Mapping the Interacting Domains between the Rabies Virus Polymerase and Phosphoprotein

The RNA polymerase of rabies virus consists of two subunits, the large (L) protein and the phosphoprotein (P), with 2,127 and 297 amino acids, respectively. When these proteins were coexpressed via the vaccinia virus-T7 RNA polymerase recombinant in mammalian cells, they formed a complex as detected by coimmunoprecipitation. Analysis of P and L deletion mutants was performed to identify the regions of both proteins involved in complex formation. The interaction of P with L was not disrupted by large deletions removing the carboxy-terminal half of the P protein. On the contrary, P proteins containing a deletion in the amino terminus were defective in complex formation with L. Moreover, fusion proteins containing the 19 or the 52 first residues of P in frame with green fluorescent protein (GFP) still bound to L. These results indicate that the major L binding site resides within the 19 first residues of the P protein. We also mapped the region of L involved in the interaction with P. Mutant L proteins consisting of the carboxy-terminal 1,656, 956, 690, and 566 amino acids all bound to the P protein, whereas deletion of 789 residues within the terminal region eliminated binding to P protein. This result demonstrates that the carboxy-terminal domain of L is required for the interaction with P.

狂犬病病毒的RNA聚合酶（RNA polymerase）由两个亚基组成，分别为大蛋白（large protein，L）与磷蛋白（phosphoprotein，P），二者的氨基酸长度分别为2127和297个残基。借助重组痘苗病毒-T7 RNA聚合酶系统在哺乳动物细胞中共表达这两种蛋白时，可通过免疫共沉淀（coimmunoprecipitation）检测到二者形成复合物。为鉴定两种蛋白中参与复合物形成的区域，研究人员对P蛋白与L蛋白的缺失突变体展开了分析。移除P蛋白羧基端半段的大片段缺失并未破坏P与L之间的相互作用；与之相反，氨基端存在缺失的P蛋白则无法与L形成复合物。此外，将P蛋白前19或前52个残基与绿色荧光蛋白（green fluorescent protein，GFP）以正确读码框融合得到的融合蛋白，仍可与L蛋白结合。上述结果表明，L蛋白的主要结合位点位于P蛋白的前19个残基区域内。本研究同时定位了L蛋白中与P蛋白相互作用的区域：包含羧基端1656、956、690及566个氨基酸残基的突变L蛋白，均能与P蛋白结合；而在该末端区域内缺失789个残基则会消除L蛋白与P蛋白的结合能力。该结果证实，L蛋白的羧基端结构域是其与P蛋白相互作用所必需的。