The crucial but insufficient role of E2s domain’s residues 490 and 492 in determining the host tropism of hepatitis E virus

Domain: neuroscience
Published: 2026-02-09
Editorial outcome: major_revision

Abstract
Hepatitis E virus (HEV) is a significant pathogen causing acute viral hepatitis globally, posing a particular threat to pregnant women. HEV infects a range of host species, with distinct genotypes exhibiting genotype-specific tropism for different host hepatocytes. The P domain of viral capsid protein plays a central role in host cell attachment, but the molecular determinants that govern its host specificity remain unclear. This study investigates the molecular mechanisms underlying HEV host tropism by using a zoonotic HEV specific antibody 6H8. An epitope involving residues 490 and 492 is identified crucial for both mAb 6H8 binding and virus-cell attachment. Structure-based mutagenesis, molecular dynamics simulations, virus-cell attachment assays, and viral infectivity assays highlight the importance of the N490 and M492 residues in maintaining the structural integrity of the 6H8 epitope, influencing host specificity. Mutations at 490 and 492 permit HEV-1’s and disrupt HEV-4’s binding and infection in porcine hepatocytes. However, they are insufficient alone for reestablishing swine infection in vivo, indicating additional factors are involved in HEV’s host tropism. Our findings suggest that N490 and M492 are critical but not sole determinants of HEV-4’s specific tropism for porcine hepatocytes and advance our understanding of HEV’s zoonotic transmission and host specificity.

Introduction
Hepatitis E virus (HEV), a positive-sense RNA virus, is a significant pathogen causing acute viral hepatitis globally. This virus poses a grave threat to pregnant women, with mortality rates reaching up to 25%1. HEV strains isolated from humans are categorized within the genus Orthohepevirus A, family Hepeviridae2,3. Phylogenetic studies have identified eight HEV genotypes, with genotypes 1 to 4 being prevalent globally, while genotypes 5 to 8 are relatively rare3. The first four genotypes are further grouped based on their primary hosts: genotypes 1 and 2 (HEV-1 and HEV-2) are exclusive to humans and non-human primates, forming the human group, while genotypes 3 and 4 (HEV-3 and HEV-4), comprising the zoonotic group, exhibit a broader host range that includes humans, swine, and other mammals4.

Since the initial discovery of HEV in swine in 1997 in the United States5, zoonotic strains have been detected in regions heavily reliant on pork, establishing swine as significant reservoirs of HEV6,7. HEV infections in swine have been reported in both developed and developing countries8,9, with notably high prevalence rates of anti-HEV antibodies and HEV RNA reaching up to 92.8%8 and 44.4%9, respectively. The transmission of zoonotic HEV to humans, particularly from HEV-3 and HEV-4 through pork products, has been extensively documented10–13. The distinct host tropism between human and zoonotic HEV groups has led to different epidemiological patterns: human group HEVs typically cause periodic outbreaks in developing countries through contaminated drinking water14, whereas zoonotic group HEVs are associated with sporadic cases in both developing and developed countries through meat consumption15,16. There is also the potential for occupational transmission among individuals in close contact with infected animals17. These differences suggest the existence of varying molecular mechanisms of host cell recognition, a topic that remains not fully understood.

The underlying viral and host factors responsible for the host range of HEV remain elusive17. Various viral factors, including open reading frame 1 (ORF1), ORF2, 5’ noncoding region (NCR), codon usage, and adaptive evolution, have been reported to influence host range determination18–27. HEV virus-like particles (VLPs) bound and entered cells in a species-specific manner, suggesting that viral entry is a vital determinant of HEV host tropism and zoonotic potential26. The outer domain of pORF2, referred to as the P domain or E2s domain, facilitates the virion’s attachment to host cells25. This suggests that the interaction of pORF2’s with host cells plays a crucial role in determining the virus’s host specificity. Previous study has emphasized the importance of pORF2, particularly the E2s domain, in enabling the virus to infect porcine kidney cells24. The chimeric virus with the E2s domain from HEV-1 integrated into the HEV-3 genome exhibited similar ability to infect swine kidney cells to that of HEV-1 and different from that of HEV-324. These observations underscore the central role of the E2s in determining HEV’s host tropism. Efforts to discern the differences in the E2s domain between human and zoonotic groups HEVs have identified amino acids with distinct patterns across the two groups, such as amino acids 497, 517, 527, 537, 554, 569, 571, 593, and 599 in pORF224. However, none of these distinguished residues have been conclusively linked to the tropism of zoonotic HEV for swine24. Whether alteration in the E2s domain could disrupt the host’s tropism of zoonotic HEV for swine has not yet been verified in vivo. Furthermore, the critical residues of the E2s domain responsible for the swine tropism of zoonotic HEV remain unknown.

Given the critical role of the E2s domain in determining HEV’s host tropism, in this study we aimed to pinpoint specific epitope that differ between human and zoonotic groups of HEV. Utilizing a zoonotic HEV-specific monoclonal antibody (mAb) 6H8 and analyzing structures of various E2s and 6H8 immune complexes, we identified an epitope located exclusively on the zoonotic HEV viral capsid. Through a combination of structure-based mutagenesis, molecular dynamics simulations, virus-cell attachment assays and viral infectivity assays, we identify key residues (S488, T489, N490, and Y532) in the E2 protein that mediate both antibody recognition and virus-cell attachment. We further show how mutations in these residues affect HEV’s ability to bind specifically to porcine hepatocytes, revealing important insights into the viral determinants of host specificity. Structural and molecular dynamics analyses reveal that the N490 residue plays a pivotal role in stabilizing the epitope’s conformation, maintaining the structural integrity of the 480- and 530-loops. Mutations at N490 or M492 disrupt the 6H8 epitope, significantly reducing binding to porcine hepatocytes and impairing viral tropism. These insights offer a deeper understanding of the viral-host interactions that underlie HEV infection.

Results
HEV-4 capsids specifically bind to porcine hepatocytes
Given that swine are the primary natural reservoir for zoonotic group HEV while no porcine hepatogenic cell lines are current available, primary porcine hepatocytes (PPH) were isolated and utilized as a model for HEV study, aligning with previous studies28,29. Due to the limited prevalence of HEV-2, we focused on native HEV-1, HEV-3 and HEV-4 viruses. Our study compared PPH with the human hepatogenic cell line HepG2 and primary human hepatocytes (PHH) to evaluate the binding and entry capabilities of HEV-1 (human group), HEV-3 (zoonotic group) and HEV-4 (zoonotic group) strains. The binding potency of the HEV-3 and HEV-4 virus to PPH was significantly higher than that of HEV-1, exceeding by more than 15-fold (p<0.01) (Fig. 1A and Fig. S1). In contrast, the binding potency of the three viruses on HepG2 cells and PHH showed no notable differences (Fig. 1B, Fig. S1, and Fig. S2). The infectivity of native viruses in PPH and HepG2 were also analyzed using immunofluorescence assay (IFA). The results showed that infectivity of HEV-3 and HEV-4 was observed in both HepG2 cells and PPH, while infectivity of HEV-1 was observed exclusively in HepG2 cells but not in PPH (Fig. 1C, Fig. S3, and Fig. S4). All viruses in both HepG2 and PPH were neutralized by mAb A286 (Fig. S3, and Fig. S4), which recognizes all four HEV genotypes equally30. We then investigated the binding and entry potential of the widely reported HEV p239 VLPs on PPH, HepG2 and PHH, respectively, using a previously established IFA25. Consistent with the findings of native viruses, both genotype 1 p239 (p239-1) and genotype 4 p239 (p239-4) demonstrated effective binding and entry into HepG2 cell and PHH within 1h and 4h, respectively (Fig. 1D and Fig. S5). In contrast, only p239-4 was found to effectively bind and enter PPH cells (Fig. 1E). The binding and entry of p239-1 were observed exclusively in HepG2 and PHH but not in PPH cells (Fig. 1D, E, and Fig. S5). The binding and entry of VLP on HepG2, PPH, and PHH could be blocked by the mAb 8G12 (Fig. S5 and Fig. S6), which recognize all four HEV genotypes equally31. These findings suggests that HEV-1 and HEV-4 exhibit similar binding and entry capabilities in human hepatocytes, yet differ markedly in their interactions with porcine hepatocytes.

