Mapping of interacting domains between the nucleocapsid protein and the phosphoprotein of vesicular stomatitis virus by using a two-hybrid system.

Specific interaction between the nucleocapsid protein (N) and the phosphoprotein (P) of vesicular stomatitis virus (VSV), an important step in the life-cycle of the virus, was studied by using a two-hybrid system. Plasmids encoding P fused with the yeast GAL4 DNA-binding domain (pGALP) and N fused with the herpes simplex virus VP16 transactivating region (pVPN) were transfected into CHO cells along with a reporter plasmid encoding chloramphenicol acetyltransferase (CAT). The ability of N and P to associate in vivo was measured by activation of the CAT gene by the VP16 transactivating region. Transfection of plasmids pGALP and pVPN resulted in a high level of CAT activity, indicating that the N and P portions of the fusion proteins associated very strongly with each other. Progressive C-terminal deletions of the P protein revealed two regions that are important for association with the N protein: the N-terminal acidic domain and the C-terminal basic domain. Phosphorylation of P protein was not required for N-P association. Various deletions and mutations of the N protein revealed the C-terminal 5 amino acids (Val-Glu-Phe-Asp-Lys), in particular the amino acids Val-Glu-Phe, to be critical for N association with P. This two-hybrid system can be used in other viral systems to study the interaction between proteins involved in transcription and replication. IMAGES:

本研究借助酵母双杂交系统（two-hybrid system），对水疱性口炎病毒（vesicular stomatitis virus, VSV）核衣壳蛋白（nucleocapsid protein, N）与磷蛋白（phosphoprotein, P）之间的特异性相互作用展开探究——该相互作用是病毒生命周期中的关键步骤。将编码与酵母GAL4 DNA结合结构域（yeast GAL4 DNA-binding domain）融合的P蛋白的质粒（pGALP），以及编码与单纯疱疹病毒VP16反式激活区域（herpes simplex virus VP16 transactivating region）融合的N蛋白的质粒（pVPN），与编码氯霉素乙酰转移酶（chloramphenicol acetyltransferase, CAT）的报告质粒共同转染至中国仓鼠卵巢细胞（CHO cells）中。通过VP16反式激活区域对CAT基因的激活水平，定量检测N与P在体内的结合能力。共转染pGALP与pVPN质粒可产生高水平的CAT活性，表明融合蛋白中的N与P结构域之间存在极强的相互作用。对P蛋白进行渐进式C端截短分析后，鉴定出两个与N蛋白结合必需的区域：N端酸性结构域与C端碱性结构域。P蛋白的磷酸化并非N-P相互作用的必要条件。对N蛋白开展各类截短与突变分析后发现，其C端5个氨基酸残基（缬氨酸-谷氨酸-苯丙氨酸-天冬氨酸-赖氨酸，Val-Glu-Phe-Asp-Lys），尤其是缬氨酸-谷氨酸-苯丙氨酸（Val-Glu-Phe）这三个残基，对N蛋白与P蛋白的结合至关重要。本研究所建立的酵母双杂交系统可应用于其他病毒系统，用于研究参与转录与复制过程的蛋白质之间的相互作用。IMAGES: