Etchison D, Smith K
Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City 66103.
J Biol Chem. 1990 May 5;265(13):7492-500.
Poliovirus infection of HeLa cells results in cleavage of the p220 subunit of eukaryotic initiation factor eIF-4F and inhibits cap-dependent initiation of protein synthesis. To examine the effect of virus-induced inhibition on the structure of initiation factor complexes involved in cap binding, the polypeptide compositions of cap affinity-purified complexes from uninfected and poliovirus-infected HeLa cells were analyzed. Monoclonal antibodies directed against p220 and an eIF-3 subunit, p170, were utilized to locate eIF-3 and eIF-4F on sucrose gradients and in fractions eluting from cap analog columns. This approach resulted in the purification of several different cap-binding complexes from different cellular subfractions and revealed significant differences in their composition after infection. The results indicate that eIF-3 and eIF-4F bind to the cap structure, possibly in the form of a complex, and that a modified form of eIF-3 alone has some cap-binding activity in the complete absence of p220, eIF-4A, and eIF-4E. Ribosome-derived complexes containing cleaved p220 are no longer associated with eIF-3 or eIF-4A, and a significant amount of cleaved p220 is associated with a unique cytoplasmic cap-binding complex. The cytoplasmic complex also contains Mr = 170,000 and 80,000 polypeptides, neither of which are major components of eIF-4F. These results demonstrate significant variation in the composition of cap-binding complexes from both infected and uninfected cells. They indicate that eIF-3 might play a direct role in cap binding and suggest that poliovirus-induced cleavage of p220 results in the release of the eIF-4A subunit from eIF-4F and abolishes an association between eIF-4F and eIF-3 which may function during the multifactor steps involved in initiation of cap-mediated translation.
脊髓灰质炎病毒感染HeLa细胞会导致真核起始因子eIF-4F的p220亚基裂解，并抑制帽依赖性蛋白质合成起始。为了研究病毒诱导的抑制作用对参与帽结合的起始因子复合物结构的影响，分析了来自未感染和脊髓灰质炎病毒感染的HeLa细胞的帽亲和纯化复合物的多肽组成。利用针对p220和eIF-3亚基p170的单克隆抗体，在蔗糖梯度以及从帽类似物柱洗脱的级分中定位eIF-3和eIF-4F。这种方法从不同细胞亚组分中纯化出了几种不同的帽结合复合物，并揭示了感染后它们在组成上的显著差异。结果表明，eIF-3和eIF-4F可能以复合物的形式与帽结构结合，并且在完全不存在p220、eIF-4A和eIF-4E的情况下，单独的一种修饰形式的eIF-3具有一些帽结合活性。含有裂解p220的核糖体衍生复合物不再与eIF-3或eIF-4A相关联，并且大量裂解的p220与一种独特的细胞质帽结合复合物相关联。细胞质复合物还含有分子量为170,000和80,000的多肽，这两种多肽都不是eIF-4F的主要成分。这些结果证明了来自感染和未感染细胞的帽结合复合物在组成上存在显著差异。它们表明eIF-3可能在帽结合中起直接作用，并表明脊髓灰质炎病毒诱导的p220裂解导致eIF-4A亚基从eIF-4F中释放，并消除了eIF-4F与eIF-3之间的关联，而这种关联可能在帽介导的翻译起始所涉及的多因子步骤中发挥作用。