The observed difference in cell tropism suggested distinct binding and entry mechanisms for HEV-1 and HEV-4. In a previous study, we demonstrated that p239-1 consistently shares binding sites with the native HEV-1 virus on HepG2 cells25. Employing a VLP-virion blocking assay akin to our previous methods, we demonstrated that both p239-1 and p239-4 exhibit a comparable ability to inhibit the binding of HEV-1 to HepG2 cells, achieving half-maximal inhibitory concentrations (IC50) of 21.13 μg/mL and 14.95 μg/mL, respectively (Fig. 1F). In contrast, p239-1 exhibited a significantly reduced capability, nearly tenfold less, in preventing HEV-4 from binding to HepG2 cells compared with p239-4 (Fig. 1G). Remarkably, p239-4 could completely prevent HEV-4’s attachment to HepG2 cells with an IC50 value of 8.88 μg/mL, whereas p239-1 could only partially (60%) block the binding of HEV-4 on HepG2 cells even at a high input concentration, resulting in an IC50 of 94.69 μg/mL. These findings suggest that p239-4 competes more effectively with HEV-4 and HEV-1 for their binding sites on HepG2 cells than p239-1, the latter cannot fully occupy HEV-4’s binding sites.

As for PPH, p239-4 demonstrated a moderate ability to block HEV-4 binding to PPH, with an IC50 of 29.28 μg/mL, while no detectable blocking ability was detected for that of p239-1 (Fig. 1H). Consequently, we hypothesize that both porcine and hominine hepatocytes contain HEV-3 and HEV-4-specific binding sites, which can be recognized by HEV-3, HEV-4, and p239-4 through unique viral determinants that are not accessible to p239-1 or HEV-1.

Characterization of a zoonotic HEV-specific mAb 6H8
To determine the specific determinant present on HEV-4, a zoonotic group HEV-specific mAb, 6H8, from a previously reported panel of mAbs against the E2s domain was selected for further analysis32. Binding assays and Western blot analyses indicated that mAb 6H8 specifically recognized the dimer (E2) and VLP (p239) forms of capsid proteins belonging to HEV-3 and HEV-4, but not to HEV-1 and HEV-2 (Fig. 2A, B and Fig. S7). Surface plasmon resonance (SPR) assays conformed that mAb 6H8 efficiently bound to the E2 domains of HEV-3 (E2-3) and HEV-4 (E2-4) with high affinities of 5.39 nM and 2.51 nM, respectively, while its binding affinities for E2-1 and E2-2 were too weak to reach the detection limit (Fig. S8). These findings were corroborated by analytical ultracentrifugation (AUC) analysis, which detected immune-complexes only in the mixture of 6H8 with E2-3 and E2-4 (∼10.06 S), but not with E2-1 and E2-2 (Fig. 2C-F).

We next evaluated the capture abilities of mAb 6H8 against native viruses using an immune capture assay as previously described32, with the mAb 8G1231 as a positive control. As expected, 6H8 demonstrated capture abilities for HEV-3 and HEV-4 but not HEV-1, while 8G12 exhibited similar capture abilities for all three examined HEV genotypes (Fig. 2G, H). Furthermore, in vitro neutralization tests with HepG2 cells revealed that 6H8 specifically neutralized HEV-3 and HEV-4, with comparable IC50 values of 19.18 µg/ml and 13.88 µg/mL in HepG2 (Fig. 2I) respectively. Conversely, 6H8 showed no significant neutralizing activity against HEV-1 in HepG2 (IC50 >2000 µg/mL) (Fig. 2I). The highest 6H8 concentration even resulted in less than 50% neutralization (Fig. 2I). 6H8 exhibited comparable neutralizing activity against HEV-3 and HEV-4 in PPH, with IC50 values of 22.03 µg/mL and 25.61 µg/mL, respectively, similar to those observed in HepG2 cells (Fig. 2J). In comparison, 8G12 displayed a consistent neutralization potency across the different HEV genotypes, with IC50 values ranging from 8.92 µg/mL to 10.68 µg/mL in HepG2 and 13.78 µg/mL to 18.30 µg/mL in PPH (Fig. 2K, L). IFA confirmed that mAb 6H8 effectively neutralized HEV-3 and HEV-4 in both PPH and HepG2 cells, but failed to neutralize HEV-1 in HepG2 cells (Fig. S3 and Fig. S4). Collectively, these outcomes validate mAb 6H8 as a neutralizing antibody specific to the zoonotic group HEV.

Crystal and cryo-EM structures of 6H8 immune complexes
We subsequently sought to determine the epitope recognized by mAb 6H8 at atomic resolution. The crystal structure of the immune-complex of E2s-4 bound with the antigen-binding fragment (Fab) of 6H8 (E2s-4:6H8) was resolved at a resolution of 2.3 Å and refined to an R-factor of 21.6% (Rfree = 23.6%) (Fig. 3A, Fig. S9, and Table S1). To verified the binding mode of 6H8 on HEV virus, we also prepared the p495 (amino acids 112-606) VLP of HEV-4 (p495-4) and complexed it with 6H8 Fab. The resultant immune complex (p495-4:6H8) was subjected to cryo-electron microscopy (cryo-EM) single-particle analysis, achieving a 4.1 Å density map (Fig. 3B, Fig. S10, and Table S2). In the asymmetric unit of the crystal structure, one dimer of E2s-4 was bound by two 6H8 Fab molecules (Fig. 3A), a configuration echoed in the cryo-EM structure, where the crystal structure fitting well within the cryo-EM density map (Fig. 3B). The 6H8 Fab exhibited a well-defined electron density map, closely resembling its crystal structure (Fig. S9). The Fab’s interaction footprint, located on the dimer’s outermost side, buries a total of ∼650 Å2 of surface area (Fig. 3C). The interaction between 6H8 and E2s-4 involved amino acids Y480, Q482, T483, S487-N490, I529-S533, Y559, N560, Y584, and Y585 of E2s-4 (Fig. 3C). The binding region of 6H8 comprised of all six complementarity determining region (CDR) loops (Fig. 3D), with key interactions facilitated by 13 hydrogen-bonding contacts (≤3.2 Å) among the main-chain and side-chain atoms (Table S3), alongside several van der Waals interactions.

Sequence analysis revealed that among the 16 epitope residues involved in 6H8 recognition, two residues (amino acids 483 and 490) differentiate between the human (S483 and G490) and zoonotic (T483 and N490) groups of HEVs (Fig. 3E and Fig. S11), suggesting these residues are crucial for 6H8’s specificity towards HEV-3 and HEV-4. The remaining epitope residues are conserved across all HEV genotypes. Additionally, our previous research identified two other neutralizing epitopes on E2s structurally31,33. Notably, the HEV-1-specific neutralizing epitope, recognized by mAb 8C11, is located in E2s-1 dimer’s groove region (Fig. 3F), with a distinctive amino acid at position 497 crucial for its specificity. Another neutralizing epitope, recognized by mAb 8G12, was mapped to the E2s dimerization region and conserved across all HEV genotypes. The epitope for mAb 6H8, distinct from those of 8C11 and 8G12, defining a novel genotype-specific antigenic determinant on zoonotic group HEVs (Fig. 3F).

