Hepatitis E: Classification and Molecular Virology

3 Introduction

3.2 Hepatitis E: Classification and Molecular Virology

The Hepatitis E Virus is a member of the family of Hepeviridae which is divided into two different genera, Orthohepevirus and Piscihepevirus. The Piscihepevirus genus consists of only the cutthroat trout virus, whereas the Orthohepevirus genus includes further four species (A to D). All HEV strains infecting humans (HEV gt 1-4 and 7) belong to the Orthohepevirus species A. Other isolates

Introduction 7 detected in animals as chicken, rats, ferrets or bats are classified into the species Orthohepevirus A–D and cannot be transmitted to humans (Smith et al., 2014; Meng, 2016).

HEV is a small, icosahedral shaped RNA virus with a single-stranded, positive-sense genome of approximately 7.2 kb. The virion measures 27–32 nm in diameter and is called ‘quasi-enveloped’

since the virus exists in an enveloped and non-enveloped form (Yin et al., 2016). Recent data imply that virions secreted into the bile are non-enveloped whereas virions found in the bloodstream are wrapped in cellular membranes (Debing, Y. et al., 2016).

The viral genome consists of three open reading frames (ORFs), which are additionally framed by untranslated regions as depicted in Figure 2. The 5’-non-coding region is capped with a 7-methylguanosine and the 3’ end is polyadenylated. ORF1 encodes the non-structural proteins that are essential for the HEV RNA replication including a methyltransferase (MT), cysteine protease (Pro), RNA helicase (Hel) and RNA-dependent RNA polymerase (Pol). The function of the Y-domain, polyproline region (PPR) and the X-domain are mainly unknown so far. An additional ORF4 in the HEV genome of genotype 1 increases the virus polymerase activity (Kamar, Izopet et al., 2017; Nimgaonkar et al., 2018). The ORF2 is translated into the 660-amino-acid virus capsid protein which is important for particle assembly and host cell binding. Montpellier et al.

reported that during infection at least 3 different forms of the capsid protein are processed of which only one is associated with infectious particles (Montpellier et al., 2018). Finally, the short ORF3 encodes for a viroporin which is involved in the release of infectious particles (Ding et al., 2017). Furthermore, the ORF3 protein interacts with a variety of other host proteins which are assumed to create a host environment that facilitates the viral life cycle within the cell (Chandra et al., 2008).

Figure 2: Genetic organization of hepatitis E virus. (Kamar, Izopet et al., 2017) (Reprint with permission granted by Springer Nature)

Introduction 8 Although HEV is a primary hepatotropic and non-cytopathic virus, several studies have shown that HEV is also able to replicate in other tissues such as neuronal cells, placental cells, kidney cells and cells of the gastrointestinal tract. (Bose et al., 2014; Drave et al., 2016; Geng et al., 2016;

Kamar et al., 2016).

The viral life cycle of HEV is only partially understood. The key steps of infection, replication, assembly and release in the hepatocytes are depicted in Figure 3. Initially the virus attaches to heparan sulfate proteoglycans on the surface and enters the cell through a receptor-dependent clathrin-mediated endocytosis (Kalia et al., 2009; Kapur et al., 2012). After uncoating, the viral RNA is translated by host proteins into the polyprotein of ORF1. Then the viral RNA polymerase is able to transcribe the positive-sense (+) RNA into a full-length negative-sense (-) transcript.

This serves as a template to produce more full-length (+) RNA as well as subgenomic (sg) RNA that comprises ORF2 and ORF3 (Varma et al., 2011). During this step the RNA helicase plays an important role in unwinding the two RNA strands (Karpe and Lole, 2010). The capsid and ORF3 protein are translated from the sgRNA and then processed within the endoplasmic reticulum (ER). Finally, the viral RNA is packaged to allow formation and assembly of new virions. It is assumed that HEV utilizes the ORF3 protein in connection with the cellular secretory machinery to establish an interaction with the endosomal sorting complexes required for transport (ESCRT) for viral release from the host cell (Nagashima et al., 2011; Nimgaonkar et al., 2018).

