Novel Phosphoprotein-Interacting Region in Nipah Virus Nucleocapsid Protein and Its Involvement in Viral Replication
Novel Phosphoprotein-Interacting Region in Nipah Virus Nucleocapsid Protein and Its Involvement in Viral Replication
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DOI：
10.1128/jvi.00339-10
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发表时间：
2010-10-01
影响因子：
5.4
通讯作者：
Kai, Chieko
中科院分区：
文献类型：
--
作者：
Omi-Furutani, Mio;Yoneda, Misako;Kai, Chieko
尼帕病毒（Nipah virus，NiV）的核衣壳（N）蛋白与磷蛋白（P）在核衣壳组装过程中的相互作用是病毒生命周期中必不可少的过程，因为只有核衣壳化的RNA基因组才能用于复制。为了确定负责N-P相互作用的区域，我们利用荧光蛋白标签来可视化活细胞中的NiV N和P蛋白，并分析它们的细胞定位。N蛋白与单体增强型青色荧光蛋白（N-ECFP）融合后呈点状分布，P蛋白与单体红色荧光蛋白（P-mRFP）融合后呈弥散分布。当两种蛋白共表达时，P-mRFP与N-ECFP点共定位。产生具有连续氨基酸缺失的N-ECFP突变体以寻找负责该N-P共定位的区域。我们发现，除了先前报道的467- 496-氨基酸（aa）区域外，aa 135 - 146负责N-P共定位。通过在未标记的N蛋白中引入丙氨酸取代，进一步研究了N-P相互作用的关键残基。对氨基酸135 - 146区域的丙氨酸扫描表明，存在对N-P相互作用和N的功能至关重要的不同区域。这是第一项研究，以可视化尼帕病毒蛋白在活细胞中，并评估与P蛋白相互作用的N蛋白的基本结构域。
The interaction of Nipah virus (NiV) nucleocapsid (N) protein with phosphoprotein (P) during nucleocapsid assembly is the essential process in the viral life cycle, since only the encapsidated RNA genome can be used for replication. To identify the region responsible for N-P interaction, we utilized fluorescent protein tags to visualize NiV N and P proteins in live cells and analyzed their cellular localization. N protein fused to monomeric enhanced cyan fluorescence protein (N-ECFP) exhibited a dotted pattern in transfected cells, while P protein fused to monomeric red fluorescent protein (P-mRFP) showed diffuse distribution. When the two proteins were coexpressed, P-mRFP colocalized with N-ECFP dots. N-ECFP mutants with serial amino acid deletions were generated to search for the region(s) responsible for this N-P colocalization. We found that, in addition to the 467- to 496-amino-acid (aa) region reported previously, aa 135 to 146 were responsible for the N-P colocalization. The residues crucial for N-P interaction were further investigated by introducing alanine substitutions into the untagged N protein. Alanine scanning in the region of aa 135 to 146 has revealed that there are distinct regions essential for the interaction of N-P and the function of N. This is the first study to visualize Nipah viral proteins in live cells and to assess the essential domain of N protein for the interaction with P protein.