失分析结果显示，N蛋白含有一段位于氨基酸残基465至481位的羧基端双向核定位信号（nuclear localization signal, NLS），而P蛋白则存在一段位于氨基酸残基40至124位的亲核区域。该N蛋白核定位信号可脱离N蛋白的原有序列上下文独立发挥功能，能够引导由谷胱甘肽S-转移酶（glutathione S-transferase, GST）与绿色荧光蛋白融合形成的人工重组蛋白完成核输入。表达与定位图谱分析实验提示，P蛋白的亲核结构域位于其N蛋白结合结构域范围内。与单独表达时的定位模式相比，N与P蛋白共表达会显著改变二者的定位特征，并使两种蛋白均转移至核亚区域。酵母双杂交与GST下拉实验验证了N-P及P-P蛋白间的相互作用，缺失分析进一步明确了二者的相互作用结构域。携带突变的N蛋白核定位信号无法借助具备核输入能力的P蛋白被转运至细胞核，推测其原因为N蛋白与P蛋白的结合掩盖了P蛋白的核定位信号。综上，本研究结果支持如下模型：N蛋白与P蛋白可独立进入细胞核，随后通过蛋白相互作用介导二者的核亚定位。