The virions get secreted with an additional envelope of ORF3 proteins and lipids. The envelope is destroyed by bile salts when released into the biliary canaliculi. In contrast, virions secreted into the bloodstream maintain the envelope (Okamoto, 2013; Debing, Y. et al., 2016).

Introduction 10 inflammation or injury. During the following icteric phase, the bilirubin level is elevated and patients might suffer from vomiting, diarrhea and itching accompanied by jaundice and dark-colored urine. These symptoms are normally self-limiting. The viral load and the elevated liver enzymes decrease without the requirement of antiviral therapy (Wedemeyer et al., 2012; Dalton et al., 2018). Nevertheless, in some cases infected individuals progress to a fulminate hepatitis with acute liver failure, which leads to mortality rates of 0,5–4% in the population without preexisting liver diseases (Blasco Perrin et al., 2015).

The clinical manifestation of HEV is dependent on the HEV genotype besides the individual host factors. Infections with the tropical genotypes 1 and 2 affect more frequently younger people (15–

35 years) (Purcell and Emerson, 2008). These infections are symptomatic in 15–20% of the cases.

Additionally, gt1 and 2 seem to show a higher manifestation rate with often a more severe progression of the disease (Rein et al., 2012). Especially pregnant women in the second or third trimester are at high risk to develop a life-threating hepatitis caused by an infection with HEV genotype 1. Besides acute liver failure, hemorrhages and stillbirth, the infection in pregnant women results in more than a quarter of the cases in mortality. The underlying pathomechanisms are still mainly unknown (Pérez‐Gracia et al., 2017). Individuals with pre-existing liver diseases more regularly experience a fulminant course of the infection and display higher mortality (Kumar and Saraswat, 2013). In high-income countries elderly males tend to get infected and develop symptoms of acute hepatitis. For the most part, individuals infected with genotypes 3 or 4 develop no symptoms and progression to acute liver failure is uncommon (Hartl et al., 2016).

In immunocompromised patients HEV replication can persist longer than 3–6 months. Only for genotype 3, 4 and 7 chronic infections have been described leading to liver fibrosis, cirrhosis and eventually chronic liver failure (Kamar et al., 2013). As the clinical course of HEV infection is dependent on the strength of the host’s immune system, most chronic hepatitis occur in solid-organ-transplanted (SOT) patients receiving a immunosuppressive therapy (Kamar et al., 2008).

Chronic infection is further associated with any immunosuppression in the context of hematological disorders, rheumatic diseases, stem cell transplantation and HIV-infected individuals.

Although HEV mainly replicates in the liver, extrahepatic manifestations are increasingly diagnosed in association with HEV infection. Most cases are reported for neurological disorders including Guillain-Barré-Syndrome, neuralgic amyotrophy and encephalitis. Furthermore, renal

Introduction 11 injuries, pancreatitis, thyroiditis, myocarditis and hematological disorders have been described as extrahepatic manifestations of HEV (Kamar et al., 2016; Pischke et al., 2017).

Figure 4: HEV infection and hepatitis E. (Kamar, Izopet et al., 2017) (Reprint with permission granted by Springer Nature)

3.4 H epatitis E: Diagnostics

HEV infections can be diagnosed either indirectly by the detection of anti-HEV antibodies (IgM and IgG) in the serum via enzyme-linked immunosorbent assays (ELISA), or directly by the detection of HEV capsid antigen or HEV RNA in the blood or stool through quantitative PCR.

To diagnose HEV-specific antibodies, several ELISA-based assays exist which vary considerably in their specificity and sensitivity, so that they must be evaluated with greatest care (Norder et al., 2016). An acute infection is marked by the detection of anti-HEV IgM in the serum. Within 2–4 weeks following virus transmission, an IgM-response can be detected in immunocompetent individuals. The antibody increase is generally accompanied by elevated liver enzymes and lasts 6–9 months. A positive anti-HEV IgG test indicates a previous HEV infection. The IgG antibodies persist for many years but do not completely protect against re-infection (Huang et al., 2010).

