Hepatitis E Virus Replication Research Articles

Phosphorylation of Ser711 residue in the hypervariable region of zoonotic genotype 3 hepatitis E virus is important for virus replication.

Hepatitis E virus (HEV) is distinct from other hepatotropic viruses because it is zoonotic. HEV-1 and HEV-2 exclusively infect humans, whereas HEV-3 and HEV-4 are zoonotic. However, the viral and/or host factors responsible for cross-species HEV transmission remain elusive. The hypervariable region (HVR) in HEV is extremely heterogenetic and is implicated in HEV adaptation. Here, we investigated the potential role of Serine phosphorylation in the HVR in HEV replication. We first analyzed HVR sequences across different HEV genotypes and identified a unique region at the N-terminus of the HVR, which is variable in the human-exclusive HEV genotypes but relatively conserved in zoonotic HEV genotypes. Using predictive tools, we identified four potential phosphorylation sites that are highly conserved in zoonotic HEV-3 and HEV-4 genomes but absent in human-exclusive HEV-1 strains. To explore the functional significance of these putative phosphorylation sites, we introduced mutations into the HEV-3 infectious clone and indicator replicon, replacing each Serine residue individually with alanine or aspartic acid, and assessed the impact of these substitutions on HEV-3 replication. We found that the phospho-blatant S711A mutant significantly reduced virus replication, whereas the phospho-mimetic S711D mutant modestly reduced virus replication. Conversely, mutations in the other three Serine residues did not significantly affect HEV-3 replication. Furthermore, we demonstrated that Ser711 phosphorylation did not alter host cell tropism of zoonotic HEV-3. In conclusion, our results showed that potential phosphorylation of the Ser711 residue significantly affects HEV-3 replication in vitro, providing new insights into the potential mechanisms of zoonotic HEV transmission.IMPORTANCEHEV is an important zoonotic pathogen, causing both acute and chronic hepatitis E and extrahepatic manifestation of diseases, such as neurological sequelae. The zoonotic HEV-3 is linked to chronic infection and neurological diseases. The specific viral and/or host factors facilitating cross-species HEV infection are unknown. The intrinsically disordered HVR in ORF1 is crucial for viral fitness and adaptation, both in vitro and in vivo. We hypothesized that phosphorylation of Serine residues in the HVR of zoonotic HEV by unknown host cellular kinases is associated with cross-species HEV transmission. In this study, we identified a conserved region within the HVR of zoonotic HEV strains but absent in the human-exclusive HEV-1 and HEV-2. We elucidated the important role of phosphorylation at the Ser711 residue in zoonotic HEV-3 replication, without altering the host cell tropism. These findings contribute to our understanding the mechanisms of cross-species HEV transmission.

Oxysterol binding protein (OSBP) contributes to hepatitis E virus replication

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus and causes primarily acute self-limiting infections. The ORF1 of the HEV genome encodes a polyprotein around 190 kDa, which contains several putative domains, including helicase and RNA-dependent RNA polymerase. The HEV-encoded helicase is a member of the superfamily 1 helicase family and possesses multiple enzymatic functions, such as RNA 5′-triphosphatase, RNA unwinding, and NTPase, which are thought to contribute to viral RNA synthesis. However, the helicase interaction with cellular proteins remains less known. Oxysterol binding protein (OSBP) is a lipid regulator that shuffles between the Golgi apparatus and the endoplasmic reticulum for cholesterol and phosphatidylinositol-4-phosphate exchange and controls the efflux of cholesterol from cells. In this study, the RNAi-mediated silencing of OSBP significantly reduced HEV replication. Further studies indicate that the HEV helicase interacted with OSBP, shown by co-immunoprecipitation and co-localization in co-transfected cells. The presence of helicase blocked OSBP preferential translocation to the Golgi apparatus. These results demonstrate that OSBP contributes to HEV replication and enrich our understanding of the HEV-cell interactions.

