Zamora Miguel, Marissen Wilfred E, Lloyd Richard E
Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, Texas 77030, USA.
J Virol. 2002 Jan;76(1):165-77. doi: 10.1128/jvi.76.1.165-177.2002.
Cleavage of eukaryotic translation initiation factor 4GI (eIF4GI) is required for shutoff of host cell translation during poliovirus (PV) infection of HeLa cells. Reports published by several groups have led to confusion whether this cleavage is mediated by viral 2A protease (2A(pro)) or a putative cellular enzyme (termed eIF4Gase) which is activated by 2A(pro) or other aspects of viral infection. Here we have further investigated eIF4Gase activities in PV-infected cells. Column purification of eIF4GI cleavage activity separated two activities which generated N-terminal cleavage products of different lengths. Both activities were detected using either native eIF4G or radiolabeled recombinant eIF4G as the substrate. Analysis of cleavage products formed by each activity on native and mutant substrates suggests that one activity cleaves eIF4G1 at or very near the 2A(pro) cleavage site and the other activity cleaves approximately 40 residues upstream of the 2A(pro) cleavage site. When PV infections in HeLa cells were supplemented with 2 mM guanidine, which indirectly limits expression of 2A(pro), two distinct C-terminal cleavage fragments of eIF4GI were detected. These C-terminal cleavage fragments of eIF4GI were purified from infected cells, and a new eIF4GI cleavage site was mapped to a unique site 43 amino acids upstream of the known 2A(pro) cleavage site. Further, eIF4GI cleavage in vivo could be blocked by addition of zVAD to PV-guanidine infections. zVAD is a broad-spectrum caspase inhibitor which had no effect on 2A(pro) cleavage activity or PV polyprotein processing. Lastly, similar types of eIF4Gase cleavage activities were also detected in uninfected cells under various conditions, including early apoptosis or during cell cycle transit. The data suggest that the same types of eIF4GI cleavage activities which are generated in PV-infected cells can also be generated in the absence of virus. Taken together, the data support a model in which multiple cellular activities process eIF4GI in PV-infected cells, in addition to 2A(pro).
在脊髓灰质炎病毒（PV）感染HeLa细胞期间，真核生物翻译起始因子4GI（eIF4GI）的切割是宿主细胞翻译关闭所必需的。几个研究小组发表的报告引发了关于这种切割是由病毒2A蛋白酶（2A(pro)）介导，还是由一种假定的细胞酶（称为eIF4Gase）介导的困惑，这种细胞酶由2A(pro)或病毒感染的其他方面激活。在这里，我们进一步研究了PV感染细胞中的eIF4Gase活性。通过柱纯化eIF4GI切割活性分离出两种活性，它们产生不同长度的N端切割产物。使用天然eIF4G或放射性标记的重组eIF4G作为底物均可检测到这两种活性。对每种活性在天然和突变底物上形成的切割产物的分析表明，一种活性在2A(pro)切割位点处或非常接近该位点切割eIF4G1，另一种活性在2A(pro)切割位点上游约40个残基处切割。当在HeLa细胞的PV感染中添加2 mM胍时，胍间接限制了2A(pro)的表达，此时检测到了eIF4GI的两个不同的C端切割片段。从感染细胞中纯化了这些eIF4GI的C端切割片段，并将一个新的eIF4GI切割位点定位到已知2A(pro)切割位点上游独特的43个氨基酸处。此外，在PV-胍感染中添加zVAD可阻断体内的eIF4GI切割。zVAD是一种广谱半胱天冬酶抑制剂，对2A(pro)切割活性或PV多聚蛋白加工没有影响。最后，在各种条件下，包括早期凋亡或细胞周期转换期间，在未感染的细胞中也检测到了类似类型的eIF4Gase切割活性。数据表明，在没有病毒的情况下，也可以产生与PV感染细胞中相同类型的eIF4GI切割活性。综上所述，数据支持一种模型，即在PV感染的细胞中，除了2A(pro)之外，还有多种细胞活性对eIF4GI进行加工处理。