However, immunocompromised patients might have delayed antibody responses or remain antibody negative caused by the immunosuppression. Therefore, immunosuppressed individuals should always be tested via direct methods. The direct detection and quantification of viral RNA via viral nucleic acid testing represents the gold standard of the diagnostics of a current HEV infection. To improve comparability of interlaboratory results, the World Health Organization introduced international standards for HEV PCR techniques in 2013 (Baylis et al., 2013).

Introduction 12 3.5 H epatitis E: Treatm ent

Most hepatitis E infections are self-limited and do not require any antiviral therapy. However, some infected patients are at high risk to progress from acute to chronic hepatitis.

Immunosuppressed individuals represent the majority of patients who do not achieve a viral clearance without treatment. Viral infection is defined as chronic after 3 months of persistent infection (Kamar et al., 2008). A reduction of the immunosuppressive drugs is the first therapeutic option for patients under immunosuppressive therapy. Especially calcineurin and mTOR inhibitors were found to elevate the risk of developing chronic HEV (Wang et al., 2014). Previous studies show that approximately one-third of patients spontaneously clear the virus after the reduction of immunosuppression (Kamar, Abravanel, Selves et al., 2010; Kamar et al., 2011). The second line of therapy is an off-label use of ribavirin (RBV) for chronic as well as acute severe hepatitis.

Kamar et al. published in 2014 a large multicentric study involving 59 chronically infected patients who received SOT. They were treated for 3 months at a median dose of 600 mg RBV per day.

After the treatment period 78% of the patients had achieved a sustained virological response, meaning undetectable HEV RNA levels in the blood 6 months after the end of therapy. Today RBV monotherapy is the only efficient treatment for chronic hepatitis. Nevertheless, treatment failure occurred in some case reports, mainly caused by a RBV dose reduction required because of side-effects as anemia (Kamar et al., 2014).

Several mechanisms of action have been described for RBV. The depletion of intracellular guanosine triphosphate (GTP) pools has been found crucial as antiviral mechanism of RBV in vitro. The inhibition of the inosine monophosphate dehydrogenase by RBV 5’monophosphat leads to a reduced production of GTP which is necessary for viral RNA synthesis (Debing, Yannick et al., 2014). Furthermore, it has been shown that RBV has a mutagenic effect on the HEV genome by inducing mismatches and nucleotide substitutions. On the one hand the increase of mutations can cause the extinction of the virus population. On the other hand, due to the higher mutation rates RBV treatment can result in the selection of viral variants with enhanced viral replication fitness (Todt, Walter et al., 2016). Todt et al., Debing et al. identified several novel single nucleotide variation sites in the viral genome emerging during RBV treatment in patients (Debing, Y., Ramiere et al., 2016; Todt, Gisa et al., 2016). Both studies describe the previously discovered G1634R mutation in the polymerase domain of HEV ORF1 protein which results in a higher replication fitness of the virus in vitro (Debing, Y. et al., 2014). The research groups correlate

Introduction 13 RBV treatment failure in patients with the mutated viral intra-host populations that might have acquired variants leading to an apparent resistance to ribavirin.

As treatment failures occur under RBV, another therapy option is the administration of PEGylated Interferon-α for 3 months. In a small patient cohort, a number of chronically infected individuals successfully cleared HEV. However, interferon therapy has to be evaluated carefully because it stimulates the immune system and can lead to acute rejection in SOT recipients (Kamar, Abravanel, Garrouste et al., 2010; Kamar, Rostaing et al., 2010; Haagsma et al., 2010).