Genetic determinants of host- and virus-derived insertions for hepatitis E virus replication

Hepatitis E virus (HEV) is a long-neglected RNA virus and the major causative agent of acute viral hepatitis in humans. Recent data suggest that HEV has a very heterogeneous hypervariable region (HVR), which can tolerate major genomic rearrangements. In this study, we identify insertions of previously undescribed sequence snippets in serum samples of a ribavirin treatment failure patient. These insertions increase viral replication while not affecting sensitivity towards ribavirin in a subgenomic replicon assay. All insertions contain a predicted nuclear localization sequence and alanine scanning mutagenesis of lysine residues in the HVR influences viral replication. Sequential replacement of lysine residues additionally alters intracellular localization in a fluorescence dye-coupled construct. Furthermore, distinct sequence patterns outside the HVR are identified as viral determinants that recapitulate the enhancing effect. In conclusion, patient-derived insertions can increase HEV replication and synergistically acting viral determinants in and outside the HVR are described. These results will help to understand the underlying principles of viral adaptation by viral- and host-sequence snatching during the clinical course of infection.

Performance of sofosbuvir and NITD008 in extrahepatic neuronal cells against HEV

Hepatitis E is an underestimated disease, leading to estimated 20 million infections and up to 70,000 deaths annually. Infections are mostly asymptomatic, but can reach mortality rates up to 25% in pregnant women or become chronic in immunocompromised patients. Hepatitis E virus (HEV) infection have been associated with a range of extrahepatic manifestations, including a spectrum of neurological symptoms. Current therapy options are limited to non-specific antivirals like ribavirin, but recently, repurposed viral polymerase inhibitors like sofosbuvir and NITD008 were described to inhibit HEV replication. Here, we evaluated the efficacy of these drugs in various neuronal-derived cell lines to determine their potency outside the liver. Our findings indicate that both drugs, especially sofosbuvir, exhibited reduced efficacy in neuronal cells compared to hepatic cells. These results should be taken into account in the development of direct-acting antivirals for HEV and their potency at extrahepatic replication sites.

Multifunctional ORF3 protein of hepatitis E virus.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen that is transmitted primarily through the fecal-oral route and can cause acute hepatitis in humans. Since HEV was identified as a zoonotic pathogen, different species of HEV strains have been globally identified from various hosts, leading to an expanding range of hosts. The HEV genome consists of a 5' noncoding region, three open reading frames (ORFs), and a 3' noncoding region. The ORF3 protein is the smallest but has many functions in HEV release and pathogenesis. In this review, we systematically summarize recent progress in understanding the functions of the HEV ORF3 protein in virion release, biogenesis of quasi-enveloped viruses, antigenicity, and host environmental regulation. This review will help us to understand HEV replication and pathogenesis mechanisms better.

A genome-wide CRISPR/Cas9 screen identifies a role for Rab5A and early endosomes in hepatitis E virus replication.

Hepatitis E virus (HEV) is a major cause of acute hepatitis worldwide. As the other positive-strand RNA viruses, it is believed to replicate its genome in a membrane-associated replication complex. However, current understanding of the host factors required for productive HEV infection is limited and the site as well as the composition of the HEV replication complex are still poorly characterized. To identify host factors required for HEV RNA replication, we performed a genome-wide CRISPR/Cas9 screen in permissive human cell lines harboring subgenomic HEV replicons allowing for positive and negative selection. Among the validated candidates, Ras-related early endosomal protein Rab5A was selected for further characterization. siRNA-mediated silencing of Rab5A and its effectors APPL1 and EEA1, but not of the late and recycling endosome components Rab7A and Rab11A, respectively, significantly reduced HEV RNA replication. Furthermore, pharmacological inhibition of Rab5A and of dynamin-2, required for the formation of early endosomes, resulted in a dose-dependent decrease of HEV RNA replication. Colocalization studies revealed close proximity of Rab5A, the HEV ORF1 protein, corresponding to the viral replicase, as well as HEV positive- and negative-strand RNA. In conclusion, we successfully exploited CRISPR/Cas9 and selectable subgenomic replicons to identify host factors of a noncytolytic virus. This approach revealed a role for Rab5A and early endosomes in HEV RNA replication, likely by serving as a scaffold for the establishment of functional replication complexes. Our findings yield insights into the HEV life cycle and the virus-host interactions required for productive infection.

Viral Biology and Immune Privilege in the Development of Extrahepatic Manifestations During Hepatitis E Virus Infection.

