Cao Jianzhong, Wu Ching-Ching, Lin Tsang Long
Department of Comparative Pathobiology, Purdue University, West Lafayette, IN, USA.
Intervirology. 2008;51(5):342-51. doi: 10.1159/000175837. Epub 2008 Nov 21.
Turkey coronavirus (TCoV) polyprotein was predicted to be cleaved into 15 non-structural proteins (nsp2 to nsp16), but none of these nsps have been characterized. TCoV nsp15 consists of 338 residues and shares 40% sequence similarity to U-specific Nidovirales endoribonuclease (NendoU) of severe acute respiratory syndrome coronavirus.
The purpose of the present study was to characterize TCoV nsp15.
The TCoV nsp15 gene was cloned into pTriEX1 and expressed as a C-terminal His-tagged recombinant protein in BL21 (DE3). The recombinant nsp15 was purified by Ni-NTA resin. Synthetic RNA substrates were used to determine the substrate specificity of the TCoV nsp15. RNA zymography was used to determine the active form of the nsp15.
The TCoV nsp15 did not cleave DNA but degraded total cellular RNA. The TCoV nsp15 cleaved single-stranded (ss) RNA at the uridylate site. The TCoV nsp15 cleaved hairpin RNA, pRNA, and double-stranded RNA (dsRNA) of infectious bursal disease virus very slowly, implying that dsRNA is not a good substrate for the TCoV nsp15. No divalent metal ion was required for in vitro enzymatic activity of the TCoV nsp15. The active form of the TCoV nsp15 was a homohexamer and disulfide bond was essential for the enzymatic activity.
The TCoV nsp15 is a NendoU but has some characteristics different from other NendoU.
土耳其冠状病毒（TCoV）多聚蛋白预计可切割成15种非结构蛋白（nsp2至nsp16），但这些nsp均未得到表征。TCoV nsp15由338个残基组成，与严重急性呼吸综合征冠状病毒的U特异性巢病毒目核糖核酸酶（NendoU）具有40%的序列相似性。
本研究旨在表征TCoV nsp15。
将TCoV nsp15基因克隆到pTriEX1中，并在BL21（DE3）中作为C端带His标签的重组蛋白表达。重组nsp15通过Ni-NTA树脂纯化。使用合成RNA底物来确定TCoV nsp15的底物特异性。RNA酶谱法用于确定nsp15的活性形式。
TCoV nsp15不切割DNA，但可降解总细胞RNA。TCoV nsp15在尿苷酸位点切割单链（ss）RNA。TCoV nsp15非常缓慢地切割传染性法氏囊病病毒的发夹RNA、pRNA和双链RNA（dsRNA），这意味着dsRNA不是TCoV nsp15的良好底物。TCoV nsp15的体外酶活性不需要二价金属离子。TCoV nsp15的活性形式是同源六聚体，二硫键对酶活性至关重要。
TCoV nsp15是一种NendoU，但具有一些与其他NendoU不同的特征。
