Mutations in hepatitis E virus ORF1 shape replication and disease severity

A new review published in the journal Discover Viruses brings together the evidence linking mutations in hepatitis E virus ORF1, the region of the viral genome that encodes its non-structural polyprotein, to changes in viral replication and to the severity of disease in infected patients. The authors, Rohini A. Nangare and Yogesh A. Karpe, synthesise virus-host interaction studies, immunological work, genome sequencing of infected patients and reverse genetics experiments to argue that ORF1 is not merely the replication machinery of the virus but a central contributor to HEV pathogenesis. Their central conclusion is that specific amino acid substitutions and insertions within ORF1 domains can enhance viral fitness and are among the factors that contribute to fulminant hepatic failure in HEV-infected patients, although the review also stresses that the evidence base remains incomplete and that many associations still require causal validation.

Hepatitis E virus is a single-stranded, positive-sense RNA virus of the family Hepeviridae and the genus Paslahepevirus, with a genome of roughly 7.2 kilobases that encodes three main open reading frames, and a fourth ORF in genotype 1 expressed during endoplasmic reticulum stress. Genotypes 1 and 2 are anthroponotic and dominate large outbreaks in developing nations, while genotypes 3 and 4 are zoonotic, transmitted to humans mainly through undercooked meat; genotype 3 is the most dominant strain in Europe and the Americas and genotype 4 is distributed in East Asia. Further genotypes originate from wild boars in Japan and from dromedary and Bactrian camels, with genotype 8 so far not found to infect humans. Acute infection is usually self-limiting, producing malaise, abdominal pain, jaundice, anorexia and nausea for a month or less, but severe complications occur in pregnant women, where mortality can reach up to 30 percent, and in immunocompromised persons, in whom chronic infection can lead to liver failure. The mechanisms underlying the severe pathology seen in pregnancy remain poorly understood.

ORF1 encodes a large non-structural polyprotein organised into recognisable domains: a methyltransferase, a Y domain, a fatty acid binding domain previously called the putative protease region, a hypervariable region, an X or macro domain, a helicase and an RNA-dependent RNA polymerase. Each of these domains has been interrogated in cell culture and biochemical systems, and the review walks through what is known and unknown about each. The methyltransferase converts GTP to m7-GTP for the 5′ cap of the viral RNA, a structure that stabilises the genome and increases translation efficiency. The Y domain was identified alongside the X domain by sequence comparison in 1992, and computational analyses have predicted a palmitoylation site and a membrane-binding alpha helix within it. Site-directed mutations at conserved residues of the Y domain in genotype 1, namely W413A, C337A and C336A, abolished HEV replication in a hepatoma cell line, underscoring that these conserved sites are critical for the viral life cycle even though the domain’s precise function is still under investigation.

The fatty acid binding domain carries perhaps the most contested history in HEV biology. Early computational work assigned the region as a putative cysteine protease involved in polyprotein processing, and several studies in vaccinia, baculovirus and bacterial expression systems reported processed products of the ORF1 polyprotein, with one group concluding that the protease processes both pORF1 and pORF2. However, the review emphasises that these experiments were conducted in heterologous systems rather than mammalian cells relevant to natural infection, and studies expressing ORF1 in mammalian cells, including pulse-chase experiments in HeLa and Huh-7 cells, failed to detect processing. Structural biology has since reshaped the picture: crystallography of a segment of the protein revealed significant structural homology with fatty acid binding domains despite low sequence similarity, together with a zinc-coordinating metal binding site, and structure-prediction modelling of genotype 3 concluded that the region lacks protease features and instead resembles a fatty acid-binding domain. Recent opinion pieces have even suggested the community abandon the term HEV protease altogether. Mutagenesis nonetheless shows the region matters: substitutions at multiple cysteine and histidine residues in this domain completely obliterated HEV replication, and studies of predicted cleavage sites for the host coagulation proteases thrombin and factor Xa found that alanine substitution at six of seven putative sites abolished viral replication in sub-genomic replicons of both genotype 1 and genotype 3.

The hypervariable region, a proline-rich stretch flanked by conserved sequences, is the most diverse part of the HEV genome and has yielded some of the most striking clinical observations. Deletion of the HVR did not affect HEV replication in one study, suggesting it is dispensable for infection itself but may serve other functions in pathogenesis. More dramatically, sequencing of HEV from a chronically infected liver-transplant patient revealed large deletions and an in-frame insertion of human ribosomal protein S19 encoding sequence within the HVR, and cell culture experiments showed that the inserted sequence conferred a replicative growth advantage. Analyses of isolates from solid organ transplant recipients found insertions of human and viral gene fragments, including fragments of the viral polymerase, the human tyrosine aminotransferase gene and the inter-trypsin inhibitor gene, while another adapted strain carried a 171-nucleotide insertion encoding human ribosomal protein S17 that benefited replication and expanded the host range of multiple HEV strains in vitro. Human ribosomal proteins S17 and S19 were later found to carry nuclear localisation sequences capable of importing the ORF1 protein, although the significance of this change in subcellular localisation remains unclear.

