Richmond K E, Chenault K, Sherwood J L, German T L
Department of Plant Pathology, University of Wisconsin-Madison, Madison, Wisconsin, 53706, USA.
Virology. 1998 Aug 15;248(1):6-11. doi: 10.1006/viro.1998.9223.
Tomato spotted wilt tospovirus (TSWV) is the type member of the plant-infecting viruses of the genus Tospovirus in the family Bunyaviridae. The three TSWV RNAs are encapsidated with nucleocapsid (N) protein to form ribonucleoprotein (RNP) which serves as the template for viral transcription and replication. Regions of the open reading frame coding for the N protein on the small (S) RNA were subcloned into pET protein expression vectors and expressed in Escherichia coli BL21 (DE3) cells. Full-length N, N amino and carboxy halves, and two N carboxy-terminal regions were expressed and isolated by metal chelate affinity chromatography. The N protein, both of its halves and the extreme carboxy-terminal region, bound cooperatively and irrespective of sequence to radiolabeled single-stranded RNA produced by runoff transcription of clones of either TSWV S RNA or cowpea chlorotic mottle virus RNA3. N protein did not bind to radiolabeled double-stranded TSWV RNA. The density of the synthetic RNase-sensitive N protein-RNA complexes was 1.32 g/ml, similar to the density of authentic Bunyaviridae RNPs. These studies are the first to indicate differences in the nucleic acid binding abilities of Tospovirus and Hantavirus nucleocapsid proteins, the only characterized nucleocapsid proteins of the family Bunyaviridae.
番茄斑萎病毒（TSWV）是布尼亚病毒科番茄斑萎病毒属植物感染病毒的典型成员。TSWV的三种RNA与核衣壳（N）蛋白包裹在一起，形成核糖核蛋白（RNP），它作为病毒转录和复制的模板。将小（S）RNA上编码N蛋白的开放阅读框区域亚克隆到pET蛋白表达载体中，并在大肠杆菌BL21（DE3）细胞中表达。通过金属螯合亲和层析表达并分离出全长N、N蛋白的氨基和羧基端片段，以及两个N蛋白羧基末端区域。N蛋白及其两个片段以及极端羧基末端区域，与通过TSWV S RNA或豇豆褪绿斑驳病毒RNA3克隆的径流转录产生的放射性标记单链RNA协同结合，且与序列无关。N蛋白不与放射性标记的双链TSWV RNA结合。合成的对核糖核酸酶敏感的N蛋白-RNA复合物的密度为1.32 g/ml，与真正的布尼亚病毒科核糖核蛋白的密度相似。这些研究首次表明番茄斑萎病毒和汉坦病毒核衣壳蛋白在核酸结合能力上存在差异，这两种蛋白是布尼亚病毒科仅有的已鉴定核衣壳蛋白。
