van Kuppeveld F J, van den Hurk P J, Zoll J, Galama J M, Melchers W J
Department of Medical Microbiology, University of Nijmegen, The Netherlands.
J Virol. 1996 Nov;70(11):7632-40. doi: 10.1128/JVI.70.11.7632-7640.1996.
The enterovirus 2B/2C cleavage site differs from the common cleavage site motif AxxQ/G by the occurrence of either polar residues at the P1' position or large aliphatic residues at the P4 position. To study (i) the putative contribution of these aberrant residues to the stability of precursor protein 2BC, (ii) the determinants of cleavage site specificity and efficiency of 3Cpro, and (iii) the importance of efficient cleavage at this site for viral replication, a mutational analysis of the coxsackie B3 virus (CBV3) 2B/2C cleavage site (AxxQ/N) was performed. Neither replacement of the P1' asparagine with a serine or a glycine nor replacement of the P4 alanine with a valine significantly affected 2B/2C cleavage efficiency, RNA replication, or virus growth. The introduction of a P4 asparagine, as can be found at the CBV3 3C/3D cleavage site, caused a severe reduction in 2B/2C cleavage and abolished virus growth. These data support the idea that a P4 asparagine is an unfavorable residue that contributes to a slow turnover of precursor protein 3CD but argue that it is unlikely that the aberrant 2B/2C cleavage site motifs serve to regulate 2B/2C processing efficiency and protein 2BC stability. The viability of a double mutant containing a P4 asparagine and a P1' glycine demonstrated that a P1' residue can compensate for the adverse effects of an unfavorable P4 residue. Poliovirus (or poliovirus-like) 2B/2C cleavage site motifs were correctly processed by CBV 3Cpro, albeit with a reduced efficiency, and yielded viable viruses. Analysis of in vivo protein synthesis showed that mutant viruses containing poorly processed 2B/2C cleavage sites were unable to completely shut off cellular protein synthesis. The failure to inhibit host translation coincided with a reduced ability to modify membrane permeability, as measured by the sensitivity to the unpermeant translation inhibitor hygromycin B. These data suggest that a critical level of protein 2B or 2C, or both, may be required to alter membrane permeability and, possibly as a consequence, to shut off host cell translation.
肠道病毒2B/2C裂解位点与常见的裂解位点基序AxxQ/G不同，原因在于P1' 位置出现极性残基或P4位置出现大的脂肪族残基。为了研究：（i）这些异常残基对前体蛋白2BC稳定性的假定贡献；（ii）3C蛋白酶裂解位点特异性和效率的决定因素；（iii）该位点高效裂解对病毒复制的重要性，对柯萨奇B3病毒（CBV3）的2B/2C裂解位点（AxxQ/N）进行了突变分析。将P1' 天冬酰胺替换为丝氨酸或甘氨酸，以及将P4丙氨酸替换为缬氨酸，均未显著影响2B/2C的裂解效率、RNA复制或病毒生长。如在CBV3 3C/3D裂解位点所见，引入P4天冬酰胺会导致2B/2C裂解严重减少并使病毒生长停止。这些数据支持以下观点：P4天冬酰胺是一个不利残基，它导致前体蛋白3CD周转缓慢，但表明异常的2B/2C裂解位点基序不太可能用于调节2B/2C的加工效率和蛋白2BC的稳定性。含有P4天冬酰胺和P1' 甘氨酸的双突变体的存活能力表明，P'I残基可以补偿不利的P4残基的负面影响。脊髓灰质炎病毒（或类脊髓灰质炎病毒）的2B/2C裂解位点基序虽效率降低但被CBV 3C蛋白酶正确加工，并产生了活病毒。体内蛋白质合成分析表明，含有加工不良的2B/2C裂解位点的突变病毒无法完全阻断细胞蛋白质合成。未能抑制宿主翻译与改变膜通透性的能力降低同时出现，这通过对不透膜的翻译抑制剂潮霉素B的敏感性来衡量。这些数据表明，可能需要达到关键水平的蛋白2B或2C，或两者兼而有之，才能改变膜通透性，并可能因此阻断宿主细胞翻译。
