Gillian A L, Nibert M L
Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison 53706, USA.
Virology. 1998 Jan 5;240(1):1-11. doi: 10.1006/viro.1997.8905.
Reovirus nonstructural protein sigma NS exhibits a ssRNA-binding activity thought to be involved in assembling the reovirus mRNAs for genome replication and virion morphogenesis. To extend analysis of this activity, recombinant sigma NS (r sigma NS) was expressed in insect cells using a recombinant baculovirus. In infected-cell extracts, r sigma NS was found in large complexes (> or = 30 S) that were disassembled into smaller, 13-19 S complexes upon treatment with RNase A. R sigma NS also bound to poly(A)-Sepharose beads both before and after purification. Treatment with high salt during purification caused r sigma NS to sediment in even smaller, 7-9 S complexes, consistent with more complete loss of RNA. To localize the RNA-binding site, limited proteolysis was used to fragment the r sigma NS protein. Upon mild treatment with thermolysin, 11 amino acids were removed from the amino terminus of r sigma NS, and the resulting protein no longer bound to poly(A). In addition, when r sigma NS in cell extracts was treated with thermolysin to generate the amino-terminally truncated from, it sedimented at 7-9 S, also consistent with the loss of RNA-binding capacity. To confirm these findings, a deletion mutant lacking amino acids 2-11 was constructed and expressed in insect cells from a recombinant baculovirus. The mutant protein in cell extracts showed greatly reduced poly(A)-binding activity and sedimented as 7-9 S complexes. These data suggest that the first 11 amino acids of sigma NS, which are predicted to form an amphipathic alpha-helix, are important for both ssRNA binding and formation of complexes larger than 7-9 S.
呼肠孤病毒非结构蛋白σNS具有单链RNA结合活性，该活性被认为参与组装呼肠孤病毒mRNA以进行基因组复制和病毒粒子形态发生。为了扩展对该活性的分析，使用重组杆状病毒在昆虫细胞中表达了重组σNS（rσNS）。在感染细胞提取物中，rσNS存在于大的复合物（≥30S）中，用核糖核酸酶A处理后会分解成较小的13 - 19S复合物。rσNS在纯化前后均与聚（A）-琼脂糖珠结合。纯化过程中用高盐处理导致rσNS沉降为甚至更小的7 - 9S复合物，这与RNA更完全的丢失一致。为了定位RNA结合位点，使用有限蛋白酶解将rσNS蛋白片段化。用嗜热菌蛋白酶轻度处理后，从rσNS的氨基末端去除了11个氨基酸，所得蛋白质不再与聚（A）结合。此外，当用嗜热菌蛋白酶处理细胞提取物中的rσNS以产生氨基末端截短形式时，它沉降在7 - 9S，这也与RNA结合能力的丧失一致。为了证实这些发现，构建了一个缺失氨基酸2 - 11的缺失突变体，并使用重组杆状病毒在昆虫细胞中表达。细胞提取物中的突变蛋白显示出聚（A）结合活性大大降低，并沉降为7 - 9S复合物。这些数据表明，σNS的前11个氨基酸预计形成一个两亲性α螺旋，对单链RNA结合和大于7 - 9S的复合物形成都很重要。
