Kean K M, Teterina N L, Marc D, Girard M
Unité de Virologie Moléculaire (CNRS UA 545), Institut Pasteur, Paris, France.
Virology. 1991 Apr;181(2):609-19. doi: 10.1016/0042-6822(91)90894-h.
It was recently suggested that the picornavirus 3C proteases are homologous to the chymotrypsin-like serine proteases. The two structural models proposed differ in one of the postulated active site residues, Glu/Asp71 or Asp85. We changed Glu71 of the poliovirus type 1 protease to Asp or Gln and Asp85 to Glu by oligonucleotide-directed site-specific mutagenesis of an infectious cDNA, and attempted to recover virus after transfection. Both Glu71 changes were lethal for the virus and proteolytic activity was abolished in vitro with the exception of the primary cleavage event at the P2/P3 junction. In contrast, the Asp85----Glu virus was viable. This mutant was temperature-sensitive for growth at 39 degrees and exhibited a minute plaque phenotype at permissive temperature. This defect correlated with low levels of viral-specific RNA and protein syntheses and slow virus growth. Proteolytic processing at the COOH-terminus of 3C was impaired, reducing the production of mature 3C and the viral replicase 3D. In addition, 3C-mediated cleavage events within the P2 region of the polyprotein seemed to occur rather inefficiently. 3C-specific processing within P1 and elsewhere within P3 was unaffected. We suggest that Asp85 does not form part of the active site of 3C, but could be important for the specific recognition of cleavage sites within P2.
最近有人提出，小核糖核酸病毒3C蛋白酶与胰凝乳蛋白酶样丝氨酸蛋白酶同源。所提出的两种结构模型在假定的活性位点残基之一，即Glu/Asp71或Asp85上有所不同。我们通过对感染性cDNA进行寡核苷酸定向位点特异性诱变，将脊髓灰质炎病毒1型蛋白酶的Glu71突变为Asp或Gln，将Asp85突变为Glu，并在转染后试图回收病毒。Glu71的两种变化对病毒都是致死性的，并且除了在P2/P3连接处的初级切割事件外，体外蛋白水解活性被消除。相比之下，Asp85突变为Glu的病毒是存活的。该突变体在39℃下生长对温度敏感，并且在允许温度下表现出微小噬斑表型。这种缺陷与病毒特异性RNA和蛋白质合成水平低以及病毒生长缓慢相关。3C羧基末端的蛋白水解加工受损，减少了成熟3C和病毒复制酶3D的产生。此外，多聚蛋白P2区域内3C介导的切割事件似乎发生得相当低效。P1内以及P3内其他位置的3C特异性加工未受影响。我们认为Asp85不是3C活性位点的一部分，但可能对P2内切割位点的特异性识别很重要。
