Hepatitis E virus (HEV) replicates and can persist in hepatocytes despite an activated cell-intrinsic antiviral response. A 2025 study (Mehnert et al., PLoS Pathog) showed that the capsid protein ORF2 is a decisive viral determinant: it dampens pattern-recognition-receptor-triggered interferon (IFN) and IFN-stimulated gene (ISG) induction in part through interaction with the kinase TBK1, and independently protects viral replication from antiviral effectors, allowing replication and the antiviral response to equilibrate rather than collapse. The study left a multi-facet open problem: the molecular mechanism of the TBK1-independent protection of replicating HEV RNA from ISG effectors; the then-unidentified ORF2–TBK1 interaction interface (the protein-structure-prediction program AlphaFold failed on the intrinsically disordered ORF2 N-terminus); paracrine functions of secreted ORF2 isoforms; the determinants of acute (HEV-1) versus chronic (HEV-3) outcomes; contributions of ORF1/ORF3 in authentic infection; species tropism via host-species TBK1; and integration with JAK/STAT refractoriness and plasmacytoid dendritic cells in vivo. A 2026 follow-up from the same group (Jordan et al., Cell Reports) substantially resolved the interface facet by showing that the disordered ORF2 N-terminus drives liquid-liquid phase separation and amyloid-like fibril formation that sequester TBK1. The surviving open problem is the mechanistic account of the remaining arms, chiefly how ORF2 shields replicating viral RNA from ISG effectors independently of TBK1 and which ISG effectors actually restrict HEV, together with the genotype/host-species, ORF1/ORF3, and in-vivo integration facets.

Background

Hepatitis E virus (HEV) is a positive-sense, single-stranded RNA virus (family Hepeviridae, genus Paslahepevirus) and a major cause of acute viral hepatitis worldwide. Its ~7.2 kb genome encodes three proteins: ORF1, the nonstructural polyprotein providing the replication machinery including the RNA-dependent RNA polymerase; ORF2, the capsid protein; and ORF3, a small phosphoprotein mediating secretion of viral progeny. Clinically, human-restricted genotypes HEV-1/HEV-2 cause acute, self-limiting hepatitis (with high mortality in pregnancy), whereas zoonotic HEV-3/HEV-4, acquired from pigs or wild boar, can establish chronic infection, fibrosis, and cirrhosis in immunocompromised individuals. The first line of intracellular defense is the cell-intrinsic antiviral response: pattern-recognition receptors (PRRs), such as the RNA helicases RIG-I and MDA5, sense viral RNA and signal to TBK1 (TANK-binding kinase 1, the central adaptor kinase of this pathway), which phosphorylates the transcription factor IRF3 to induce type I and type III interferons (IFNs), while parallel signaling through the IκB kinase (IKK) complex activates inflammatory genes dependent on the transcription factor NF-κB. Secreted IFN acts in autocrine and paracrine fashion through JAK/STAT (the Janus kinase/signal transducer and activator of transcription pathway) to induce hundreds of IFN-stimulated genes (ISGs), many with direct antiviral effector functions. Unlike hepatitis C virus, HEV does not cleave PRR adaptors, yet HEV was known to replicate and persist in the presence of a sustained type III IFN response (Yin et al. 2017), and persistently infected cultures become refractory to exogenous IFN through continuous JAK/STAT activation; the mechanisms underlying this tolerance were poorly understood. ORF2 itself is multifunctional and exists in several isoforms: the capsid form packaged into virions that leave the cell quasi-enveloped (cloaked in host-derived membrane rather than a canonical viral envelope), abundantly secreted glycosylated (ORF2g) and cleaved (ORF2c) forms proposed to act as antigenic decoys (Yin et al. 2018), and a nuclear form; an N-terminal arginine-rich motif (ARM) is the master regulator of isoform fate (Hervouet et al. 2022). Overexpression studies had reported that ORF2 blunts IFN induction via its ARM, in part through functional interaction with TBK1 (Lin et al. 2019), and inhibits RIG-I-mediated IFN responses (Hingane et al. 2020). Mehnert et al. (2025) established these antagonisms in authentic full-length HEV infection of immunocompetent hepatocyte systems (hepatoma cells and stem-cell-derived hepatocyte-like cells, which support pan-genotype replication; Wu et al. 2018). Deleting ORF2 from the viral genome (ΔORF2) heightened IFN/ISG induction and collapsed replication at an early infection bottleneck; ORF2 dampened ISG responses in both infected cells and uninfected bystanders as shown by single-cell RNA sequencing (scRNA-seq); and ORF2 protected replication from exogenous type I and type III IFN even when TBK1-dependent induction was pharmacologically bypassed — implying a second, TBK1-independent evasion arm that shields replicating viral RNA from ISG effectors (candidate mechanisms discussed: formation or protection of viral replication compartments sheltering viral RNA from effectors such as ISG nucleases, or direct antagonism of specific ISG products). The paper also reported that ORF2 engages TBK1 directly, that this interaction is reduced by ARM mutation, and that the protein-structure-prediction program AlphaFold could not predict the interface because the ORF2 N-terminus is intrinsically disordered (a protein segment lacking stable folded structure). A direct sequel from the same group (Jordan et al. 2026) showed that this disordered N-terminus drives liquid-liquid phase separation (LLPS, formation of protein-rich membraneless condensates) and the assembly of ordered, amyloid-like stacked tubular fibrils in infected cells, and that these fibrils sequester TBK1 to dampen antiviral responses and enhance replication — revealing the mesoscale biophysical nature of the ORF2–TBK1 engagement and explaining why atomic-interface prediction failed. Plasmacytoid dendritic cells (pDCs), the professional IFN-producing cells of the immune system, add a further layer: in co-culture, pDCs sense HEV-replicating cells in a contact- and Toll-like receptor 7 (TLR7, an endosomal innate sensor of viral RNA)-dependent manner, and glycosylated ORF2 potentiates this recognition while nuclear ORF2 activates IRF3 (Joshi et al. 2025), so how hepatocyte-intrinsic ORF2 antagonism interfaces with professional immune cells in vivo remains to be established.

Problem Statement

Determine the molecular mechanisms by which hepatitis E virus (HEV) ORF2-mediated evasion of cell-intrinsic antiviral responses enables persistent viral replication, beyond the now-demonstrated sequestration of TBK1 by ORF2 amyloid-like fibrils. Concretely: (i) by what molecular mechanism does ORF2 (the viral capsid protein) protect replicating HEV RNA from the antiviral functions of interferon-stimulated genes (ISGs) independently of TBK1 (TANK-binding kinase 1), and which ISG effectors in fact restrict HEV replication; (ii) do differential ORF2-TBK1/fibril interactions across HEV genotypes (HEV-1 versus HEV-3) or across host species (human, pig, rabbit TBK1) contribute to the distinct clinical outcomes — acute self-limiting versus chronic infection — and to HEV species tropism; (iii) what paracrine immune functions do the secreted ORF2g and ORF2c isoforms (the glycosylated and cleaved secreted forms of the capsid protein) exert beyond the reported modulation of plasmacytoid dendritic cell recognition, and what contributions do the ORF1 polyprotein's processing products and ORF3 make to immune evasion in authentic infection with viral mutants in immunocompetent cells; and (iv) how does ORF2-mediated antagonism integrate with the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling refractoriness observed in persistently infected cells, and how do these mechanisms operate in vivo in the presence of professional IFN-producing cells such as plasmacytoid dendritic cells. Facets (i)–(iv) are all open facets of the same problem.

References

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