The hepatitis E virus capsid protein ORF2 counteracts cell-intrinsic antiviral responses to enable persistence in hepatocytes

  1. Ann-Kathrin Mehnert
  2. Sebastian Stegmaier
  3. Carlos Ramirez
  4. Vladimir Gonçalves Magalhães
  5. Carla Siebenkotten
  6. Jungen Hu
  7. Ana Luisa Costa
  8. Daniel Kirrmaier
  9. Michael Knop
  10. Xianfang Wu
  11. Thibault Tubiana
  12. Carl Herrmann
  13. Marco Binder
  14. Viet Loan Dao Thi

Reviewed by

Read the full article See related articles

Discuss this preprint

Start a discussion What are Sciety discussions?

Listed in

Log in to save this article

Abstract

Hepatitis E virus (HEV) is a significant human pathogen causing both acute and chronic infections worldwide. The cell-intrinsic antiviral response serves as the initial defense against viruses and has been shown to be activated upon HEV infection. HEV can replicate in the presence of this response, but the underlying mechanisms remain poorly understood.

Here, we investigated the role of the HEV structural proteins ORF2 and ORF3 in immunocompetent cells. Mechanistically, we validated that ectopic ORF2, but not ORF3, interfered with antiviral and inflammatory signaling downstream of pattern recognition receptors, in part through interaction with the central adaptor protein TANK binding kinase 1. In the full-length viral context, ORF2 contributed to a reduced antiviral response and consequently, more efficient viral replication. In addition, we discovered a protective mechanism mediated by ORF2 that shielded viral replication from antiviral effectors. Using single-cell RNA-sequencing, we confirmed that the presence of ORF2 in infected cells dampened antiviral responses in both actively infected cells and bystanders. As a consequence, we found that early in the infection process, the progression of authentic HEV infection relied on the presence of ORF2, facilitating a balance between viral replication and the antiviral response within immunocompetent cells. Altogether, our findings shed new light on the multifaceted role of ORF2 in the HEV life cycle and improve our understanding of the determinants that may contribute to HEV persistence.

Significance statement

Hepatitis E virus (HEV) is an important yet often underestimated pathogen. Depending on the genotype, HEV infections can progress to chronicity, but the underlying mechanisms remain poorly understood. To gain insight into potential determinants, we investigated how HEV evades the body’s first line of defense, the cell-intrinsic antiviral response. We discovered that the HEV capsid protein ORF2 is crucial in limiting this response by interfering with antiviral signaling pathways and shielding viral replication from immune effectors. This balance between viral replication and the antiviral response contributes to persistent HEV infection in immunocompetent cells. Our findings reveal a new role for the HEV capsid protein in the viral life cycle and highlight it as an important target for novel therapeutic approaches.

Article activity feed

  1. Review Commons

    Note: This response was posted by the corresponding author to Review Commons. The content has not been altered except for formatting.

    Learn more at Review Commons

    Reply to the reviewers

    The authors do not wish to provide a response at this time

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #3

    Evidence, reproducibility and clarity

    In the manuscript entitled " The hepatitis E virus capsid protein ORF2 counteracts cell-intrinsic antiviral responses to enable persistence in hepatocytes ", Ann-Kathrin Mehnert interrogated that HEV pORF2 can inhibit host antiviral response. They found interaction of HEV ORF2 and TBK1. The finding is interesting and echoed with some previous studies that ORF2 can inhibit innate immunity.

    The study could benefit from the consideration of some major and specific points, as indicated below:

    Major issues:

    1. The researchers used p6, a cell-adapted clone, which was isolated form a chronic HEV patient. As previous studies suggested, p6 may behave differently than wild-type strains. Did the authors tried other HEV strains, as they used ips-induced model that was reported supportive to wild-type HEV?
    2. Figure 1F, ORF2 can interact with TBK1 as showed. But the prediction from Alphafold is weak. Also, could the author more evidence than the co-IP?
    3. Figure 2C and 2D, at 5 dpi, one can observed a stronger antiviral response, but at 7 dpi, no obvious difference was observed. Could the authors comment on this? 4．Figure 2H and 2I, detailed description of how the authors measured the positive cells should be provided. Did the authors selected whole plate of cells for counting? As showed in Figure 2H, the signals of IF were stronger at 5 and 7 dpi when compared at 3 dpi, but why the proportion of positive cells was reduced in Figure 2I?
    4. The study emphasized the function of ORF2 on HEV "persistence". However, this cannot be fully supported by cell models. In future, study on chronic HEV infection animal models may be conducted.
    5. The authors study ORF2 in whole. It will be of benefit to the readers that the authors could specified the function of secreted ORF2 and ORF2 capsid in the current study.

