In our study, we characterized the anti-HEV effect of the natural compound silvestrol as part of the screening approach for novel HEV inhibitors. Silvestrol is already known from literature as potent antitumor as well as antiviral agent and was originally found in a screen for eukaryotic initiation factor 4A (eIF4A) activity inhibitors by Bordeleau et al. (Bordeleau et al., 2008). They showed that silvestrol inhibits translation initiation by modulating the eIF4A helicase and thereby alters the drug sensitivity of cancer cells. The compound seems to induce dimerization and it increases the binding affinity between eIF4A helicase and RNA. This results in a dysregulation of the ribosome recruitment and in an inability to bind the translations preinitiation complex (Bordeleau et al., 2008; Cencic et al., 2009). Subsequently, different research groups demonstrated a significant therapeutic activity of silvestrol on mouse tumor models targeting the translation machinery in cancer (Kogure, Takayuki et al., 2013; Patton et al., 2015). From that it became clear, that the eIF4A helicase represents an essential drug target that is involved in the host cellular respectively oncogenic protein synthesis.
Similar to cancer cells, most viruses including HEV rely on the host cellular machinery using the translation mechanisms to synthesize their own viral proteins. The HEV mRNA is capped at the 5’ end and contains a short 5’ untranslated region (UTR). This indicates that the virus is dependent on cap-recognizing proteins for an efficient RNA replication. In accordance with that, Zhou et al. previously showed that HEV indeed requires the host cellular eIF4F complex for RNA translation (Zhou et al., 2015). As a heterotrimeric protein complex, eIF4F is composed of the DEAD-box helicase eIF4A, a 5ʹ mRNA cap-binding subunit eIF4E and the large scaffolding protein eIF4G. The function of the complex is to recruit the mRNA to the ribosomes and to facilitate the scanning of the 5’ UTRs (Bhat et al., 2015). This process is referred to as ‘translation initiation’ of the eIF4F complex and is the first and most rate-limiting step of translation. During the translation initiation the eIF4A RNA helicase unwinds RNA secondary structures in the 5’ -UTRs of mRNAs. By this mechanism, silvestrol interferes with the translation. Sadlish et al.
identified eIF4A mutants that are resistant to silvestrol, which further proved that eIF4A has to be the molecular target of silvestrol (Sadlish et al., 2013).
As HEV is expected to require the cellular translation machinery including the eIF4F complex in order to initiate the translation of the viral proteins, we investigated the effects of silvestrol on the HEV life cycle. First, we explored the inhibitory effects of silvestrol in a HEV replicon system
Discussion and Outlook 25 with the human hepatoma cell line Huh7.5. Our following observations that silvestrol inhibited the replication of different subgenomic HEV replicons in a dose-dependent manner were consistent with the earlier findings (Bordeleau et al., 2008; Cencic et al., 2009). The different HEV replicons included a gt1 construct, a gt3-based wild board isolate, the most used gt3 construct p6 and a variant of p6 with a mutation in the polymerase region (G1634R), that leads to a higher replication fitness. For all constructs we observed an inhibitory effect at low nanomolar concentrations of silvestrol. Whereas the inhibitory concentrations 50 (IC50) of silvestrol ranged between 4 and 6.6 nM, the IC50 for RBV were 1000-fold higher. It is remarkable that for the same inhibitory effect we needed far less silvestrol than RBV since RBV is the mostly used HEV inhibitor. Then the inhibition efficiency of silvestrol on HEV replication was compared to a replicon construct based on HCV. HCV translation requires an internal ribosomal entry side (IRES) and is therefore independent of the eIF4A helicase cap-driven translation initiation. That explains the modest inhibition of HCV replication in our experiments and supports the expected mode of action of silvestrol to modulate the eIF4A helicase. As silvestrol targets host factors, it was important to monitor the cell viability under treatment. Throughout the whole study we assessed silvestrol toxicity using a MTT assay and determined viable cells via visual cell counting to exclude severe antiproliferative properties of silvestrol. After 24h treatment we observed only small toxic effects and the cytotoxic concentration 50 (CC50) was never reached. Longer incubation periods caused a decreasing cell metabolic activity although after 42h the combined values of IC50 and CC50 still resulted in a selectivity index of about 8.6 for the p6 strain. These findings lead to the prediction that within a therapeutic window silvestrol can be applied to efficiently inhibit HEV replication at non-toxic concentrations.
To further confirm our results, we performed viral infection experiments. In two different assay setups simulating the whole HEV viral life cycle, we observed inhibitory effects of silvestrol on infectious viral particle production and HEV RNA copy numbers. In the first HEV infection assay HepG2/C3A cells were infected with cell culture-derived infectious HEV (HEVcc) and the impact of silvestrol at different concentrations and incubation periods was tested. The most efficient inhibition was reached with 20 nM and 50 nM silvestrol at a late time point. Surprisingly the overall antiviral activity of silvestrol was less prominent in that assay than in the before described experiments with Huh7.5 cells. Therefore, a direct comparison of the antiviral effect was performed on HEVp6 replication in HepG2/C3A and Huh7.5 cells. The results confirmed our observations and showed a shift of the dose-response curves with a higher effectivity of silvestrol in transfected Huh7.5. Although the reasons for these differences are not immediately apparent, they may be
Discussion and Outlook 26 related to the complex mode of action of silvestrol. On the one hand the translation of the viral RNA is inhibited and on the other hand the translation of host cellular mRNAs. Because the host cellular mRNAs vary from cell line to cell line, the differences of silvestrol’s effectivity might be explained.