Structural basis for the genotype-specific reactivity of 6H8
We proceeded to experimentally confirm the importance of interface amino acids of the E2 domain involved in 6H8’s specific binding through a series of structure-based mutagenesis analysis. Each of the 16 residues involved in interactions—480, 482-483, 487-490, 529-533, 559-560, and 584-585—were mutated to alanine for subsequent examination. The dimerization of these mutant proteins was verified using SDS-PAGE, and their binding activities to mAb 6H8 were assessed through enzyme-linked immunosorbent assay (ELISA). Mutations at S488A, T489A, N490A and Y532A completely abolished the binding of mAb 6H8 (Fig. 4A), highlighting the indispensable role of S488, T489, N490 and Y532 in mAb 6H8 binding. Among these four key residues, S488 and T489 are in close contact with both the heavy and light chains of mAb 6H8 (Fig. 4B, C). Y532 primarily engages with the heavy chain and forms an interaction network containing several hydrogen bonds and π-π interactions (Fig. 4D). Intriguingly, despite the absence of hydrogen bonds between the zoonotic group-specific residue N490 and mAb 6H8, its alteration led to a total loss of binding capability (Fig. 4A). The crystal structure revealed that N490 is in hydrogen bond liaison with another crucial epitope residue Y532, suggesting that it contributes to the conformational fidelity of the mAb 6H8 epitope (Fig. 4E). Consequently, conformational perturbations induced by the N490A mutation may compromise the epitope’s structural integrity on E2s-4, culminating in its diminished binding to mAb 6H8, as demonstrated by the outcomes of the N490A experiment (Fig. 4A).

To investigate the role of the binding site recognized by mAb 6H8 in virus-cell attachment, we individually mutated amino acids 488, 489, 490 and 532 to alanine in both p239-1 and p239-4 VLPs to evaluate their cell-binding sensitivities. The attachment of these mutated VLPs to HepG2 and PPH cells was assessed by staining with an anti-ORF2 antibody, followed by confocal microscopy analysis to determine their binding efficiencies (Fig. S12). In PPH cells, mutations at residues 488, 490 and 532 of p239-4 completely abolished binding, while residue 489 did not. In contrast, for HepG2 cells, mutations at residues 488 and 532 in both p239-1 and p239-4 disrupted binding, whereas mutations at residues 489 and 490 didn’t significantly affect binding compared to the wild type. Remarkably, although mutations at residue 490 in both p239-1 and p239-4 did not alter their attachment to HepG2 cells, the same mutation in p239-4 led to a marked reduction in binding to PPH cells. This indicates that residue 490 plays a crucial role not only in the specific interaction with mAb 6H8 but also in determining the viral tropism toward porcine hepatocytes.

The epitope of 6H8 is primarily located on the 480-loop (amino acids 481-492) and 530-loop (amino acids 530-534). Within these two loops, three amino acids (483, 490 and 492) differ between the human and zoonotic groups of HEVs. Specifically, in zoonotic group HEV, threonine (T), asparagine (N) and methionine (M) in residues 483, 490 and 492, are substituted by serine (S), glycine (G) and valine (V) in the human group, respectively (Fig. 3E and Fig. S11). Of these three residues, amino acid 483 was shown not to play a role in binding to 6H8 (Fig. 4A), whereas residue 490, which is crucial for both the antibody’s recognition and attachment to porcine hepatocytes. Given the proximity of residue 492 to both of epitope key residues 490 and 532, we conducted a comprehensive set of reverse mutations on the E2-1 and E2-4 proteins, respectively, including both single and paired mutations specifically targeting residues 490 and 492 (Table S3). For the E2-1 mutants, altering residue 490 to asparagine (G490N) single-handedly restored 6H8’s reactivity, whereas mutating 492 to methionine (V492M) had no such effect. However, pairing these mutations (G490N/V492M) enhanced the reactivity towards 6H8 by 2.6 times more than the single G490N mutation (Table S4 and Fig. 4F). Structural analyses of both the wild-type and mutant E2s-1 proteins (E2s-1-G490N/V492M) (Table S1) revealed subtle differences in both loop regions and the positions of key residues 490, 492, and 532 (Fig. 4H). The structural conformation of the 480- and 530-loop regions (6H8 epitope region) in the structure of E2s-1-G490N/V492M is more similar to that in E2s-4 than to E2s-1, as evaluated by root mean square deviations (RMSDs) of 0.227 and 0.458 Å. A notable hydrogen bond interaction between the side chain of N490 and Y532 was observed, akin to that observed in E2s-4, which is deemed essential for maintaining the specific conformation of the 480- and 530-loops, thereby facilitating 6H8’s distinctive recognition.

Similar reverse mutations were conducted on the E2s-4 protein to evaluate their impact on binding to 6H8. Consistently, only proteins mutated at N490 to glycine (N490G), whether alone or in combination with other mutations, exhibited a complete loss of reactivity towards 6H8 (Fig. 4G). Further structural analysis of the double mutant E2s-4 (E2s-4-N490G/M492V) compared to the wild-type E2s-4 revealed, as anticipated, that its conformation in the 480- and 530-loops region was more closely aligned with that of E2s-1 than with the original E2s-4 structure (Fig. 4I). Additionally, these mutations disrupted the above-mentioned hydrogen bond and π-π interaction network around residue 532 (Fig. 4I). This evidence definitively establishes residue N490 in HEV-4 as a critical determinant for the specific interaction between 6H8 and the viral capsid, with M492 playing a supportive role in maintaining the conformation critical for 6H8 epitope recognition.

N490 and M492 play a crucial role in maintaining epitope stability of 6H8
To further investigate the impact of mutations at residues 490 and 492 on 6H8 binding across different HEV genotypes, we conducted molecular dynamics (MD) simulations using diverse E2s protein structures with different configurations at these positions. Eight resolved or modeled structures, E2s-1, E2s-1-G490N, E2s-1-V492M, E2s-1-G490N/V492M, E2s-4, E2s-4-N490G, E2s-4-M492V and E2s-4-N490G/M492V, were subjected in 1000 nanoseconds (ns) MD simulations. The convergence of root mean square deviation (RMSD) values below 2 Å indicates sufficient structural equilibrium and suitable for further analysis (Fig. S13). To quantitatively compare the flexibility of different structures, we calculated the root mean square fluctuation (RMSF) for each residue based on the MD simulation trajectories. The RMSF profiles revealed significant structural differences between structures possessing N490 (E2s-4, E2s-1-G490N/492M, E2s-4-M492V and E2s-1-G490N, which bind to 6H8) and those with G490 (E2s-1, E2s-1-V492M, E2s-4-N490G and E2s-4-N490G/M492V, which do not bind to 6H8) (Fig. 5A). Structures with N490 exhibit markedly lower flexibility in the 480- and 530-loop regions, which mainly constitute the 6H8 epitope, compared to structures with G490 (Fig. 5A and Fig. S14). The increased flexibility observed in the 6H8 epitope region in G490-containing structures suggests that residue 490 plays a crucial role in stabilizing the epitope configuration.

