Wirblich C, Sibilia M, Boniotti M B, Rossi C, Thiel H J, Meyers G
Federal Research Centre for Virus Diseases of Animals, Tübingen, Germany.
J Virol. 1995 Nov;69(11):7159-68. doi: 10.1128/JVI.69.11.7159-7168.1995.
Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size.
兔出血症病毒是杯状病毒科的一种正链RNA病毒，它编码一种类胰蛋白酶半胱氨酸蛋白酶，作为一个大的多蛋白的一部分。在大肠杆菌中表达时，该蛋白酶通过在其N端和C端的蛋白水解切割从较大的前体中释放出来。两个切割位点通过对切割产物的N端序列分析来确定。蛋白酶N端的切割在第1108和1109位的EG二肽处高效发生。蛋白酶C端的切割在第1251和1252位的ET二肽处低效发生。为了研究该蛋白酶的切割特异性，在蛋白酶N端边界切割位点的P2、P1和P1'位置引入了氨基酸替换。该分析表明，P1位置的氨基酸是底物识别的最重要决定因素。该位置仅允许谷氨酸、谷氨酰胺和天冬氨酸。在P1'位置，甘氨酸、丝氨酸和丙氨酸是该蛋白酶的优选底物，但也允许一些具有较大侧链的氨基酸。P2位置的替换对切割效率影响很小。ORF1多蛋白C端一半的无细胞表达表明，该蛋白酶催化RNA聚合酶和衣壳蛋白连接处的切割。第1767和1768位的EG二肽被确定为假定的切割位点。我们的数据表明，兔出血症病毒编码一种类胰蛋白酶半胱氨酸蛋白酶，其在功能和特异性方面与3C蛋白酶相似，但在大小方面与2A蛋白酶更相似。
