Castelló Alfredo, Sanz Miguel Angel, Molina Susana, Carrasco Luis
Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Facultad de Ciencias, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain.
J Mol Biol. 2006 Feb 3;355(5):942-56. doi: 10.1016/j.jmb.2005.11.024. Epub 2005 Nov 28.
The infection of baby hamster kidney (BHK) cells by Sindbis virus gives rise to a drastic inhibition of cellular translation, while under these conditions the synthesis of viral structural proteins directed by the subgenomic 26S mRNA takes place efficiently. Here, the requirement for intact initiation factor eIF4G for the translation of this subgenomic mRNA has been examined. To this end, SV replicons that contain the protease of human immunodeficiency virus type 1 (HIV-1) or the poliovirus 2A(pro) replacing the sequences of SV glycoproteins have been constructed. BHK cells electroporated with the different RNAs synthesize protein C and the corresponding protease at late times. Notably, the proteolysis of eIF4G by both proteases has little effect on the translation of the 26S mRNA. In addition, recombinant viable SVs were engineered that encode HIV-1 PR or poliovirus 2A protease under the control of a duplicated late promoter. Viral protein synthesis at late times of infection by the recombinant viruses is slightly affected in BHK cells that contain proteolysed eIF4G. The translatability of SV genomic 49S mRNA was assayed in BHK cells infected with a recombinant virus that synthesizes luciferase and transfected with a replicon that expresses poliovirus 2Apro. Under conditions where eIF4G has been hydrolysed significantly the translation of genomic SV RNA was deeply inhibited. These findings indicate a different requirement for intact eIF4G in the translation of genomic and subgenomic SV mRNAs. Finally, the translation of the reporter gene that encodes green fluorescent protein, placed under the control of a second duplicate late promoter, is also resistant to the cleavage of eIF4G. In conclusion, despite the presence of a cap structure in the 5' end of the subgenomic SV mRNA, intact eIF4G is not necessary for its translation.
辛德毕斯病毒感染幼仓鼠肾（BHK）细胞会导致细胞翻译受到强烈抑制，而在这些条件下，由亚基因组26S mRNA指导的病毒结构蛋白合成却能高效进行。在此，研究了完整的起始因子eIF4G对该亚基因组mRNA翻译的需求。为此，构建了含有1型人类免疫缺陷病毒（HIV-1）蛋白酶或脊髓灰质炎病毒2A（pro）替代SV糖蛋白序列的SV复制子。用不同RNA进行电穿孔的BHK细胞在后期合成蛋白C和相应的蛋白酶。值得注意的是，两种蛋白酶对eIF4G的蛋白水解对26S mRNA的翻译影响很小。此外，构建了重组活SV，其在重复的晚期启动子控制下编码HIV-1蛋白酶或脊髓灰质炎病毒2A蛋白酶。在含有被蛋白水解的eIF4G的BHK细胞中，重组病毒感染后期的病毒蛋白合成受到轻微影响。在感染了合成荧光素酶的重组病毒并转染了表达脊髓灰质炎病毒2Apro的复制子的BHK细胞中，检测了SV基因组49S mRNA的可翻译性。在eIF4G被显著水解的条件下，基因组SV RNA的翻译受到深度抑制。这些发现表明，基因组和亚基因组SV mRNA的翻译对完整eIF4G的需求不同。最后，置于第二个重复晚期启动子控制下的编码绿色荧光蛋白的报告基因的翻译也对eIF4G的切割具有抗性。总之，尽管亚基因组SV mRNA的5'端存在帽结构，但其翻译并不需要完整的eIF4G。
