Bodjo S C, Lelenta M, Couacy-Hymann E, Kwiatek O, Albina E, Gargani D, Libeau G, Diallo A
Animal Production Unit, FAO/AIEA Agriculture & Biotechnology Laboratory, IAEA Laboratories, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria.
Virus Res. 2008 Jan;131(1):23-32. doi: 10.1016/j.virusres.2007.08.005. Epub 2007 Sep 17.
For Mononegavirales, the template for transcription and replication is not the naked RNA but the nucleoprotein (N) encapsidated genomic and anti-genomic RNA. Because of this central role in the replication of these viruses, N has been the subject of numerous structural and functional mapping studies. Here, we report on the cloning of the Peste des Petits Ruminants virus (PPRV) N gene into the baculovirus vector and the expression of the protein in insect cells. By electron microscopy observation, we have shown that this recombinant PPRV N forms nucleocapsid-like particles in insect cells in the absence of other PPRV proteins, as reported for other paramyxoviruses. As it is known that the formation of these particles is first linked to the self-assembly of N, we have made several deletions in the PPRV N gene and expressed these mutants in insect cells. Analysis of these proteins by immunoprecipitation and electron microscopy observation enabled us to map the N-N interaction domains into two regions of PPRV N: aa 1-120 and 146-241. The fragment aa 121-145, which is not conserved within the morbillivirus group, is also required for the formation/stability of the nucleocapsid helical structure.
对于单股负链RNA病毒目成员来说，转录和复制的模板不是裸露的RNA，而是被核蛋白（N）包裹的基因组RNA和反基因组RNA。由于N在这些病毒的复制过程中发挥核心作用，因此它一直是众多结构和功能图谱研究的对象。在此，我们报告了将小反刍兽疫病毒（PPRV）的N基因克隆到杆状病毒载体中，并在昆虫细胞中表达该蛋白的过程。通过电子显微镜观察，我们发现，与其他副粘病毒的情况一样，这种重组PPRV N在没有其他PPRV蛋白的情况下，能在昆虫细胞中形成核衣壳样颗粒。由于已知这些颗粒的形成首先与N的自组装有关，我们对PPRV N基因进行了多处缺失，并在昆虫细胞中表达了这些突变体。通过免疫沉淀和电子显微镜观察对这些蛋白进行分析，使我们能够将N-N相互作用结构域定位到PPRV N的两个区域：氨基酸1-120和146-241。氨基酸121-145片段在麻疹病毒属内并不保守，它也是核衣壳螺旋结构形成/稳定所必需的。