Hepatitis E virus (HEV) exhibits tropism toward hepatocytes and thus affects the liver; however, HEV may also affect other tissues, including the heart, kidneys, intestines, testicles, and central nervous system. To date, the pathophysiological links between HEV infection and extrahepatic manifestations have not yet been established. Considering that HEV infects multiple types of cells, the direct effects of virus replication in peripheral tissues represent a plausible explanation for extrahepatic manifestations. In addition, since the immune response is crucial in the development of the disease, the immune characteristics of affected tissues should be revisited to identify commonalities explaining the effects of the virus. This review summarizes the most recent advances in understanding the virus biology and immune-privileged status of specific tissues as major elements for HEV replication in diverse organs. These discoveries may open avenues to explain the multiple extrahepatic manifestations associated with HEV infection and ultimately to design effective strategies for infection control.

The hepatitis E virus ORF1 hypervariable region confers partial cyclophilin dependency.

Hepatitis E virus (HEV) is an emerging pathogen responsible for more than 20 million cases of acute hepatitis globally per annum. Healthy individuals typically have a self-limiting infection, but mortality rates in some populations such as pregnant women can reach 30 %. A detailed understanding of the virus lifecycle is lacking, mainly due to limitations in experimental systems. In this regard, the cyclophilins are an important family of proteins that have peptidyl-prolyl isomerase activity and play roles in the replication of a number of positive-sense RNA viruses, including hepatotropic viruses such as hepatitis C virus (HCV). Cyclophilins A and B (CypA/B) are the two most abundant Cyps in hepatocytes and are therefore potential targets for pan-viral therapeutics. Here, we investigated the importance of CypA and CypB for HEV genome replication using sub-genomic replicons. Using a combination of pharmacological inhibition by cyclosporine A (CsA), and silencing by small hairpin RNA we find that CypA and CypB are not essential for HEV replication. However, we find that silencing of CypB reduces replication of some HEV isolates in some cells. Furthermore, sensitivity to Cyp silencing appears to be partly conferred by the sequence within the hypervariable region of the viral polyprotein. These data suggest HEV is atypical in its requirements for cyclophilin for viral genome replication and that this phenomenon could be genotype- and sequence-specific.

Inhibition of HEV Replication by FDA-Approved RdRp Inhibitors.

Hepatitis E virus (HEV) is primarily a hepatotropic virus that is responsible for acute hepatitis E in the general population and for chronic hepatitis in immunocompromised individuals. In the absence of a globally accessible vaccine, pegylated interferon-α and ribavirin are the only antiviral agents available for the treatment of chronic patients. As viral RNA-dependent RNA polymerases (RdRps) are indispensable for RNA replication, they are considered potential drug targets. In this study, we screened some well-known RdRp inhibitor molecules, notably, favipiravir, sofosbuvir, remdesivir, filibuvir, and tegobuvir. Of these, monotherapy with favipiravir and sofosbuvir inhibited the RdRp activity with an IC50 value of 10.2 ± 4.9 and 5.2 ± 2.9 μM, respectively, compared to the reference drug ribavirin (3.5 ± 1.6 μM). Further investigation of the combination therapy showed a reduction in viral RNA copy numbers by approximately 90%. Therefore, favipiravir has an additive effect when used with sofosbuvir. Therefore, we propose that favipiravir is a promising anti-HEV drug that can be used in combination with sofosbuvir.

Janus kinase-inhibition modulates hepatitis E virus infection

Hepatitis E virus (HEV) usually causes a self-limiting disease, but especially immunocompromised individuals are at risk to develop a chronic and severe course of infection. Janus kinase (JAK) inhibitors (JAKi) are a novel drug class for the treatment of autoimmune inflammatory rheumatic disease (AIRD). As JAKs play a key role in innate immunity, viral infections and reactivations are frequently reported during JAKi treatment in AIRD patients. The aim of this study was to characterize the influence of JAKis on HEV replication. To this end, we evaluated liver enzymes of an AIRD patient under JAKi therapy with hepatitis E. Further, experiments with HEV (Kernow-C1 p6) were performed by infection of primary human hepatocytes (PHHs) followed by immunofluorescence staining of viral markers and transcriptomic analysis. Infection experiments in PHHs displayed an up to 50-fold increase of progeny virus production during JAKi treatment and transcriptomic analysis revealed induction of antiviral programs during infection. Upregulation of interferon-stimulated genes (ISG) was perturbed in the presence of JAKis, concomitant with elevated HEV RNA levels. The obtained results suggest that therapeutic JAK inhibition increases HEV replication by modulating the HEV-triggered immune response. Therefore, JAKi treatment and the occurrence of elevated liver enzymes requires a monitoring of potential HEV infections.

