Mayer Daniel, Baginsky Sacha, Schwemmle Martin
Department of Virology, Institute for Medical Microbiology and Hygiene, University of Freiburg, Germany.
Proteomics. 2005 Nov;5(17):4483-7. doi: 10.1002/pmic.200402095.
The biochemical purification and analysis of viral ribonucleoprotein complexes (RNPs) of negative-strand RNA viruses is hampered by the lack of suitable tags that facilitate specific enrichment of these complexes. We therefore tested whether fusion of the tandem-affinity-purification (TAP) tag to the main component of viral RNPs, the nucleoprotein, might allow the isolation of these RNPs from cells. We constitutively expressed TAP-tagged nucleoprotein of Borna disease virus (BDV) in cells persistently infected with this virus. The TAP-tagged bait was efficiently incorporated into viral RNPs, did not interfere with BDV replication and was also packaged into viral particles. Native purification of the tagged protein complexes from BDV-infected cells by two consecutive affinity columns resulted in the isolation of several viral proteins, which were identified by MS analysis as the matrix protein, the two forms of the nucleoprotein and the phosphoprotein. In addition to the viral proteins, RT-PCR analysis revealed the presence of viral genomic RNA. Introduction of further protease cleavage sites within the TAP-tag significantly increased the purification yield. These results demonstrate that purification of TAP-tagged viral RNPs is possible and efficient, and may therefore provide new avenues for biochemical and functional studies of these complexes.
负链RNA病毒的病毒核糖核蛋白复合体（RNP）的生化纯化和分析因缺乏有助于特异性富集这些复合体的合适标签而受到阻碍。因此，我们测试了将串联亲和纯化（TAP）标签与病毒RNP的主要成分核蛋白融合，是否能从细胞中分离出这些RNP。我们在持续感染博尔纳病病毒（BDV）的细胞中组成性表达TAP标签的BDV核蛋白。TAP标签的诱饵蛋白有效地掺入病毒RNP中，不干扰BDV复制，并且也被包装到病毒颗粒中。通过两个连续的亲和柱从BDV感染的细胞中对标记蛋白复合体进行天然纯化，得到了几种病毒蛋白，通过质谱分析鉴定为基质蛋白、两种形式的核蛋白和磷蛋白。除了病毒蛋白，逆转录聚合酶链反应（RT-PCR）分析显示存在病毒基因组RNA。在TAP标签内引入更多蛋白酶切割位点显著提高了纯化产量。这些结果表明，纯化TAP标签的病毒RNP是可行且高效的，因此可能为这些复合体的生化和功能研究提供新途径。