To underline the specificity of silvestrol on HEV replication, pluripotent stem cell-derived hepatocyte-like cells (HLCs) were used. These cells are not cancer-derived cell lines and do not have alterations in metabolic, innate immune and apoptotic responses. Therefore, HLCs offer a more physiologically relevant cell culture system (Wu et al., 2018). The cells were infected with patient-derived HEV from genotypes 1–4 and treated with silvestrol post-infection. No cytotoxicity due to treatment was observed but a clear reduction of HEV RNA copy numbers in infected HLCs for all four different genotypes. This assay proved a pangenotypic effect of the compound and an even more pronounced inhibition of HEV infection in these cells.
The results raised the question whether treatment with silvestrol would also inhibit HEV infection in vivo. As mentioned above silvestrol was originally discovered as anti-cancer compound and in the beginning mainly characterized in anti-cancer studies. Several research groups showed that silvestrol effectively inhibits tumor growth in vivo without evidence of toxicity (Cencic et al., 2009; Lucas et al., 2009; Alinari et al., 2012; Kogure, T. et al., 2013). The studies demonstrated how crucial the translational control in cancer development and progression is (Silvera et al., 2010). The treatment with silvestrol seemed to affect translation initiation in the tumor cells, leading to a reduced progression of tumor spread in the different animal models.
The study of HEV infection was hampered for a long time because of the absence of a small animal model. Ordinary mice cannot be infected with HEV and only lately researchers are able to infect immune-deficient human liver chimeric mice with HEV. This animal model allows the preclinical in vivo evaluation of novel antiviral compounds (Sayed et al., 2017). Based on the antitumor in vivo studies, the chronically infected animals were treated intraperitoneal with silvestrol for either six or ten days. The administration of silvestrol was effective within the first days, resulting in a rapid decline of HEV RNA levels in the feces compared to the control group. Although there was only a small number of animals tested, this first successful in vivo study validated the promising therapeutic potential of silvestrol and can lead to further silvestrol-based assays to improve established antiviral strategies.
Discussion and Outlook 27 Previously to our study, Biedenkopf et al. published a paper on the inhibitory effects of silvestrol on Ebola virus (EBOV) replication. Like HEV and all other viruses, EBOV protein synthesis depends on the cellular translation machinery. Biedenkopf et al. showed for the first time that specifically targeting the host factor eIF4A with silvestrol leads to a sufficient inhibition of the viral propagation before any effects on cellular functions occur (Biedenkopf et al., 2017). Further studies also reported antiviral effects of silvestrol on other RNA viruses, including corona- and picornaviruses, Zika and Chikungunya (Müller, C. et al., 2018; Elgner et al., 2018; Henss et al., 2018).
In contrast to direct-acting antivirals that target viral proteins, molecules like silvestrol interfere with cellular factors involved in the viral life cycle. On the one hand, targeting host factors bears the risk to inhibit essential metabolic processes required for cellular homeostasis. On the other hand, agents that inhibit host factors have the advantage that they are less susceptible to escape mutations by viruses. That feature can hamper the evolution of viral evasion mechanisms and prevent viral resistance (Müller, K. et al., 2012; Zeisel et al., 2015). Especially HEV is a virus where escape mutations play a major role whether a treatment leads to viral clearance or not.
Deep sequencing results revealed that several HEV RNA mutations were the reason for ribavirin resistance and treatment failure in patients (Debing, Y., Ramiere et al., 2016; Lhomme et al., 2016; Todt, Gisa et al., 2016). Generally, RNA viruses like HEV consist of so-called mutant clouds.
In HEV infected individuals the viral population contains a repertoire of HEV variants that comprises genomes that are similar but not-identical. High replication rates and high error-rates of the RdRP are the source of those mutant clouds. The mutations facilitate virus adaptation and evasion of selective constraints like antiviral agents. (Domingo et al., 2012). Using a combination of drugs with different targets and mechanisms of action, may prevent viral resistance in the course of HEV infection. Moreover, possible synergistic antiviral effects of two compounds have the advantage of a reduction in the necessary doses, minimizing the risk of toxicity and side effects.
Therefore, we evaluated a combinatory treatment of silvestrol and the known HEV-inhibitor ribavirin. To analyze the interactions between the two compounds, two different models were used. The calculated combination indices as well as the three-dimensional surface plot indicated that the combination of silvestrol with RBV has additive antiviral effects in the HEV replication system. Although clear synergistic effects could not be observed, the combination may offer an option in case of treatment failure as soon as an optimal ratio of the drugs in vivo is identified.
Discussion and Outlook 28 In the course of our study the new compound silvestrol was identified which inhibits HEV efficiently in vitro as well as in vivo. Perhaps the still limited treatment options of HEV infections reflect the long-lasting ignorance of HEV as a global pathogen. Currently, ribavirin is the only effective antiviral drug against HEV. Particularly for pregnant women there is a great need for alternative treatments. Unfortunately, specifically ribavirin is contraindicated in pregnant women, who disproportionately suffer from the symptoms of HEV infection. By investigating the anti-HEV properties of silvestrol and additive effects with RBV, our study tried to make a valuable contribution to the field of drug research and future treatment strategies of hepatitis E. Targeting the host factors eIF4A during cap-dependent translation initiation is a promising broad-spectrum antiviral strategy. Both, in our HEV cell culture systems as well as in the animal model system, viral replication of different HEV genotypes was blocked without major toxic side effects. However, many aspects about possible adverse effects in humans are so far completely unknown and there is still a long way to go until silvestrol might be used as proper drug against Hepatitis E, Ebola or other viruses, where antiviral effects are observed.
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