Variable Outcomes of Hepatitis E Infections in Patients with Hemato-Oncologic Diseases

Introduction: The hepatitis E virus (HEV) represents an important cause of viral hepatitis and could cause chronic infections in immunocompromised patients. However, data about immunocompromised patients other than solid organ transplant recipients are limited. Methods: We identified patients from a laboratory database and retrospectively compiled and analyzed clinical as well as laboratory data in detail. Results: Overall, 22 severely immunosuppressed patients, excluding solid organ transplant recipients, were identified. Four patients did not experience viral clearance (one without and three despite ribavirin therapy). Three patients acquired the infection after allogeneic hematopoietic stem cell transplantation (alloHSCT) and recovered spontaneously, whereas another patient, infected prior to alloHSCT, developed a chronic infection. Four patients failed to clear HEV, resulting in fatal liver failure in 2 patients. The CD4+ cell counts increased in all but 1 patient attaining a sustained virological response (SVR), as compared to patients with clinical failure. Severe immunoglobulin deficiency did not appear to obviate the control of HEV. Six of ten (60%) patients with and nine of 12 (75%) patients without ribavirin therapy achieved an SVR. Conclusions: Upfront ribavirin therapy does not appear mandatory in patients without CD4+ lymphopenia, but a prolonged HEV replication carries the risk of liver failure. Our data suggest that chronic HEV infections could cause T-cell exhaustion, which might be overruled with ribavirin therapy.

The PRMT5/WDR77 complex restricts hepatitis E virus replication.

Hepatitis E virus (HEV) is one of the main pathogenic agents of acute hepatitis in the world. The mechanism of HEV replication, especially host factors governing HEV replication is still not clear. Here, using HEV ORF1 trans-complementation cell culture system and HEV replicon system, combining with stable isotope labelling with amino acids in cell culture (SILAC) and mass spectrometry (MS), we aimed to identify the host factors regulating HEV replication. We identified a diversity of host factors associated with HEV ORF1 protein, which were putatively responsible for viral genomic RNA replication, in these two cell culture models. Of note, the protein arginine methyltransferase 5 (PRMT5)/WDR77 complex was identified in both cell culture models as the top hit. Furthermore, we demonstrated that PRMT5 and WDR77 can specifically inhibit HEV replication, but not other viruses such as HCV or SARS-CoV-2, and this inhibition is conserved among different HEV strains and genotypes. Mechanistically, PRMT5/WDR77 can catalyse methylation of ORF1 on its R458, impairing its replicase activity, and virus bearing R458K mutation in ORF1 relieves the restriction of PRMT5/WDR77 accordingly. Taken together, our study promotes more comprehensive understanding of viral infections but also provides therapeutic targets for intervention.

Soluble ORF2 protein enhances HEV replication and induces long-lasting antibody response and protective immunity in vivo.

The HEV is a small positive-sense RNA virus that encodes a cytoplasmic form of the capsid protein (ORF2c), essential for virion structure, and a secreted glycosylated form (ORF2s) that accumulates at high titer in serum and can mask neutralizing epitopes. We explored the contribution of ORF2s to HEV replication and its role in generating antibodies against ORF2 in a nonhuman primate model. We used a recombinant HEV genotype 3 variant that does not express ORF2s due to the introduction of stop codons (ORF2s mut ). Rhesus macaques (RMs) were given intrahepatic injections of infectious wildtype HEV (ORF2s wt ) RNA or a variant lacking ORF2s expression (ORF2s mut ). The replication of the ORF2s mut virus was delayed by ~2 weeks compared with ORF2s wt , and peak titers were nearly tenfold lower. Reversions of the 3 mutations that blocked ORF2s expression were not detected in the ORF2s mut genomes, indicating genetic stability. However, serum antibodies against ORF2 were transiently detected in RMs infected with ORF2s mut , whereas they were long-lasting in RMs infected with ORF2s wt . Moreover, RMs infected with ORF2s mut were more susceptible to reinfection, as evidenced by the viral RNA detected in fecal samples and the expansion of HEV-specific CD8 + T cells. These findings indicate that ORF2s may be dispensable for viral replication in vivo but is required for long-lived antibody-mediated responses that protect against HEV re-exposure.

