Zhao Y, Thomas C, Bremer C, Roy P
Department of Biochemistry, University of Oxford, United Kingdom.
J Virol. 1994 Apr;68(4):2179-85. doi: 10.1128/JVI.68.4.2179-2185.1994.
Genome segment 8 (S8) of bluetongue virus serotype 10 (BTV-10) encodes the nonstructural protein NS2. This protein, which has single-stranded RNA (ssRNA) binding capacity, is found in BTV-infected cells in the form of virus inclusion bodies (VIBs). To identify the domain(s) important for RNA binding and oligomerization of the protein, a number of deletions were made in regions of the gene that code for either the amino or carboxy terminus of the protein. The modified genes were cloned into and expressed from baculovirus vectors based on Autographa californica nuclear polyhedrosis virus. Truncated NS2 proteins were individually analyzed for the ability to bind ssRNA and to form VIBs. The results indicated that the carboxy terminus of the protein is involved neither in RNA binding nor in the formation of VIBs. The amino terminus of NS2 was shown to be essential for ssRNA binding but not for NS2 protein oligomerization. Point mutations that involved the substitution of various charged residues at the amino terminus of NS2 were generated and tested for the ability to bind ssRNA. The results showed that the arginines at amino acid residues 6 and 7 and the lysine at residue 4, but not the glutamic acid at residue 2, are involved in ssRNA binding.
蓝舌病病毒10型（BTV-10）的基因组片段8（S8）编码非结构蛋白NS2。这种具有单链RNA（ssRNA）结合能力的蛋白，以病毒包涵体（VIBs）的形式存在于BTV感染的细胞中。为了确定该蛋白中对RNA结合和寡聚化重要的结构域，在编码该蛋白氨基末端或羧基末端的基因区域进行了一系列缺失。将修饰后的基因克隆到基于苜蓿银纹夜蛾核型多角体病毒的杆状病毒载体中并表达。对截短的NS2蛋白分别进行结合ssRNA和形成VIBs能力的分析。结果表明，该蛋白的羧基末端既不参与RNA结合也不参与VIBs的形成。NS2的氨基末端被证明对ssRNA结合至关重要，但对NS2蛋白寡聚化并非如此。产生了涉及NS2氨基末端各种带电荷残基取代的点突变，并测试其结合ssRNA的能力。结果表明，氨基酸残基6和7处的精氨酸以及残基4处的赖氨酸，而非残基2处的谷氨酸，参与ssRNA结合。