To further assess the impact of residue 490 on the structure of the 6H8 epitope, we examined the structural changes of the 480- and 530-loops during MD simulation by calculating the RMSD of the backbone atoms in this region relative to their initial conformations. Structures with N490 (E2s-4, E2s-1-G490N/V492M, E2s-4-M492V, and E2s-1-G490N) maintain lower average RMSD values of 1.14 Å, 1.44 Å, 1.17 Å and 1.60 Å, respectively, indicating the stable conformations necessary for 6H8 binding (Fig. 5B and Fig. S13). In contrast, structures with G490 (E2-1, E2-4-N490G/M492V, E2-1-V492M and E2-4-N490G) exhibit significantly higher deviation with average RMSD values of 4.19 Å, 2.61 Å, 2.65 Å and 2.96 Å, respectively (Fig. 5B and Fig. S13). The observed differences in the deviation of the 6H8 epitope region during MD simulations suggest that structures with G490 undergo significant conformational changes in the 6H8 epitope region, especially 480-loop, which may lead to the loss of the conformation necessary for 6H8 binding. To quantify the extent of epitope deflection, we defined the 480-loop deviation angles measured between the alpha carbon (Cα) of residue 488 in the different MD simulation frames, relative to the midpoint of the two Cα atoms of residues 486 and 491 in the initial frame. The N490-containing structures exhibit significant stability on the 480-loop during MD simulations, with average 480-loop deviation angles lower than 20°, substantially lower than those of the G490-containing structures (all greater than 25°) (Fig. 5C). Moreover, during the three replicate 1000 ns simulation periods, the G490-containing structures exhibits greater deflection in 480-loop, with significantly longer durations of marked deflection (exceeding 40°) compared to the N490-containing structures (Fig. 5D). These findings confirm that N490 plays a pivotal role in maintaining the stability of 6H8 epitope, while its substitution with glycine lead to destabilization and disruption of the epitope conformation primarily by increasing deflection in 480-loop. This evidence is consistent with our structural data, which indicate that a crucial hydrogen bond between N490 and Y532 in E2s-4, which bridges the 480- and 530-loops, is absent in G490-containing structures. Taken together, these results suggest that the loss of N490-Y532 hydrogen bond in the G490-containing structures lead to significant deflection and conformational changes in the 6H8 epitope, particularly in the 480-loop, ultimately diminishing 6H8’s binding capacity.

Additionally, for the G490-containing structures, we found that E2s-1 and E2s-4-N490G/M492V, both containing V492, exhibited greater maximum degrees of deflection (greater than 100°) in the 480-loop compared to that of E2s-1-V492M and E2s-4-N490G, which contain M492 (Fig. 5D). This observation suggests that methionine at position 492 can partially mitigate the 480-loop deviations caused by the absence of asparagine at position 490. Given that N490-Y532 hydrogen bond is crucial for epitope stability, while residue 492 does not directly interact with 6H8, we analyzed the occupancy of N490-Y532 hydrogen bond over the simulation trajectory in those N490-containting structures. E2s-1-G490N/V492M and E2s-4 exhibited significantly higher occupancy rates of the N490-Y532 hydrogen bond (24.8% and 21.7%, respectively) than E2s-1-G490N (4.8%) and E2s-4-M492V (3.6%) (Fig. S15). This indicates the supportive role of M492 in maintaining the N490-Y532 hydrogen bond, thereby enhancing the binding of 6H8 to E2s.

In summary, the MD simulations revealed that N490 in HEV-4 E2s plays a crucial role in stabilizing the conformation of both 480- and 530-loops by formed an important hydrogen bond with Y532, thereby maintaining the stability and integrity of the 6H8 epitope. M492, although not as crucial as N490, also contributes to the stabilization of these loops, collectively influencing the binding affinity of 6H8.

N490 and M492 determine HEV-4’s specific binding and infection in porcine hepatocytes
The involvement of N490 and M492 in determining host tropism was further elucidated using an IFA with recombinant p239 VLPs and their mutants. In the p239-4 mutants, both p239-4-N490G and the double mutant p239-4-N490G/M492V fail to bind to PPH cells, whereas p239-4-M492V maintained the binding ability (Fig. 6A and Fig. S16). In contrast, for the p239-1 mutants, only p239-1-G490N/V492M, but not p239-1-G490N or p239-1-V492M, successfully bound to PPH cells (Fig. 6A). Fluorescence quantification further verified that although p239-4-M492V bound to PPH, demonstrating a positive signal by IFA, its fluorescence intensity was twofold lower than that of the wild-type p239-4 (Fig. 6B). On the other hand, both p239-1-G490N/V492M and wild-type p239-4, each incorporating N490 and M492, exhibited comparable binding levels to PPH cells (Fig. 6B). Notably, all VLPs displayed similar fluorescence intensities when binding to HepG2 cells, consistent with the IFA results (Fig. 6A, C, and Fig. S17). These findings together suggest that altering asparagine at position 490 to glycine in p239-4 nullified its attachment ability to porcine hepatocytes, whereas restoring p239-1’s attachment to these cells required simultaneous mutations at both positions 490 and 492 (G490N and V492M).

To further elucidate the role of residues 490 and 492 at the virus level, we rescued the 490/492 double-mutant viruses using reverse genetics technology as previously reported34. The wild-type and mutant HEV viruses were produced and purified from the lysate of cells transfected with the RNA of recombinant viral genomes (Fig. S18). The viral titers of HEV-1, HEV-1-G490N/V492M, HEV-4, and HEV-4-N490G/M492V were 3.32×103, 3.91×103, 2.28×103, and 1.91×103 focus-forming units (FFU)/mL, respectively (Fig. S19). The specific infectivity, defined as the RNA copy number per infectious event, was comparable among all wild-type and mutant HEV viruses, ranging from 2.56×104 to 5.24×104 copies/FFU. The binding abilities of 6H8 to both wild-type and mutant viruses were assessed using two independent methods, yielding similar results: HEV-1-G490N/V492M virus obtained the 6H8 epitope, and HEV-4-N490G/M492V virus completely destroyed the 6H8 epitope (Fig. 7A, B). These results were consistent with those obtained from p239 VLPs (Fig. 6A–C).

The binding abilities of wild-type and mutant virions to different hepatocytes were then measured using quantified real-time reverse transcription PCR (RT-PCR). As expected, all wild-type viruses and their mutants showed similar binding efficiencies with HepG2 cells (Fig. 7C). Mutant virus HEV-1-G490N/V492M and wild-type HEV-4 demonstrated equivalent binding efficacies on PPH cells (Fig. 7C). Conversely, the HEV-4-N490G/M492V virus exhibited significant reduced binding ability to PPH cells, resembling that of wild-type HEV-1 (Fig. 7C). In contrast, both HEV-4-N490G/M492V and wild-type HEV-1 viruses exhibited lower binding potency with PPH than with HepG2 cells (Fig. 7D). Nonetheless, HEV-1-G490N/V492M and wild-type HEV-4 showed similar binding potency with both HepG2 and PPH cells (Fig. 7D).

The infectivity of wild-type and mutant viruses in different hepatocytes was further assessed using ELISA- and IFA-based infection assays as previously reported17,35. All wild-type and their corresponding mutant viruses showed similar infectivity in HepG2 cells (Fig. 7E–H). However, HEV-1 and HEV-4-N490G/M492V showed undetectable infectivity in PPH (Fig. 7E, and Fig. 7G, H), whereas HEV-1-G490N/V492M and HEV-4 demonstrated equivalent infectivity in PPH (Fig. 7E, and Fig. 7G, H). Both HEV-1-G490N/V492M and HEV-4 displayed similar infectivity in PPH and HepG2 cells, while HEV-1 and HEV-4-N490G/M492V showed reduced infectivity in PPH compared to HepG2 cells (Fig. 7F). All wild-type and mutant viruses in PPH and HepG2 could be neutralized by mAb A286 (Fig. 7E, G, Fig. S3, and Fig. S4). In contrast, mAb 6H8 effectively neutralized HEV-4 and HEV-1-G490N/V492M in both cell types, but failed to neutralize HEV-1 and HEV-4-N490G/M492V in HepG2 cells (Fig. 7E, G, Fig. S3, and Fig. S4).

Collectively, these findings imply that the G490N and V492M substitutions in the HEV-1 virus lead to a loss of genotype-specific antigenic determinant and tropism for porcine hepatocytes.

