Differential Mechanisms for the Involvement of Polyamines and Hypusinated eIF5A in Ebola Virus Gene Expression

Abstract

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.

Difluoromethylornithine, a Decarboxylase 1 Inhibitor, Suppresses Hepatitis B Virus Replication by Reducing HBc Protein Levels

“…In addition, hypusination of eIF5A, a unique posttranslational modification of an aminobutyl moiety from the spermidine at the Lys50 site via deoxyhypusine synthase (DHPS), is necessary for viral protein translation (Olsen and Connor, 2017). As a result, reducing the levels of spermidine by DFMO treatment can also inhibits the expression of EBOV minigenome (Olsen et al, 2016(Olsen et al, , 2018. In our research, we revealed that DFMO reduces the HBc protein levels by promoting its ubiquitination (Figure 6), as a consequence, inhibiting HBV capsids levels and DNA replication (Figure 3).…”

Diverse Functions of Polyamines in Virus Infection

“…In addition to roles in polymerase activity, CHIKV, ZIKV, and HCV translation rely on polyamines, as DFMO-treated (polyamine depleted via ODC1 inhibition) cells exhibited reduced viral translation [43,47]. Further work in Ebola virus (EBOV) and Marburgvirus (MARV) highlighted that polyamines function in transcription from viral genomes, but the translation of these transcripts relied on polyamines through eIF5A hypusination ( Figure 3) [48,49]. The knockdown of eIF5A or inhibition of the enzymes involved in its hypusination resulted in significant decreases in the accumulation of viral proteins.…”

“…A recent review summarizes the promise of these molecules as antiviral agents [85]. Studies by Olsen and colleagues [48,49] demonstrated the efficacy of these inhibitors against Ebola virus infection, and prior work showed that HIV-1 is similarly sensitive to this group of inhibitors [86]. In sum, several polyamine-targeting molecules show activity against viruses, and further work into their mechanisms of action, toxicity, and in vivo activity is required for the possibility of targeting polyamines to treat or prevent viral infection.…”

Current status of small molecule drug development for Ebola virus and other filoviruses

“…Hsp90 inhibitors demonstrate inhibition of EBOV in cell culture, likely through the destabilization of EBOV L [23,62]. It has also been demonstrated that inhibition of polyamine biosynthesis, such as by 2-difluoromethylornithine (DFMO), and hypusination, by N1-guanyl-1,7-diamineheptane (GC7) and ciclopirox, reduces EBOV replication [63,64].…”