The X or macro domain, conserved across eukaryotes, bacteria and archaea, binds poly(ADP-ribose) and shows low ADP-ribose phosphatase activity, and substitution mutations at predicted catalytic residues N809 and H812 abolished HEV replication. The macro, helicase and protease domains have all been shown to interact with the host ferritin light chain in yeast two-hybrid assays, and follow-up work found that the macro domain lowers ferritin secretion in a hepatoma cell line without affecting cellular ferritin levels, leading the authors of that work to propose that the interaction may suppress innate immunity. On the clinical side, comparison of quasispecies in solid organ transplant patients found that those with chronic infection carried far more complex and diverse sequences in the macrodomain and polyproline region than patients who recovered, suggesting that diversity in these regions may be linked with persistence and hinting at a possible biomarker role for screening chronic infection in immunocompromised patients.

The helicase, a superfamily 1 enzyme with seven signature motifs, hydrolyses nucleotide triphosphates, unwinds RNA duplexes with 5′ overhangs in a 5′ to 3′ direction and carries RNA triphosphatase activity needed for cap synthesis. Mutations L1110F and V1120I, found in the helicase domain of HEV from patients with fulminant hepatic failure, affected replication when introduced into an HEV replicon in Huh 7-derived S10-3 cells, although recombinant helicase proteins carrying these mutations showed no change in biochemical activity relative to wild type, pointing instead to possible alterations in virus-host protein interactions. A separate mutation, V239A, was possibly linked with increased severity in severe genotype 3 infections, while the V1213A mutation introduced into genotype 1 to 4 replicons decreased luciferase activity, indicating reduced replication, yet did not correlate with severity of genotype 3 disease, a reminder that mutation effects are genotype-dependent.

The RNA-dependent RNA polymerase, containing the conserved GDD motif required for catalytic activity, has accumulated the largest catalogue of clinically relevant mutations. Sequencing of genotype 1 patients found that F179S in the methyltransferase significantly differed across fulminant hepatic failure and acute hepatitis cases, while in a larger cohort the D29N and V27A mutations in the methyltransferase region were linked with acute liver failure, including in pregnant women, and a contradictory H105R mutation was linked with low viremia. In the polymerase itself, G1634R identified in chronically infected patients appears associated with enhanced replication in the human liver and reduced effectiveness of ribavirin treatment, and N1530T and C1483W correlated with high viral titer in patients with acute liver failure. Yet the picture is not uniform: several ribavirin-resistance-associated mutations failed to show enhanced resistance or improved replication fitness in vitro, and the K1383N mutation actually reduced replication and increased susceptibility to ribavirin. The A1343V substitution, reported in patients receiving sofosbuvir, reduces sensitivity to that drug in vitro. The review concludes that RdRp sequencing could offer clinicians valuable predictions, while cautioning that drug metabolism and host immune responses may also contribute to treatment failure.

Taken together, the review positions ORF1 mutations as one contributing factor among several in the development of fulminant hepatic failure, alongside viral-host interactions that modulate the host immune response and, in pregnancy, hormonal changes that alter cell-mediated immunity. It also acknowledges substantial gaps: most functional studies have relied on recombinant proteins expressed in heterologous systems that may not recapitulate native folding or post-translational modifications, the biological relevance of ORF1 function during active replication in vivo remains poorly defined, and genomic associations must be validated through reverse genetics to confirm direct causal relationships. With no specific antiviral approved for HEV, ribavirin reserved for chronic infection and prohibited in pregnancy because it is a potent teratogen, and resistance mutations already documented, the authors argue that mutation-specific resistance patterns and next-generation sequencing may eventually support personalised second-line therapies, provided the genotype-specific pathogenic significance of each mutation is first established experimentally.

Subject of Research: Mutations in the hepatitis E virus ORF1 non-structural polyprotein and their effects on viral replication and disease severity

Article Title: Impact of hepatitis E virus ORF1 mutations on virus replication and disease severity

Article References: Nangare, R. A., & Karpe, Y. A. (2026). Impact of hepatitis E virus ORF1 mutations on virus replication and disease severity. Discover Viruses, 3(1), Article 15. https://doi.org/10.1007/s44370-026-00050-3

Image Credits: AI Generated

DOI: 10.1007/s44370-026-00050-3

Keywords: hepatitis E virus, ORF1, viral replication, fulminant hepatic failure, RNA-dependent RNA polymerase, methyltransferase, hypervariable region, macro domain, helicase, ribavirin resistance, genotype 1, genotype 3