N490 and M492 are crucial but not the sole factors for HEV-4 infection in miniature swine
Although HEV-1-G490N/V492M virus retained binding abilities to porcine hepatocytes in vitro and HEV-4-N490G/M492V exhibited a loss of this capacity, the infectivity of these two mutant viruses in swine in vivo remains undetermined. To assess the impact of N490 and M492 on cross-species infectivity, experiments were conducted with both wild-type and mutant HEVs in both miniature swine (Bama miniature swine) and cynomolgus monkeys (Macaca fascicularis), respectively, following previously established protocols18,31,36,37. The experimental design includes four groups of cynomolgus monkeys infected with either HEV-1, HEV-4, HEV-1-G490N/V492M or HEV-4-N490G/M492V, alongside four corresponding groups of miniature swine (Fig. 7I, J). After infection, stool and serum samples were collected biweekly to detect HEV RNA via RT-PCR and to measure total anti-HEV antibodies in serum. All monkeys exhibited obvious HEV viremia, with onset between 1.0- and 1.5-weeks post-infection, persisting for 2.5-5.0 weeks (Fig. 7I). Fecal virus shedding commenced 0.5 weeks post-infection and ceased between 3.5-6.5 weeks (Fig. 7I). Anti-HEV antibodies were detectable in all groups at 1.5-3.0 weeks post-infection and remained detectable throughout the study (Fig. 7I). These findings demonstrate that both wild-type and mutant HEVs effectively infect cynomolgus monkeys, indicating that the mutations at residues 490 and 492 do not compromise the viruses’ infective capabilities in non-human primates.

Bama miniature swine have been shown to be susceptible to HEV-3 and HEV-4, but not to HEV-137. In our tests, only those Bama miniature swine infected with wild-type HEV-4 exhibited clear signs of HEV viremia, viral shedding, and anti-HEV antibody responses (Fig. 7J). The duration of viremia and viral shedding was ∼5.5–6.0 weeks and 5.0–6.0 weeks, respectively. The anti-HEV antibody responses in swine infected with HEV-4 appeared later and lasted for a shorter duration compared to those in monkeys infected with HEV-4. Of note, the groups exposed to HEV-1, HEV-4-N490G/M492V and HEV-1-G490N/V492M exhibited no detectable fecal virus shedding, HEV viremia, or anti-HEV antibody seroconversion throughout the study, indicating a lack of infection by these viruses in swine (Fig. 7J). The diminished infectivity of the HEV-4-N490G/M492V mutant in swine underscores the pivotal role of residues N490 and M492 in HEV-4’s capability to infect swine. Contrary to expectations, the HEV-1-G490N/V492M mutant, despite regaining binding affinity and infectivity on porcine hepatocytes (Fig. 7C, E, G, H), failed to initiate infection in Bama miniature swine (Fig. 7I), suggesting that altering residues 490 and 492 alone is insufficient to reestablish HEV-1 infection in this species. These findings suggest that additional factors, beyond residues 490 and 492, may collectively influence HEV-4’s specific tropism for swine. Overall, this study emphasizes that while residues 490 and 492 play a critical role in HEV-4 infectivity in swine, they are not solely decisive.

Methods
Ethics statement
Primary human hepatocytes were obtained under ethical approval from the China Ethics Committee of Registering Clinical Trials (approval number: ChiECRCT20210471). Donor identities were blinded to the researchers by using anonymized sample identifiers, and all studies were conducted using de-identified samples. All animal experiments were approved by the Institutional Animal Care and Use Committee and Laboratory Animal Management Ethics Committee of Xiamen University. All experiments were performed in accordance with the relevant guidelines and regulations of Xiamen University.

Cell lines
HepG2 (HB-8065) cells were obtained from the American Type Culture Collection (ATCC, MD, USA) and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, CA, USA) supplemented with 10% fetal bovine serum (FBS) (GIBCO, CA, USA).

Viruses, antibodies, and proteins
The genotype 1 virus (strain Xinjiang, GenBank accession no. L08816.1), genotype 3 virus (strain JRC-HE3, GenBank: accession no. AB630971.1), and genotype 4 virus (strain Ch-S-1, GenBank: accession no. EF077630) were isolated from stool samples obtained from rhesus monkeys challenged with HEV. The stool samples containing virus were suspended in 10% weight/volume (w/v) of phosphate-buffered saline (PBS) containing 1% w/v bovine serum albumin (BSA). The suspension was centrifuged at 10,000 × g for 20 min, and the supernatant was harvested, filtered with a 0.22 μm membrane filter, and stored at -20°C until use. HEV-1 strain Sar-55 (GenBank accession no. AF444002) and HEV-4 strain D32 (GenBank accession no. PP475173.1) were generated from infectious cDNA clones by plasmid-based reverse genetics technology as described previously with some modifications34. The mAb 6H8 was screened using a murine mAb preparation protocol as previously reported32,60. The E2 (amino acids 394-606), E2s (amino acids 455–602) and p239 (amino acids 368-606) proteins were recombinantly expressed and purified as previously described61. A series of mutants were generated by site-directed mutagenesis using PCR and expressed following the same procedure as the wild-type proteins32.

Primary Porcine Hepatocyte Isolation and Culture
The isolation and culture of PPH were performed using a modified two-step method of collagenase perfusion followed by differential centrifugation28,29,58. In brief, the liver of miniature swine was perfused with perfusion solutions containing collagenase via the portal vein. Hepatocytes were dispersed and centrifuged in medium three times at 100 × g. After centrifugation, the hepatocytes were resuspended and seeded in collagen-coated culture plates in William’s medium E (containing 1 µM dexamethasone, 0.1 mM insulin, 20 ng/mL epidermal growth factor (EGF), 20 ng/mL fibroblast growth factor (FGF), 20 ng/mL hepatocyte growth factor (HGF), and 10% FBS and maintained at 37°C in the presence of 5% CO2.

Primary Human Hepatocyte Culture
Cryopreserved PHH were obtained from Liwo Biotechnology (Shanghai, China) and seeded at a density of 1×10⁶ viable cells per well in collagen-coated 6-well plates (BD Biosciences, CA, USA). After 4 h-attachment at 37°C with 5% CO2, cells were maintained in DMEM/F12 medium (Gibco, CA, USA) supplemented with the following components: 2% B27 supplement minus vitamin A (Shanghai Yuanye, Shanghai, China), 1 mM N-acetyl-L-cysteine (Sigma-Aldrich, MO, USA), 10 mM nicotinamide (Sigma-Aldrich, MO, USA), 1% L-alanyl-L-glutamine dipeptide (GlutaMAX), 1% penicillin, 1% streptomycin. Growth factors were added at the following concentrations: 50 ng/mL EGF (Sinobiological, Beijing, China), 100 ng/mL FGF10 (Sinobiological, Beijing, China), 25 ng/mL HGF (Sinobiological, Beijing, China), and 50 ng/mL FGF7 (Thermo Fisher Scientific). Small-molecule inhibitors were also included: 10 μM Y-27632 ROCK inhibitor (Enzo Life Sciences, New York, USA), 100 nM A83-01 (TopScience, Shanghai, China), and 3 μM CHIR-99021 (TopScience, Shanghai, China). Cultures were maintained at 37°C in a humidified with 5% CO2, and the medium was changed daily.

