Noueiry Amine O, Diez Juana, Falk Shaun P, Chen Jianbo, Ahlquist Paul
Institute for Molecular Virology. Howard Hughes Medical Institute, University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
Mol Cell Biol. 2003 Jun;23(12):4094-106. doi: 10.1128/MCB.23.12.4094-4106.2003.
Previously, we used the ability of the higher eukaryotic positive-strand RNA virus brome mosaic virus (BMV) to replicate in yeast to show that the yeast LSM1 gene is required for recruiting BMV RNA from translation to replication. Here we extend this observation to show that Lsm1p and other components of the Lsm1p-Lsm7p/Pat1p deadenylation-dependent mRNA decapping complex were also required for translating BMV RNAs. Inhibition of BMV RNA translation was selective, with no effect on general cellular translation. We show that viral genomic RNAs suitable for RNA replication were already distinguished from nonreplication templates at translation, well before RNA recruitment to replication. Among mRNA turnover pathways, only factors specific for deadenylated mRNA decapping were required for BMV RNA translation. Dependence on these factors was not only a consequence of the nonpolyadenylated nature of BMV RNAs but also involved the combined effects of the viral 5' and 3' noncoding regions and 2a polymerase open reading frame. High-resolution sucrose density gradient analysis showed that, while mutating factors in the Lsm1p-7p/Pat1p complex completely inhibited viral RNA translation, the levels of viral RNA associated with ribosomes were only slightly reduced in mutant yeast. This polysome association was further verified by using a conditional allele of essential translation initiation factor PRT1, which markedly decreased polysome association of viral genomic RNA in the presence or absence of an LSM7 mutation. Together, these results show that a defective Lsm1p-7p/Pat1p complex inhibits BMV RNA translation primarily by stalling or slowing the elongation of ribosomes along the viral open reading frame. Thus, factors in the Lsm1p-7p/Pat1p complex function not only in mRNA decapping but also in translation, and both translation and recruitment of BMV RNAs to viral RNA replication are regulated by a cell pathway that transfers mRNAs from translation to degradation.
此前，我们利用高等真核生物正链RNA病毒雀麦花叶病毒（BMV）在酵母中复制的能力，证明酵母LSM1基因是将BMV RNA从翻译过程招募至复制过程所必需的。在此，我们拓展了这一观察结果，表明Lsm1p以及Lsm1p - Lsm7p/Pat1p去腺苷酸化依赖性mRNA脱帽复合体的其他组分对于BMV RNA的翻译也是必需的。BMV RNA翻译的抑制具有选择性，对一般细胞翻译没有影响。我们发现，适用于RNA复制的病毒基因组RNA在翻译时就已与非复制模板区分开来，远早于RNA被招募至复制过程。在mRNA周转途径中，BMV RNA翻译仅需要对去腺苷酸化的mRNA脱帽具有特异性的因子。对这些因子的依赖性不仅是BMV RNA非多聚腺苷酸化性质的结果，还涉及病毒5'和3'非编码区以及2a聚合酶开放阅读框的综合作用。高分辨率蔗糖密度梯度分析表明，虽然突变Lsm1p - 7p/Pat1p复合体中的因子会完全抑制病毒RNA翻译，但在突变酵母中与核糖体相关的病毒RNA水平仅略有降低。通过使用必需翻译起始因子PRT1的条件等位基因进一步验证了这种多核糖体关联，该等位基因在存在或不存在LSM7突变的情况下均显著降低了病毒基因组RNA与多核糖体的关联。总之，这些结果表明，有缺陷的Lsm1p - 7p/Pat1p复合体主要通过使核糖体沿病毒开放阅读框的延伸停滞或减慢来抑制BMV RNA翻译。因此，Lsm1p - 7p/Pat1p复合体中的因子不仅在mRNA脱帽中起作用，还在翻译中起作用，并且BMV RNA的翻译和招募至病毒RNA复制均受一个将mRNA从翻译转移至降解的细胞途径调控。
