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 the block in gene expression upon hypusine depletion was post-transcriptional， as minigenome reporter mRNA transcribed by the EBOV polymerase accumulated normally in the presence of drug treatment where protein did not. When thi... More
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 the block in gene expression upon hypusine depletion was post-transcriptional， 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 of， but not synthesis of， EBOV transcripts. In contrast， depletion of polyamines with DFMO resulted in a strong block in the accumulation of EBOV polymerase-produced mRNA， indicating a distinct mechanism of polyamine suppression of EBOV gene expression. Supplementing with exogenous polyamines after DFMO treatment restored mRNA accumulation and luciferase activity. These data indicate cellular polyamines are required for two distinct aspects of the EBOV lifecycle. 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. Ebolavirus 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 of， but not the production of， EBOV mRNAs. These findings suggest that effectively targeting this pathway could provide a biphasic block of EBOV replication.