Virus Rescue
The infectious cDNA clone of HEV-1 strain Sar-55, pSK-HEV-2 (GenBank accession no. AF444002), was donated by Emerson34. HEV-4 strain D32 (GenBank accession no. PP475173.1) was isolated from the bile of swine in a slaughterhouse in Xiamen, China. Standard molecular techniques were used to assemble a full-length cDNA clone of the D32 strain from cDNA fragments that were produced by RT-PCR. HEV-4-N490G/M492V was generated in the backbone of D32 by replacing the residues asparagine at 490 and methionine at 492 of ORF2 with glycine and valine, respectively. And HEV-1-G490N/V492M was generated in the backbone of Sar-55 by replacing the residues glycine at 490 and valine at 492 of ORF2 with asparagine and methionine, respectively. The wild-type and mutant HEV stocks were produced by plasmid-based reverse genetics technology as described previously with some modifications34. Briefly, plasmid pSK-HEV-2 and D32 were both linearized with the restriction enzyme BglII downstream of the poly(A) tail of HEV. In vitro transcribed capped RNA transcripts were produced with the mMESSAGE mMACHINE T7 Transcription Kit (Invitrogen, CA, USA) at 20°C for 16 h using 2 μg of linearized DNA. In vitro-transcribed capped RNA transcripts were then transfected into S10-3 cells (a subclone of human hepatoma Huh-7 cells). The transfected cells were cultured at 37°C in DMEM containing 10% FBS for 15 to 20 days and then lysed in distilled H2O by three repeated cycles of freeze and thaw. After centrifugation for 30 min at 10,000 × g to remove cell debris, the supernatant was harvested as HEV stock. Viral titers were quantified by real-time RT-PCR as described below. To quantify HEV infectious titers, HepG2/C3A cells were seeded in 96-well plates at 2×104 cells per well one day prior to the infection assay. Cells were infected with viruses for 8 h. Five days post-infection, cells were analyzed by immunofluorescence assay as described below. The number of FFU was counted according to a previous report28.

Real-time reverse transcription PCR
Viral RNA levels were quantified using real-time RT-PCR as previously reported62. Real-time RT-PCR was carried out using a commercial One-Step RT-PCR Kit (Genmagbio, Beijing, China) on a CFX96TM Real-Time System and C1000TM thermal cycler device (Bio-Rad, CA, USA). Plasmids with known viral copy numbers were serially diluted to generate standard curves for HEV RNA quantification.

SPR assay
The binding affinities of mAb 6H8 for different genotype E2 proteins were measured by SPR on BIAcore 3000 (GE Healthcare, USA) at 25°C in HBS (10 mM HEPES, pH 7.4, and 150 mM NaCl). One channel of a CM-5 sensor chip was coated with 290 response units (RU) of mAb 6H8, and the other flow cell was left uncoated and blocked as a reference channel. Serially diluted antigen was flowed through the mAb 6H8-coated channel and the reference channel at 30 µL/min for 200 s followed by a 700-second dissociation phase. Sensorgrams of serial concentrations were globally fitted using a 1:1 Langmuir model.

Indirect ELISA
The antigen (100 ng per well) was coated onto 96-well microplates and incubated at 37°C for 2 h. After blocking with blocking reagent (PBS containing 2% BSA), the antigen-coated microplates were washed once with wash buffer (PBS with 0.5% Tween-20, PBST). Serially diluted mAbs were added to the antigen-coated plates and incubated with antigen at 37°C for 30 min. After five washes with PBST buffer, the bound antibodies were detected with horseradish peroxidase (HRP)-conjugated goat anti-mouse (GAM) antibody at 37°C for 30 min. Subsequently, the plates were washed five times and the color was developed with 100 µL of tetramethylbenzidine (TMB) substrate. The optical density (OD) was examined with a microplate reader (Autobio, Zhengzhou, China) at 450 nm with a reference of 620 nm.

Western blotting
Western blotting was conducted in accordance with previously described methods60. Briefly, recombinant E2 proteins with or without boiling were electrophoresed on SDS-PAGE gel and subsequently transferred to nitrocellulose membranes (Fig. 2B). The membranes were blocked with 5% skim milk in PBS for 30 min and washed with PBST. Subsequently, the membranes were incubated sequentially with mAb 6H8 followed by HRP-conjugated GAM antibody. The color was developed using SuperSignal West Femto Maximum Sensitivity Substrate (PIERCE, MA, USA) and the reaction signals were determined using ImageQuant LAS 4000 mini (GE Healthcare, Little Chalfont, UK).

Analytical ultracentrifugation
Sedimentation velocity experiments were performed at 20°C on a Beckman XL-A AUC, equipped with absorbance optics and an An60-Ti rotor, as previously described63. Briefly, all samples were diluted to ∼1.0 OD280 in a buffer containing 20 mM HEPES (pH 7.5) and 150 mM NaCl, in a 1.2-cm light length cell. The rotor speed was set at 40,000 rpm. E2 proteins and mAb 6H8 were pre-incubated at a molar ratio of 2:1 at 37°C for 2 h to allow for any possible interactions. Sedimentation coefficient values were obtained using the c(s) method with the Sedfit software kindly provided by Dr. Schuck P (National Institutes of Health, MA, USA).

Immune capture assay
The immune capture assay was performed based on real-time RT-PCR assay and ELISA as previously described32,64. The plates were coated with 900 ng of mAbs per well. After a single wash, the antibody-coated plates were blocked with 350 µL of blocking reagent for 2 h at 37°C. Then, the plates were washed once and incubated with a 200-μL aliquot of HEV suspension at 37°C for 2 h. For the real-time RT-PCR assay, after rinsing five times with wash buffer, 200 μL of TRIzol was added to each well and incubated at 4°C for 10 min. HEV RNA was purified, and the viral RNA levels captured by the mAbs were measured with a real-time RT-PCR assay as described above. For the ELISA assay, plates were rinsed five times with wash buffer, and 200 μL of HRP-conjugated mAb no. 4 solution was added to each well, followed by incubation at 37°C for 30 min. After five additional washes, 100 μL of TMB substrate solution was added to each well and incubated for 15 min at 37°C. Absorbance was measured using a microplate reader.

Detection of HEV antigen by ELISA
The antigen in the cell supernatants was detected using a commercial ELISA kit (Wantai, Beijing, China) and an in-house sandwich ELISA. The antigen detection kits were developed with mAb 12F12 as the capture antibody and mAb no. 4 as the detection antibody64 and can be used for research purposes only. The detection of HEV antigen using the commercial ELISA kit was performed according to the manufacturer’s instructions.

The protocol for the in-house sandwich ELISA was the same as described previously64, except that mAb 6H8 was used as the capture antibody instead of mAb 12F12. Briefly, 96-well ELISA plates were coated with mAb 6H8 (500 ng per well) and incubated overnight at 4°C. The plates were washed once with PBST and subsequently blocked with blocking reagent. Samples (50 μL) were then added to the wells and incubated at 37°C for 60 min. A 100-μL aliquot of no. 4-HRP solution was added directly to each well without washing. Following an additional 30 min incubation, the plates were washed five times with PBST. Color development and OD measurement were performed as described above.

HEV cell-binding assay
HepG2 cells, PPH, and PHH were seeded into 96-well plates or 6-well plates and then treated with one of the following three assays. For the binding assay, serially 2-fold-diluted (initial 64 copies per cell) HEV virions were directly added to both HepG2 cells and PPH or 1000 copies per cell HEV virions were incubated with PHH. In the p239 competition infection assay, both HepG2 cells and PPH were pre-incubated with serial 2-fold dilutions of p239 (starting at 320 μg/mL) and then incubated with ∼64 copies per cell of HEV virions. In the neutralizing activity assay, the virus was pre-incubated with 2-fold serially diluted mAbs or PBS at 37°C for 60 min prior to incubation with HepG2 and PPH. The following procedures were the same for all three assays. After incubation at 37°C for 60 min, the infected cells were washed three times with PBS and the samples were harvested. HEV RNA was purified and quantified by real-time RT PCR.

