Dual Mechanism for the Translation of Subgenomic mRNA from Sindbis Virus in Infected and Uninfected Cells

Infection of BHK cells by Sindbis virus (SV) gives rise to a profound inhibition of cellular protein synthesis, whereas translation of viral subgenomic mRNA that encodes viral structural proteins, continues for hours. To gain further knowledge on the mechanism by which this subgenomic mRNA is translated, the requirements for some initiation factors (eIFs) and for the presence of the initiator AUG were examined both in infected and in uninfected cells. To this end, BHK cells were transfected with different SV replicons or with in vitro made SV subgenomic mRNAs after inactivation of some eIFs. Specifically, eIF4G was cleaved by expression of the poliovirus 2A protease (2Apro) and the alpha subunit of eIF2 was inactivated by phosphorylation induced by arsenite treatment. Moreover, cellular location of these and other translation components was analyzed in BHK infected cells by confocal microscopy. Cleavage of eIF4G by poliovirus 2Apro does not hamper translation of subgenomic mRNA in SV infected cells, but bisection of this factor blocks subgenomic mRNA translation in uninfected cells or in cell-free systems. SV infection induces phosphorylation of eIF2��, a process that is increased by arsenite treatment. Under these conditions, translation of subgenomic mRNA occurs to almost the same extent as controls in the infected cells but is drastically inhibited in uninfected cells. Notably, the correct initiation site on the subgenomic mRNA is still partially recognized when the initiation codon AUG is modified to other codons only in infected cells. Finally, immunolocalization of different eIFs reveals that eIF2 �� and eIF4G are excluded from the foci, where viral RNA replication occurs, while eIF3, eEF2 and ribosomes concentrate in these regions. These findings support the notion that canonical initiation takes place when the subgenomic mRNA is translated out of the infection context, while initiation can occur without some eIFs and even at non-AUG codons in infected cells.辛德毕斯病毒（Sindbis virus, SV）感染叙利亚仓鼠肾细胞（BHK cells）后，会强烈抑制宿主细胞的蛋白质合成，而编码病毒结构蛋白的病毒亚基因组mRNA（subgenomic mRNA）的翻译却可持续数小时。为进一步阐明该亚基因组mRNA的翻译机制，研究人员分别在感染与未感染的细胞中，探究了部分真核起始因子（eIFs）的需求，以及起始密码子AUG的必要性。 为此，研究人员先灭活部分真核起始因子，随后用不同的辛德毕斯病毒复制子（replicons）或体外制备的辛德毕斯病毒亚基因组mRNA转染BHK细胞。具体操作包括：通过表达脊髓灰质炎病毒（poliovirus）2A蛋白酶（2Apro）切割真核起始因子4G（eIF4G）；通过亚砷酸盐处理诱导磷酸化，使真核起始因子2（eIF2）的α亚基失活。此外，研究人员还通过共聚焦显微镜（confocal microscopy），分析了SV感染的BHK细胞中各类翻译相关组分的细胞定位。 脊髓灰质炎病毒2A蛋白酶对eIF4G的切割，并不会阻碍SV感染细胞中亚基因组mRNA的翻译，但该因子的裂解会阻断未感染细胞或无细胞体系（cell-free systems）中的亚基因组mRNA翻译。SV感染可诱导eIF2α发生磷酸化，亚砷酸盐处理可进一步增强该磷酸化过程。在此条件下，感染细胞中亚基因组mRNA的翻译水平与对照组几乎无差异，但未感染细胞中的翻译则被显著抑制。 值得注意的是，仅在感染细胞中，当亚基因组mRNA的起始密码子AUG被突变为其他密码子时，其正确的翻译起始位点仍可被部分识别。最后，不同真核起始因子的免疫定位结果显示，eIF2α与eIF4G被排除在病毒RNA复制灶之外，而eIF3、肽链延伸因子2（eEF2）与核糖体则聚集于这些区域。 上述研究结果表明，在非感染环境下，亚基因组mRNA的翻译遵循经典的翻译起始机制；而在感染细胞中，其翻译可无需部分真核起始因子，甚至可在非AUG密码子处起始。