FOOT-AND-MOUTH-DISEASE VIRUS LEADER PROTEINASE - PURIFICATION OF THE LB FORM AND DETERMINATION OF ITS CLEAVAGE SITE ON EIF4-GAMMA
FOOT-AND-MOUTH-DISEASE VIRUS LEADER PROTEINASE - PURIFICATION OF THE LB FORM AND DETERMINATION OF ITS CLEAVAGE SITE ON EIF4-GAMMA
复制标题
DOI：
10.1128/jvi.68.9.5677-5684.1994
复制
发表时间：
1994-09-01
影响因子：
5.4
通讯作者：
SKERN, T
中科院分区：
文献类型：
--
作者：
KIRCHWEGER, R;ZIEGLER, E;SKERN, T
许多小核糖核酸病毒在感染细胞中引起细胞内mRNAs翻译的急剧减少，而不影响其自身RNA的翻译。在鼻病毒、肠道病毒和口疮病毒感染过程中，蛋白质合成起始因子eIF-4伽马发生特异性蛋白分解，显然导致核糖体无法结合带帽的mRNAs。在人类鼻病毒和肠道病毒中，eIF-4伽马的裂解是由病毒2A型蛋白酶进行的；在口蹄疫病毒(即口蹄疫病毒)中，主导酶负责这一反应。在此，我们描述了在大肠杆菌中表达的前导蛋白Lb形式的纯化。纯化的Leader或2A型蛋白水解酶在体外获得的EIF-4γ的初级切割产物与体内感染时发现的产物在电泳上没有区别。然而，在体外还观察到ELF-4伽马蛋白与先导蛋白和人鼻病毒22A型蛋白水解酶的结合。通过自动Edman降解纯化的C-末端裂解产物测序，确定了兔网织红细胞中eIF-4-γ的前导蛋白的裂解位点。切割位点位于Gly-479和Arg-480之间，因此不同于鼻病毒和肠道病毒2A型蛋白酶的切割位点，后者在Arg-486和Gly-487之间切割。
Many picornaviruses cause a dramatic decrease in the translation of cellular mRNAs in the infected cell, without affecting the translation of their own RNA. Specific proteolysis of protein synthesis initiation factor eIF-4 gamma occurs during infection with rhinoviruses, enteroviruses, and aphthoviruses, apparently leading to an inability of the ribosomes to bind capped mRNAs. Cleavage of eIF-4 gamma in human rhinoviruses and enteroviruses is carried out by the viral 2A proteinase; in aphthoviruses (i.e., foot-and-mouth disease viruses), the leader proteinase is responsible for this reaction. We describe here the purification to homogeneity of the Lb form of the leader proteinase expressed in Escherichia coli. The primary cleavage products of eIF-4 gamma obtained in vitro with purified leader or 2A proteinase are electrophoretically indistinguishable from those found during infection in vivo. However, additional proteolysis products of elF-4 gamma are observed with the leader proteinase and the human rhinovirus type 2 2A proteinase in vitro. The cleavage site of the leader proteinase in eIF-4 gamma from rabbit reticulocyte was determined by sequencing the purified C-terminal cleavage product by automated Edman degradation. The cleavage site is between Gly-479 and Arg-480 and thus differs from that of rhinovirus and enterovirus 2A proteinases, which cleave between Arg-486 and Gly-487.