Department of Microbiology and National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA.
Department of Microbiology and National Emerging Infectious Diseases Laboratories, Boston University, Boston, Massachusetts, USA
J Virol. 2018 Sep 26;92(20). doi: 10.1128/JVI.01260-18. Print 2018 Oct 15.
Polyamines and hypusinated eIF5A have been implicated in the replication of diverse viruses; however, defining their roles in supporting virus replication is still under investigation. We have previously reported that Ebola virus (EBOV) requires polyamines and hypusinated eIF5A for replication. Using a replication-deficient minigenome construct, we show that gene expression, in the absence of genome replication, requires hypusinated eIF5A. Additional experiments demonstrated that the block in gene expression upon hypusine depletion was posttranscriptional, as minigenome reporter mRNA transcribed by the EBOV polymerase accumulated normally in the presence of drug treatment where protein did not. When this mRNA was isolated from cells with low levels of hypusinated eIF5A and transfected into cells with normal eIF5A function, minigenome reporter protein accumulation was normal, demonstrating that the mRNA produced was functional but required hypusinated eIF5A function for translation. Our results support a mechanism in which hypusinated eIF5A is required for the translation, but not synthesis, of EBOV transcripts. In contrast, depletion of polyamines with difluoromethylornithine (DFMO) resulted in a strong block in the accumulation of EBOV polymerase-produced mRNA, indicating a different mechanism of polyamine suppression of EBOV gene expression. Supplementing with exogenous polyamines after DFMO treatment restored mRNA accumulation and luciferase activity. These data indicate that cellular polyamines are required for two distinct aspects of the EBOV life cycle. The bifunctional requirement for polyamines underscores the importance of these cellular metabolites in EBOV replication and suggests that repurposing existing inhibitors of this pathway could be an effective approach for EBOV therapeutics. Ebola virus is a genetically simple virus that has a small number of proteins. Because of this, it requires host molecules and proteins to produce new infectious virus particles. Though attention is often focused on cellular proteins required for this process, it has recently been shown that cellular metabolites such as polyamines are also necessary for EBOV replication. Here we show that polyamines such as spermine and spermidine are required for the accumulation of EBOV mRNA and that eIF5A, a molecule modified by spermidine, is required for the translation, but not the production, of EBOV mRNAs. These findings suggest that effectively targeting this pathway could provide a biphasic block of EBOV replication.
多胺和 Hypusinated eIF5A 已被牵连到多种病毒的复制中；然而，定义它们在支持病毒复制中的作用仍在研究中。我们之前曾报道过埃博拉病毒（EBOV）需要多胺和 Hypusinated eIF5A 来进行复制。使用复制缺陷的小基因组长达 1.2kb 的 RNA 结构，我们表明在没有基因组复制的情况下，基因表达需要 Hypusinated eIF5A。进一步的实验表明，在 Hypusine 耗尽时基因表达的阻断是转录后，因为在药物处理存在的情况下，EBOV 聚合酶转录的小基因组长达 1.2kb 的 RNA 正常积累，而蛋白质则没有。当这种 mRNA 从小基因组长达 1.2kb 的 RNA 中分离出来并转染到具有正常 eIF5A 功能的细胞中时，小基因组长达 1.2kb 的 RNA 结构的报告蛋白积累正常，这表明产生的 mRNA 是有功能的，但需要 Hypusinated eIF5A 功能来进行翻译。我们的结果支持这样一种机制，即 Hypusinated eIF5A 是 EBOV 转录物翻译所必需的，但不是合成所必需的。相比之下，用二氟甲基鸟氨酸（DFMO）耗竭多胺会导致 EBOV 聚合酶产生的 mRNA 积累受到强烈阻断，这表明多胺对 EBOV 基因表达的抑制作用有不同的机制。DFMO 处理后补充外源性多胺可恢复 mRNA 积累和荧光素酶活性。这些数据表明，细胞多胺是 EBOV 生命周期的两个不同方面所必需的。多胺的双功能需求突出了这些细胞代谢物在 EBOV 复制中的重要性，并表明重新利用该途径现有的抑制剂可能是一种有效的 EBOV 治疗方法。埃博拉病毒是一种遗传简单的病毒，只有少数几种蛋白质。正因为如此，它需要宿主分子和蛋白质来产生新的传染性病毒颗粒。尽管人们通常关注的是这一过程所需的细胞蛋白，但最近有研究表明，细胞代谢物如多胺也对 EBOV 复制是必要的。在这里，我们表明，多胺如 spermine 和 spermidine 是 EBOV mRNA 积累所必需的，并且 eIF5A，一种被 spermidine 修饰的分子，是 EBOV mRNAs 翻译所必需的，但不是产生所必需的。这些发现表明，有效地靶向该途径可以提供 EBOV 复制的双相阻断。
