Parks G D, Palmenberg A C
Institute for Molecular Virology, University of Wisconsin, Madison 53706.
J Virol. 1987 Dec;61(12):3680-7. doi: 10.1128/JVI.61.12.3680-3687.1987.
Most proteolytic cleavages within the picornavirus polyproteins are carried out by viral protease 3C. For encephalomyocarditis virus, the protease 3C-catalyzed processing occurs between Gln-Gly or Gln-Ser amino acid pairs which are flanked by proline residues, but the sequence-specific constraints on recognition and cleavage by the enzyme are not completely understood. To examine alternative cleavage site sequences, we constructed a cDNA plasmid which expresses the viral L-P1-2A capsid precursor in vitro and introduced site-specific mutations into the Gln-Gly pair at the VP3/VP1 junction. The altered protein substrates were tested for cleavage activity in assays with protease 3C. The encephalomyocarditis virus 3C processed Gln-Ala as efficiently as its natural sites but did not cleave Gln-Val, Gln-Glu, Lys-Gly, Lys-Ala, Lys-Val, Lys-Glu, or Pro-Gly combinations. Displacement of the flanking proline residue by an engineered insertion slowed but did not prevent cleavage at this site. Also, a mutant defective in processing at the VP3/VP1 junction was unable to form 14S pentameric assembly intermediates in vitro.
微小核糖核酸病毒多聚蛋白内的大多数蛋白水解切割是由病毒蛋白酶3C进行的。对于脑心肌炎病毒，蛋白酶3C催化的加工过程发生在由脯氨酸残基侧翼的Gln-Gly或Gln-Ser氨基酸对之间，但该酶识别和切割的序列特异性限制尚未完全了解。为了研究替代切割位点序列，我们构建了一个在体外表达病毒L-P1-2A衣壳前体的cDNA质粒，并在VP3/VP1连接处的Gln-Gly对中引入了位点特异性突变。用蛋白酶3C检测改变后的蛋白质底物的切割活性。脑心肌炎病毒3C切割Gln-Ala的效率与其天然位点相同，但不切割Gln-Val、Gln-Glu、Lys-Gly、Lys-Ala、Lys-Val、Lys-Glu或Pro-Gly组合。通过工程插入取代侧翼脯氨酸残基会减慢但不会阻止该位点的切割。此外，在VP3/VP1连接处加工有缺陷的突变体在体外无法形成14S五聚体组装中间体。
