Direct cleavage of eIF4G by poliovirus 2A protease is inefficient in vitro
Direct cleavage of eIF4G by poliovirus 2A protease is inefficient in vitro
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DOI：
10.1006/viro.1998.9172
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发表时间：
1998-06-05
期刊：
影响因子：
3.7
通讯作者：
Lloyd, RE
中科院分区：
文献类型：
--
作者：
Bovee, ML;Lamphear, BJ;Lloyd, RE
先前，柯萨奇病毒B4（CVB4）和人鼻病毒2型（HRV 2）的纯化的重组2A蛋白酶（2A（pro））显示切割源自人或兔eIF4G的合成肽以及从兔网织红细胞纯化的eIF4G蛋白。这些结果与先前支持脊髓灰质炎病毒感染的HeLa细胞中的eIF4G切割活性需要由脊髓灰质炎病毒（PV）2A（pro）激活的细胞因子的观点的证据相反。在本研究中，重组PV 2A（pro）显示在直接切割反应中切割兔或人eIF4G或其衍生肽，但切割4G衍生肽的效率比脊髓灰质炎病毒多聚蛋白衍生肽低100倍。在这些实验中，需要2A（pro）相对于eIF4G蛋白高达25倍摩尔过量以引起大于50%的切割。还在与PV 2A（pro）相同的条件下在肽切割测定中测试了CVB4 2A（pro），并且发现CVB4 2A（pro）以与PV 2A（pro）相似的效率切割所有eIF4G底物。最后，利用在CVB4和HRV 2蛋白酶的切割位点处含有G486 E取代的重组eIF4G的切割反应导致PV 2A（pro）的切割显著降低，与先前用HRV 2和CVB4 2A（pro）观察到的降低类似，证实所有三种病毒2A蛋白酶识别eIF4G上的相同切割位点。这些数据显示PV 2A（pro）可以在体外直接切割eIF4G，其效率与CVB 2A（pro）的效率相似，但是eIF4G的切割效率比源自脊髓灰质炎病毒多蛋白上的真实2A切割位点的肽的切割效率低约1000倍。(C)北京：科学出版社.
Previously, the purified recombinant 2A proteases (2A(pro)) of coxsackievirus B4 (CVB4) and human rhinovirus type 2 (HRV2) were shown to cleave synthetic peptides derived from human or rabbit eIF4G as well as eIF4G protein purified from rabbit reticulocytes. These results were in contrast to previous evidence which supported the view that eIF4G cleavage activity in poliovirus-infected HeLa cells required a cellular factor(s) activated by poliovirus (PV) 2A(pro). In the present study, recombinant PV 2A(pro) was shown to cleave either rabbit or human eIF4G or their derived peptides in direct cleavage reactions, but cleaved the 4G-derived peptides with 100-fold lower efficiency than with a peptide derived from the poliovirus polyprotein. in these experiments, up to 25-fold molar excess of 2A(pro) over eIF4G protein was required to cause greater than 50% cleavage. CVB4 2A(pro) was also tested in peptide cleavage assays under the same conditions as PV 2A(pro) and was found to cleave all eIF4G substrates with efficiencies similar to PV 2A(pro). Finally, cleavage reactions utilizing recombinant eIF4G containing a G486E substitution at the cleavage site for CVB4 and HRV2 proteases resulted in drastically reduced cleavage by PV 2A(pro), similar to the reduction previously seen with HRV2 and CVB4 2A(pro), confirming that all three viral 2A proteases recognize the same cleavage site on eIF4G. These data show that PV 2A(pro) can directly cleave eIF4G in vitro with efficiencies similar to those of CVB 2A(pro), but cleavage efficiency of eIF4G is approximately 1000-fold lower than cleavage of a peptide derived from the authentic 2A cleavage site on the poliovirus polyprotein. (C) 1998 Academic Press.