Binding and penetration of VLP
HepG2, PPH, and PHH cells were pre-cultured on coverslips. Subsequently, p239 wild type or p239 mutants were directly incubated with the cells at 37°C for 1 h or 4 h. For blocking controls, p239 wild-type or p239 mutants were pre-incubated with mAb 8G12 (200 μg/mL) at 37°C for 1 h prior to co-incubation with the cells. The binding and entry efficiency of VLPs was analyzed by immunofluorescence assay as described below.

Immunofluorescence assay
For immunofluorescence assay, cells were washed three times with PBS, fixed with 4% (w/v) paraformaldehyde (Sigma Aldrich, MO, USA), and permeabilized with PBS containing 0.3% Triton X-100 for 15 min. Subsequently, samples were blocked with 5% BSA in PBS for 1 h at room temperature. For VLP binding and entry assays, cells were incubated with mAbs 15B2 and 8C11 for 30 min at room temperature. For HEV virus infection assays, cells were incubated with mAbs 15B2 and 8G12 for 60 min at room temperature. After washing with PBS, fluorescein isothiocyanate-conjugated goat anti-mouse (GAM) IgG (Molecular Probes, CA, USA) was added and incubated for 30 min. Following five washes with PBS, samples were stained with 0.5% 4’,6-diamidino-2-phenylindole (DAPI) (Invitrogen, CA, USA), and fluorescence signals were analyzed with an Opera Phenix, or Operetta CLS High-Content Analysis system (PerkinElmer, MA, USA).

Viral infection detected by ELISA
Viral infection assay based on ELISA were performed as previously described35. HepG2/C3A cells or PPH were seeded into 96-well plates at a density of 3×104 cells per well one day before infection. Cells were inoculated with HEV (1000 copies per cell) at 37°C for 12 h. For neutralizing assays, HEV wild-type or mutants were pre-incubated with mAb A286 or mAb 6H8 (200 μg/mL) at 37°C for 1 h prior to incubation with the cells. Cells were washed three times with PBS and refed with complete medium supplemented with 2% DMSO. After incubation at 37°C for 3 days, cell supernatants were collected and tested for HEV antigen using a commercial ELISA kit (Wantai, Beijing, China).

Virus infection detected by immunofluorescence assay
Cells were seeded on 96-well glass-bottom plates (PerkinElmer, Inc., MA, USA) one day prior to viral infection. Viruses were diluted in culture medium containing 4% polyethylene glycol 8000 (PEG8000) to achieve 1000 copies per cell and then incubated with cells at 37°C for 6 h. For neutralization assays, viruses were pre-incubated with mAb A286 or mAb 6H8 (200 μg/mL) at 37°C for 1 h before infection. Cells were washed three times with PBS to remove unbound viral particles and subsequently cultured in complete medium supplemented with 2% DMSO for 5 days to allow viral replication and protein expression. Viral infection efficiency was evaluated using IFA as described above.

Crystallization and structural determinations
The Fab fragment of mAb 6H8 was digested by papain and purified using anion-exchange chromatography with DEAE-5PW (TOSOH, Tokyo, Japan). The E2s-4:6H8 Fab complex was incubated at 37°C for 2 h in a 1:1.5 molar ratio and then purified by size exclusion chromatography with G5000PWxl (TOSOH, Tokyo, Japan) using a buffer composed of 20 mM HEPES (pH 7.0). The E2s-4:6H8 Fab complex was concentrated to 8.5 mg/mL and used in crystallization experiments. The crystallization condition for E2s-4:6H8 Fab was 0.1 M Tris-HCl buffer (pH 8.5) with 25% w/v polyethylene glycol (PEG) 3350. All E2s proteins were concentrated to 13 mg/mL in crystallization experiments. Both E2s-1 and E2s-4-N490G/M492V were crystallized with a reservoir solution containing 0.1 M Bis-Tris buffer (pH 5.5) and 2 M (NH4)2SO4. The crystals of E2s-1-G490N/V492M were grown from a reservoir solution comprising 0.1 M NaAc and 2 M (NH4)2SO4. The reservoir solution for the crystals of 6H8 Fab was 20% PEG 1500. All crystals were grown using the vapor diffusion technique in hanging drops at 21°C, and 20% 2,3-Butanediol or 30% glycerol supplemented with their respective reservoir solutions were used as the cryoprotectant.

The X-ray diffraction data were collected at 100 K at the Shanghai Synchrotron Radiation Facility (SSRF) beam line 17U and processed using HKL2000 software. Subsequently, the structures were resolved by molecular replacement using PHASER65. The model building was carried out in COOT66, refined using the programs CNS67 and PHENIX68, and analyzed with PROCHECK69. Statistics for the data collection and structure refinement are summarized in Table S1.

Cryo-EM and three-dimensional (3D) reconstruction of HEV-4 VLP:6H8 Complex
The HEV-4-p495:6H8 immune-complex was prepared by mixing of HEV-4 p495 VLP with oversaturated 6H8 Fab, following the corresponding crystal structure of the E2s-4:6H8 complex, with molar ratios of 1:1.2 for E2s monomer and 6H8 Fab. The complex was then incubated at 37°C for 2 h. A 3 μL sample aliquot was deposited onto a glow-discharged Quantifoil holey carbon grid (R2/2, 200 mesh, Quantifoil MicroTools). After 6 s of blotting to remove excess sample, the grid was plunge-frozen in liquid ethane using a Vitrobot Mark IV (Thermo Fisher Scientific). Images were recorded on a Falcon II direct detector camera (Thermo Fisher Scientific) equipped on a Tecnai F30 microscope (Thermo Fisher Scientific) at 300 kV, with a nominal magnification of 93,000, corresponding to a pixel size of 1.109 Å, and with defocus settings determined to be between 1.0 and 3.0 μm. For each image, the total electron dose was ∼25 e/Å2, with an exposure time of 1 s. Data were automatically collected using Thermo Fisher EPU software. A total of 1,066 micrographs were obtained and the contrast transfer function parameters of each micrographs were determined using Gctf70. Micrographs with excessive drift or astigmatism were discarded before reconstruction. Particles were manually picked using the program Eman271. The origin and orientation parameters for each of these particle images were estimated using model-based procedures, and an initial model was generated with the random model method72. After several rounds of reference-free 2D and 3D classifications using Relion73,74, a total of 5,901 good particles were selected for final 3D refinement with Relion. The map resolution was determined using the gold standard criteria of 0.143 Fourier shell correlation (FSC) cut-off73. Local resolution variations were estimated using the program ResMap75. The crystal structure was fitted into the corresponding map using the “fit in map” tool in UCSF Chimera76. Figures were prepared with PyMol77 and UCSF Chimera.

Molecular dynamics simulation
The MD models were derived base on the crystal structures of the of E2s-1, E2s-4, E2s-1-G490N/V492M, and E2s-4-N490G/M492V available in this paper. Another four single-site substitution structures (E2s-1-G490N, E2s-1-V492M, E2s-4-N490G and E2s-4-M492V) was modeled in the mutation wizard of PyMOL77 based on the above crystal structures. Each E2s model was subjected to a 1000-ns simulation using the GROMACS 2024.2 software package78, with CHARMM36 force field79. The protein was solvated in a cubic box with a minimum distance of 1.0 nm between the protein surface and the box edge using the TIP3P water model. Sodium (Na+) and chloride (Cl-) ions were added to neutralize the system and to achieve a final ion concentration of 150 mM. The systems were minimized for 1000 steepest-descent steps until the maximum force on any atom was less than 1000 kJ/mol/nm. After energy minimization, the system was equilibrated in two phases: a 200 picosecond (ps) NVT (constant number of particles, volume, and temperature) simulation at 310 K using the Nose-Hoover thermostat, followed by a 1 ns NPT (constant number of particles, pressure, and temperature) simulation at 1 bar using the Berendsen barostat. Subsequent production MD was performed NPT conditions at 310 K and 1 bar using the Parrinello-Rahman barostat. Long-range electrostatic interactions were calculated using the Particle Mesh Ewald (PME) method with a 1.2 nm cutoff for Coulomb interactions and van der Waals interactions. The LINCS algorithm was employed to constrain all bond lengths, enabling the use of a 2 femtosecond (fs) timestep. Trajectory data were saved every 10 ps for subsequent analysis. Analysis of the simulation trajectories, including RMSD and RMSF, was performed using GROMACS package. Visual molecular dynamics (VMD)80 and MDAnalysis81 were used to visualize and analyze the trajectories.