Only recently, a study of Dao Thi et al. showed that sofosbuvir, normally used as potent inhibitor of the hepatitis C virus in clinics, has also some antiviral activity against HEV in vitro (Dao Thi et al., 2016). The results were promising, indicating that sofosbuvir might also act against chronic HEV infection in patients. So far only few case reports exist and in those sofosbuvir did not show such efficacy (Kamar, Wang et al., 2017; Van der Valk et al., 2017; Donnelly et al., 2017). Thus, further studies are needed to evaluate the potential benefit of sofosbuvir for the therapy of chronic HEV in vivo.

An effective vaccine against HEV, labelled ‘Hecolin’ or HEV 293 is licensed only for China, where it is available since 2012. While a preventive strategy for endemic regions and high-risk groups as pregnant women is urgently needed, the WHO has reviewed the HEV 293 vaccine as not sufficiently evaluated concerning its safety in pediatric, elderly and other subpopulations and recommends a phase IV trial despite a “reassuring phase I, II and III” (WHO, 2014; Zhang et al., 2015).

Aim of the thesis 14

4 A im of the thesis

Infections with the Hepatitis E virus present an increasing health problem worldwide. They cause high rates of morbidity and mortality among vulnerable subpopulations such as immunocompromised individuals, pregnant women and persons with pre-existing liver injuries. No direct-acting and non-teratogenic therapies against HEV infections are currently available. As described above, only ribavirin and type I interferon represent effective treatment options although they have severe side effects and are contra-indicated in pregnant women.

Throughout our project we aimed to address this problem and screened for new HEV inhibitors.

In the process, the natural compound silvestrol was identified. Silvestrol belongs to the rocaglate derivates and can be isolated from the plant species Aglaia foveolata of the Mahogany family (Kim et al., 2007). The compound bears a unique cyclo-penta[b]tetrahydrobenzofuran skeleton and is described as an efficient and specific inhibitor of the DEAD-box RNA helicase eIF4A (Bordeleau et al., 2008). The helicase is part of the eIF4F complex that is able to initiate cap-dependent translation in eukaryotes.

The aim of our study was to evaluate the anti-HEV activity of silvestrol in HEV experimental model systems in vitro as well as in vivo. We wanted to analyze how efficient silvestrol inhibits the replication of different HEV gts in cell culture. The Hepatitis C virus (HCV) was used as reference because the virus drives the translation initiation cap-independent and should be less susceptible to silvestrol. Additionally, we performed parallel experiments with RBV in order to compare the inhibitory effects and characteristics. We further wanted to investigate the antiviral effect of silvestrol on cell culture derived infectious viral particles and on primary HEV isolates.

Therefore, we assessed the infectious virus production and HEV RNA copy number respectively under silvestrol treatment. In close collaboration with the research group of Philip Meuleman from the Ghent university we aimed to proof the antiviral effect of silvestrol also in an HEV animal model system. Using human liver chimeric mice which can be chronically infected with HEV, we wanted to study if a treatment with silvestrol can also lead to a decline of HEV infection in vivo.

In the last part we planned to investigate the combinatory activity of silvestrol with RBV. Since so far RBV is the most effective treatment option against HEV, we hoped to find synergistic antiviral effects in combining RBV with silvestrol.

Manuscript 15

5 M anuscript

TH E N A TU R A L COM POU N D SILV ESTR OL IN H IBITS H EPA TITIS E V IU R S (H EV ) R EPLICA TION IN V ITR O A N D IN V IV O

Todt, D.^§; Moeller, N.^§; Praditya, D.; Kinast V.; Friesland M.; Engelmann M.; Verhoye L.;

Sayed I.M.; Behrendt, P.; Dao Thi, V.L.; Meuleman, P.; Steinmann E. Antiviral Research. 157, 151-158 (2018) https://doi.org/10.1016/j.antiviral.2018.07.010.

§ equal contribution

Reprint with permission granted by Elsevier in accordance with the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license;

https://creativecommons.org/licenses/by-nc-nd/4.0/.

Discussion and Outlook 24

6 Discussion and Outlook

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

Im Dokument Characterization of the antiviral activity of silvestrol against Hepatitis E virus infection (Seite 8-0)