Unravelling the Tripartite Interactions Among Hepatitis E Virus RNA, miR-140 and hnRNP K

In the present investigation, we have identified the functional significance of the highly conserved miR-140 binding site on the Hepatitis E Virus (HEV) genome. Multiple sequence alignment of the viral genome sequences along with RNA folding prediction indicated that the putative miR-140 binding site has significant conservation for sequence and secondary RNA structure among HEV genotypes. Site-directed mutagenesis and reporter assays indicated that an intact sequence of the miR-140 binding site is essential for HEV translation. Provision of mutant miR-140 oligos carrying same mutation as on mutant HEV successfully rescued mutant HEV replication. In vitro cell-based assays with modified oligos proved that host factor-miR-140 is a critical requirement for HEV replication. Biotinylated RNA pulldown and RNA immunoprecipitation assays proved that the predicted secondary RNA structure of the miR-140 binding site allows the recruitment of hnRNP K, which is a key protein of the HEV replication complex. We predicted the model from the obtained results that the miR-140 binding site can serve as a platform for recruitment of hnRNP K and other proteins of HEV replication complex only in the presence of miR-140.

Extrahepatic Replication Sites of Hepatitis E Virus (HEV)

Hepatitis E virus (HEV) is an emerging viral disease known to cause acute viral hepatitis globally. Various genotypes of HEV have been identified that produce genotype specific lesions depending on the HEV targeted population. Pregnant or immunosuppressed individuals develop significantly more severe hepatitis E in comparison to the general population. In the last 40 years, we discovered that the tropism of HEV is not restricted to the liver, and virus replication was demonstrated in multiple organs. Out of the 10 body systems described in humans, HEV produces lesions causing a broad range of extrahepatic clinical manifestations in each of them. Affected body systems include nervous and musculoskeletal, cardiovascular, digestive, endocrine, integumentary, renal, respiratory, immune, and reproductive systems producing systemic lesions. All extrahepatic signs are caused by either direct HEV replication in these tissues, or indirectly by various immune mediated mechanisms. Extrahepatic replication features of HEV allowed it to cross the placental barrier, blood–brain barrier (BBB), and blood–testis barrier (BTB) that do not typically grant entry to viruses in general. Thus, in this review, we summarized the extrahepatic replication sites of HEV, listed the body systems where HEV invaded, and described multiple animal models including immunocompetent and immunosuppressed that were used to study the extrahepatic replication sites of HEV.

Combined inhibitory effect of interferon and antiserum on avian hepatitis E virus

Epidemiologic investigations in recent years have shown that the detection rate of avian hepatitis E virus (HEV) in chicken flocks is increasing in China. Nevertheless, effective prevention and control measures are still lacking. In this study, specific pathogen-free (SPF) chicken serum against HEV was prepared using recombinant HEV open reading frames (ORF2 and ORF3) proteins as immunogens. An SPF chicken infection model was established by intravenous inoculation of chick embryos. Swab samples were collected at 7, 14, 21, and 28 d of age and used to detect avian HEV load, along with other indicators, by fluorescence quantitative real-time reverse transcription polymerase chain reaction (RT-qPCR) assay. The therapeutic effects on blocking vertical HEV transmission were observed, by using the methods of antibody application alone, mixed, or combined application of each of the 2 antibodies with type I interferon. The results showed that type I interferon alone or in combination with antiserum reduced the positive rate of HEV from 100 to 62.5% and 25%, respectively. However, the avian HEV-positivity rate was reduced to 75, 50, and 37.5% after type I interferon was used alone or in combination with antisera against ORF2 and ORF3, respectively. The inhibitory effect of type I interferon alone or in combination with an antiserum, on HEV replication was more significant in cells than in vivo. In this study, the inhibitory effect of type I interferon alone or in combination with an antiserum on avian HEV replication was observed in vitro and in vivo, providing the necessary technical reserve for disease prevention and control.

In Vitro Replication of Swine Hepatitis E Virus (HEV): Production of Cell-Adapted Strains.

The hepatitis E caused by the virus HEV of genotypes HEV-3 and HEV-4 is a zoonotic foodborne disease spread worldwide. HEV is currently classified into eight different genotypes (HEV-1-8). Genotypes HEV-3 and HEV-4 are zoonotic and are further divided into subtypes. Most of the information on HEV replication remains unknown due to the lack of an efficient cell cultivation system. Over the last couple of years, several protocols for HEV cultivation have been developed on different cell lines; even if they were troublesome, long, and scarcely reproducible, they offered the opportunity to study the replicative cycle of the virus. In the present study, we aimed to obtain a protocol ready to use viral stock in serum free medium that can be used with reduced time of growth and without any purification steps. The employed method allowed isolation and cell adaptation of four swine HEV-3 strains, belonging to three different subtypes. Phylogenetic analyses conducted on partial genome sequences of in vitro isolated strains did not reveal any insertion in the hypervariable region (HVR) of the genomes. A limited number of mutations was acquired in the genome during the virus growth in the partial sequences of Methyltransferase (Met) and ORF2 coding genes.

