Functional and Genetic Studies of the Substrate Specificity of Coronavirus Infectious Bronchitis Virus 3C-Like Proteinase
Functional and Genetic Studies of the Substrate Specificity of Coronavirus Infectious Bronchitis Virus 3C-Like Proteinase
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DOI：
10.1128/jvi.02490-09
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发表时间：
2010-07-01
影响因子：
5.4
通讯作者：
Liu, Ding Xiang
中科院分区：
文献类型：
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作者：
Fang, Shouguo;Shen, Hongyuan;Liu, Ding Xiang
冠状病毒（CoV）3C样蛋白酶（3CLpro）位于非结构蛋白5（nsp 5）中，在11个特定位点加工复制酶多聚蛋白1a和1ab（pp 1a和pp 1ab），产生12种成熟的非结构蛋白（nsp 5至nsp 16）。结构和生物化学研究表明，在P1位置的一个保守的谷氨酰胺残基是绝对需要有效的切割。本文通过体外切割试验和反向遗传学方法研究了传染性支气管炎病毒（IBV）3CLpro切割位点P1位氨基酸替换对切割效率和病毒复制的影响。我们的结果表明，在nsp 4 -5/Q2779、nsp 5 -6/Q3086、nsp 7 -8/Q3462、nsp 8 -9/Q3672和nsp 9 -10/Q3783位点的P1-Asn取代、在nsp 8 -9/Q3672位点的P1-Glu取代和在nsp 15 -16/Q6327位点的P1-His取代是耐受的，并且允许恢复感染性突变病毒，尽管具有不同程度的生长缺陷。相反，在nsp 6 -7/Q3379、nsp 12 -13/Q4868、nsp 13 -14/Q5468和nsp 14 -15/Q5989位点处的P1-Asn取代，以及在nsp 15 -16/Q6327位点处的P1-Pro取代，消除了相应位置处的3CLpro介导的切割，并阻断了感染性病毒的恢复。这些致命突变对RNA合成的影响的分析表明，加工中间体，如nsp 6 -7，nsp 12 -13，nsp 13 -14，nsp 14 -15和nsp 15 -16前体，可能在负链基因组RNA复制中起作用，而成熟蛋白可能是亚基因组RNA（sgRNA）转录所需的。更有趣的是，当将在nsp 15 -16位点携带Q6327 N突变的IBV感染性cDNA克隆引入细胞时，选择具有P166 S或P166 L突变的突变体3CLpro。这两种突变中的任何一种都被证明可以显著增强3CLpro介导的nsp 15 -16位点的切割效率，并补偿对感染性病毒恢复的不利影响。
Coronavirus (CoV) 3C-like proteinase (3CLpro), located in nonstructural protein 5 (nsp5), processes the replicase polyproteins 1a and 1ab (pp1a and pp1ab) at 11 specific sites to produce 12 mature nonstructural proteins (nsp5 to nsp16). Structural and biochemical studies suggest that a conserved Gln residue at the P1 position is absolutely required for efficient cleavage. Here, we investigate the effects of amino acid substitution at the P1 position of 3CLpro cleavage sites of infectious bronchitis virus (IBV) on the cleavage efficiency and viral replication by in vitro cleavage assays and reverse genetic approaches. Our results demonstrated that a P1-Asn substitution at the nsp4-5/Q2779, nsp5-6/Q3086, nsp7-8/Q3462, nsp8-9/Q3672, and nsp9-10/Q3783 sites, a P1-Glu substitution at the nsp8-9/Q3672 site, and a P1-His substitution at the nsp15-16/Q6327 site were tolerated and allowed recovery of infectious mutant viruses, albeit with variable degrees of growth defects. In contrast, a P1-Asn substitution at the nsp6-7/Q3379, nsp12-13/Q4868, nsp13-14/Q5468, and nsp14-15/Q5989 sites, as well as a P1-Pro substitution at the nsp15-16/Q6327 site, abolished 3CLpro-mediated cleavage at the corresponding position and blocked the recovery of infectious viruses. Analysis of the effects of these lethal mutations on RNA synthesis suggested that processing intermediates, such as the nsp6-7, nsp12-13, nsp13-14, nsp14-15, and nsp15-16 precursors, may function in negative-stranded genomic RNA replication, whereas mature proteins may be required for subgenomic RNA (sgRNA) transcription. More interestingly, a mutant 3CLpro with either a P166S or P166L mutation was selected when an IBV infectious cDNA clone carrying the Q6327N mutation at the nsp15-16 site was introduced into cells. Either of the two mutations was proved to enhance significantly the 3CLpro-mediated cleavage efficiency at the nsp15-16 site with a P1-Asn substitution and compensate for the detrimental effects on recovery of infectious virus.