    Minor issues:

    1. Figure 3A, this is an elegant design. More data may provide for the validation of the formation of the virions.
    2. Figure 1, data should be provided for the successful expression of HEV-1 or HEV-3 ORF2, and ORF3.
    3. line 219, the current evidence that supported this statement is weak, especially for ORF2.
    4. Suppl Figure 3F-3H, statistical analysis is needed
    5. Suppl Figure 3F-3H, it seems that when no treatment was admistrated, the level of ISG15 in ΔORF2 group was higher than those of the WT and ΔORF3 group. Could the authors comment?
    6. Figure 3D and 3E, the starting time of the detection is not aligned.
    7. Figure 3F, scale bar is missing.
    8. In M&M, statistical method should be provided with more details and cover all the experiments used.

    Significance

    In the manuscript entitled " The hepatitis E virus capsid protein ORF2 counteracts cell-intrinsic antiviral responses to enable persistence in hepatocytes ", Ann-Kathrin Mehnert interrogated that HEV pORF2 can inhibit host antiviral response. They found interaction of HEV ORF2 and TBK1. The finding is interesting and echoed with some previous studies that ORF2 can inhibit innate immunity.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    Summary: Authors described the protective mechanism mediated by ORF2 that protects viral replication from the antiviral responses. They have utilized the advanced single-cell RNA sequencing to decipher the dampened antiviral responses in the presence of ORF2 HEV. I believe the study is important for the HEV literature and believe that the manuscript can be considered for publication after authors (1) rewrites the results and discussions separately until the journal wants it to be together. (2) answer the below questions.

    Minor comments:

    Line 69, 71 - I have never seen in any paper including reference in this way!

    Line 72 and 73 - missing reference!

    Line 92, 93 - missing reference!

    Line 95 to 99 - missing references!

    Major comments:

    I would like the authors to answer few questions:

    1. Did the authors study only the P6 HEV genome? Have they done anything comparative with the other strain to understand if the proposed mechanism is not the strain specific?
    2. Can the authors explain why we do not see any band in the Fig. 1F B-actin?

    Significance

    The paper uses advanced technique as single cell RNA seq to understand the mechanism of ORF2 assisting in the HEV replication.

    The study is well designed.

    This study will add up to understand some of the persistence infection seen in solid organ transplant patients. This study gives a mechanistic overview of HEV avoidance of antiviral response.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this study, the authors investigated how HEV ORF2 interferes with host antiviral responses to sustain viral infection. They employed several models, including pattern recognition receptors (PRRs) KO cell lines, immunodeficient cells and stem cell-derived models, to prove that: 1) ORF2 is essential for viral replication and 2) ORF2 dampens the interferon and inflammatory signaling pathways. They confirmed the interaction between ORF2 and TBK1, a central mediator of innate immune responses and identified residues in ORF2 that affect its interaction with TBK1. Finally, through single cell RNAseq, they demonstrated that ORF2 is a viral antagonist that inhibits host ISG expression in both infected and bystander cells. Interestingly, the sets of genes that are upregulated in WT vs ORF2-deficient virus infected cells are not entirely identical, suggesting that ORF2 may also modulate host gene expression in addition to suppressing the immune response. This research confers new immune antagonism mechanisms mediated by HEV capsid for sustainable HEV replication in host cells and provides potential therapeutic targets for HEV treatments.

    Major comments:

    1. The authors conclude from Figure 1 that the HEV ORF2 protein antagonizes both antiviral and inflammatory signaling pathways. The authors comprehensively investigated PRRs-mediated activation of type I interferon by viruses or poly(I:C) through overexpression of MDA5, RIG-I and TLR3. However, they only investigated the impact of ORF2 on host inflammatory response through evaluating the levels of TNFAIP3 RNA in the presence of MDA5 overexpression. It would be informative if the authors also check for NFkB activation/phosphorylation and expression of classical pro-inflammatory cytokines such as IL-1b and IL6. Interestingly, changes in IFNB secretion after ORF2 overexpression appear more dramatic compared to changes in IFNB1 RNA levels (compare Figure 1A-C with Supplementary Figure 1A and C). Are the IFN-beta protein expression changes statistically significant in Supplementary Figure 1?
    2. Changes in the IFN response do not always translate into changes in the viral RNA levels. In Figure 2B-D, the authors attributed the higher induction of IFNL1 and ISG15 on day 5 to the absence of ORF2 inhibition. However, the expression of these two genes drops to the same levels as the ones in WT viral RNA-electroporated cells on day 7, which is strange as ORF2-deficient viral RNA levels continue to be inhibited on day 7. This is different from the stem cell derived hepatocytes infected with the trans-complementation viruses in Figure 3G-H where there are significant differences in ISG15 levels between WT and ORF2-deficient virus infected cells on both days 5 and 7. To support their hypothesis, the authors need to further confirm the sudden upregulated antiviral activity on day 5 in electroporated HepG2/C3A cells by testing JAK/STAT phosphorylation and type I interferon secretion.
    3. The authors used different hepatocyte systems coupled with viral RNA electroporation or trans-complementation virus infection to investigate ORF2-mediated interference of the IFN pathway, which is highly complementary. However, while the electroporation of viral RNA into HepG2/C3A (Figure 2B-D) and infection of stem cell-derived hepatocytes with trans-complementation viruses (Figure 3F-H) result in similar upregulation of ISG expression on day 5, that wasn't observed in HepG2/C3A cells infected with trans-complementation viruses (Figure 3C-E) on day 5. The authors need to discuss the discrepancy among these different systems. Since the ORF2-deficient trans-complementation virus still brings in ORF2 proteins from the producer cells but cannot generate new ORF2 proteins, do ORF2 proteins from these two different sources have different functions in different hepatocyte systems? In addition, other than the data points that are shown to be not significantly different in Figure 3D-E, are any of the other data points significantly different?
    4. The single cell RNAseq data are very informative and revealed two interesting groups of genes. First, the ISGs that are further induced in the cells infected with ORF2-deficient HEV compared to cells infected with WT HEV (Figure 4N) are likely suppressed by ORF2. Second, the ISGs that are uniquely induced in the absence of ORF2 are different from the genes that are uniquely induced by WT HEV (Supplementary Table 2), suggesting that ORF2 may also modulate host gene expression. The authors can further characterize these two groups of ISGs by performing gene knockdown or knockout and investigating whether ORF2 directly interacts with these ISG products to determine the functional consequences of their upregulation. Related to that, are there other gene expression changes beyond ISG signatures which would suggest that ORF2 can regulate host gene expression? Figure 4A-C only shows comparisons for WT or ORF2-deficient vs. uninfected cells. The authors can perform GO and KEGG analyses to see if certain biological processes/pathways are enriched among the WT vs ORF2-deficient HEV induced genes. Further characterization of these genes (ISGs or not) would shed light on the novel roles of ORF2 in both immune antagonism and gene regulation and greatly increase the significance of the study.
    5. In Supplementary Figure 3F-H, the authors used BX795 to inhibit TBK1 (a target of ORF2) and found decreases in IFNL1 and ISG15 expression whether cells are electroporated with WT, ORF2-deficient, or ORF3-deficient viral RNA. However, this does not correlate with the data in Figure 2E-G where TBK1 inhibition results in significant differences in viral RNA levels only in the absence of ORF2 or ORF3. These results would suggest that the effects of TBK1 inhibition on viral RNA levels is independent of changes in the IFN/ISG expression levels.

    Significance

    The study addresses a long-standing question in the field about the immune antagonism activities of HEV ORF2 and ORF3 which previous studies have conflicting results on. The strength of this study is the use of complementary approaches such as ORF2 trans complementation system and single cell sequencing, and more relevant models such as stem cell derived hepatocytes to rigorously dissect the role of newly synthesized ORF2 protein in immunocompetent cell context. The manuscript is well written and would appeal to researchers in the HEV and innate immunity fields. However, the significance of the study is dampened by changes in the IFN response not always correlate with the inconsistency of ORF2-mediated inhibitory effects in different models and the still poorly defined mechanism of ORF2 suppression of the IFN pathway. The study would make conceptual advance if the authors can address the discrepancies in their findings and perform additional characterization to determine the functional consequences of ORF2-mediated immune suppression and gene regulation.

    My expertise is in innate immunity and host-virus interactions.

  5. Version published to 10.1101/2025.02.03.636239 on bioRxiv