Liver Organoid Potential Application for Hepatitis E Virus Infection.

Despite the advances in hepatitis E virus (HEV) cell infection models' development, HEV infection efficacy in these cell models is still low, which hampers the further study of molecular mechanism of HEV infection and replication and even the interaction between HEV and host. Along with the advances in the technology for liver organoids generation, major efforts will be made to develop liver organoids for HEV infection. Here, we summarize the entire new and impressive cell culture system of liver organoids and discuss their potential application in HEV infection and pathogenesis. Liver organoids can be generated from tissue-resident cells isolated from biopsies of adult tissues or from iPSCs/ESCs differentiation, which can expand the large-scale experiments such as antiviral drug screening. Different types of liver cells working together can recapitulate the liver organ maintaining the physiological and biochemical microenvironments to support cell morphogenesis, migration, and response to viral infections. Efforts to optimize the protocols for liver organoids generation will speed up the research for HEV infection and pathogenesis and even the antiviral drug identification and evaluation.

Targeting proteostasis of the HEV replicase to combat infection in preclinical models

Appropriate treatment options are lacking for hepatitis E virus (HEV)-infected pregnant women and immunocompromised individuals. Thus, we aimed to identify efficient anti-HEV drugs through high-throughput screening, validate them invitro and invivo (in a preclinical animal study), and elucidate their underlying antiviral mechanism of action. Using appropriate cellular and rodent HEV infection models, we studied a critical pathway for host-HEV interactions and performed a preclinical study of the corresponding antivirals, which target proteostasis of the HEV replicase. We found 17 inhibitors that target HEV-HSP90 interactions by unbiased compound library screening on human hepatocytes harboring an HEV replicon. Inhibitors of HSP90 (iHSP90) markedly suppressed HEV replication with efficacy exceeding that of conventional antivirals (IFNα and ribavirin) invitro. Mechanistically, iHSP90 treatment released the viral replicase ORF1 protein from the ORF1-HSP90 complex and triggered rapid ubiquitin/proteasome-mediated degradation of ORF1, resulting in abrogated HEV replication. Furthermore, a preclinical trial in a Mongolian gerbil HEV infection model showed this novel anti-HEV strategy to be safe, efficient, and able to prevent HEV-induced liver damage. In this study, we uncover a proteostatic pathway that is critical for host-HEV interactions and we provide a foundation from which to translate this new understanding of the HEV life cycle into clinically promising antivirals. Appropriate treatment options for hepatitis E virus (HEV)-infected pregnant women and immunocompromised patients are lacking; hence, there is an urgent need for safe and effective HEV-specific therapies. This study identified new antivirals (inhibitors of HSP90) that significantly limit HEV infection by targeting the viral replicase for degradation. Moreover, these anti-HEV drugs were validated in an HEV rodent model and were found to be safe and efficient for prevention of HEV-induced liver injury in preclinical experiments. Our findings substantially promote the understanding of HEV pathobiology and pave the way for antiviral development.

De novo modelling of HEV replication polyprotein: Five-domain breakdown and involvement of flexibility in functional regulation

Hepatitis E virus (HEV), a major cause of acute viral hepatitis, is a single-stranded, positive-sense RNA virus. As such, it encodes a 1700-residue replication polyprotein pORF1 that directs synthesis of new viral RNA in infected cells. Here we report extensive modeling with AlphaFold2 of the full-length pORF1, and its production by in vitro translation. From this, we give a detailed update on the breakdown into domains of HEV pORF1. We also provide evidence that pORF1's N-terminal domain is likely to oligomerize to form a dodecameric pore, homologously to what has been described for Chikungunya virus. Beyond providing accurate folds for its five domains, our work highlights that there is no canonical protease encoded in pORF1 and that flexibility in several functionally important regions rather than proteolytic processing may serve to regulate HEV RNA synthesis.