Multiple sequence alignment
All available HEV protein sequences were retrieved from GenBank as of June 2025. Redundant and incomplete sequences were removed prior to analysis. For subsequent analyses, the region corresponding to the E2s domain was extracted from each aligned sequence. Sequence logo plots were created using WebLogo (https://weblogo.berkeley.edu)82. The extracted E2s domain alignments were submitted to WebLogo to generate graphical representations indicating amino acid conservation and variability at each position.

Animal study
Cynomolgus monkeys (Macaca fascicularis) and Bama miniature swine were infected with HEV, and serial serum and stool samples were collected as previously reported36,37. Briefly, serum samples from cynomolgus monkeys (1.5–3 years old) and Bama miniature swine (2 months old) were screened for anti-HEV antibodies (anti-HEV IgM and anti-HEV IgG for cynomolgus monkeys; anti-HEV Ab for Bama miniature swine) (Wantai, Beijing, China), ALT (Maker Biotechnology, Sichuan, China), and HEV RNA prior to inoculation. Stool samples were suspended in 10% w/v PBS and screened for HEV RNA. HEV RNA was purified from 50 μL of each sample and quantified using real-time RT PCR as described above. Cynomolgus monkeys and Bama miniature swine with normal ALT levels and undetectable anti-HEV antibodies and HEV RNA were included in this study. Then, monkeys or swine (n = 2 per group) were inoculated with 1×107 copies of wild-type or mutant HEV, respectively. Serum samples were tested for anti-HEV antibodies, HEV RNA, and ALT, and stool samples were tested for HEV RNA. The results of the anti-HEV antibodies assays are shown as a signal-to-cutoff ratio (S/CO). Animal experimental protocols were conducted in accordance with the guideline of the Xiamen University Institutional Committee for the Care and Use of Laboratory Animals.

Statistical analysis
Statistical analysis was carried out using GraphPad Prism version 8.0.1 (GraphPad Software Inc, CA, USA) and Origin 2021. IC50 and EC50 were calculated using a nonlinear regression model using GraphPad Prism. Data are presented as mean ± SD in Fig. 1A, B, F–H, and Fig. 7E–G, and as mean values in Fig. 2A, G, H, Fig. 4A, F, G, and Fig. 7A–D. Statistical analysis was performed with unpaired t-test (Fig. 1A, B) or two-way ANOVA (Fig. 1F–H). P values were calculated from the two-tailed test, and p values of 0.05 or lower were considered statistically significant. P values are indicated by an asterisk (*) in plots: ns: not significant; *p < 0.05, **p < 0.01; ***p < 0.001; ****p < 0.0001.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled “Molecular Determinants of Hepatitis E Virus Host Tropism” investigates the molecular mechanisms underlying the host specificity of Hepatitis E virus (HEV) by focusing on the viral capsid protein E2 and its interaction with host cells. The study identifies key residues in the E2 protein that influence host specificity and virus-cell attachment, using a combination of structure-based mutagenesis, molecular dynamics simulations, and virus-cell attachment assays.\n\nMajor Concerns:\n• The study’s findings on the role of residues N490 and M492 in HEV host tropism are not sufficiently novel. Previous studies have already highlighted the importance of the E2s domain in HEV host specificity, and the current work does not significantly advance this understanding.\n• The use of primary porcine hepatocytes (PPH) as a model for HEV study is problematic due to the lack of standardization and potential variability in cell isolation and culture conditions. This raises concerns about the reproducibility and reliability of the results.\n• The molecular dynamics simulations, while extensive, do not provide conclusive evidence for the proposed mechanisms. The simulations should be complemented with additional experimental validation to strengthen the claims.\n• The inoculums used in Figure 1 have not been characterized, as genome copy numbers are used instead of infection units or FFU. This approach is rather unspecific for determining HEV infection events, especially given that the authors appear capable of directly visualizing HEV via immunofluorescence.\n• The in vivo experiments using Bama miniature swine and cynomolgus monkeys do not convincingly demonstrate the role of residues N490 and M492 in HEV host tropism. The failure of the HEV_1_G490N/V492M mutant to infect swine, despite regaining binding affinity to porcine hepatocytes, suggests that other factors are involved in host specificity. This undermines the central hypothesis of the study.\n• The manuscript overinterprets the significance of the identified residues in determining HEV host tropism. The data do not support the conclusion that N490 and M492 are the primary determinants of HEV-4’s specific tropism for porcine hepatocytes. Additional factors and mechanisms are likely involved, which are not addressed in the study.\n• The study does not provide sufficient mechanistic insights into how the identified residues influence HEV host specificity. The proposed role of the N490-Y532 hydrogen bond in stabilizing the epitope conformation is speculative and requires further experimental validation.\n• The manuscript does not adequately discuss the limitations of the study, including the potential variability in primary cell cultures, the limitations of molecular dynamics simulations, and the need for additional in vivo validation. The manuscript would benefit from a more thorough review of the existing literature to better contextualize the findings.\n• The title “Molecular Determinants of Hepatitis E Virus Host Tropism” is rather unspecific and overstated and should be more precise.\n• For some figures, it is unclear how often an experiment was conducted. It is essential to report in every figure legend how many technical or biological replicates were included. The authors should clearly state this as it is important to evaluate the robustness of the results. Additionally, it is often unclear how many cells were imaged or quantified or if images were taken from random areas. Information on this is also important for scientific soundness.\n\nSpecific Comments:\n• Line 45: A more widely used term for the HEV capsid protein is P domain rather than E2.\n• Line 52 and later in the manuscript: Change HEV_1 and HEV_4 to HEV-1 and HEV-4.\n• Spelling and grammar checking suggested.\n\nFigures and Experimental Controls:\n• Figure 1: It is recommended to use primary human hepatocytes (PHH) as a more appropriate control for comparison to primary porcine hepatocytes (PPH). In panels A and B, inclusion of HEV-3 would provide a more comprehensive comparison.\n• Figure 1C, D: Incorporating negative controls (untreated samples) and additional controls such as serum or other blocking agents would enhance the interpretability of the results.\n• Figure 1E-F: The absence of blocking controls in these panels is noted. Including such controls would strengthen the conclusions drawn from these experiments.\n• Figure 2G: It is suggested to add an ELISA-based readout in addition to the PCR-based readout to provide a more robust analysis.\n• Line 188: Correct the reference from Fig 2G to Fig 2H.\n• Figure 2H, I: Presenting neutralization data on authentic virus across different HEV genotypes would be beneficial. An immunofluorescence assay (IFA)-based readout, accompanied by respective IF images, would provide visual confirmation of the results.\n• Supplementary Figures: Move Fig S5 to the main figures to ensure critical data is prominently displayed.\n• Alanine Scanning: The study would benefit from alanine scanning of residues 488, 489, 490, and 532 on authentic virus and assess neutralization capacity. Additionally, investigate attachment not only in HepG2 cells but also in PHH to provide a more comprehensive analysis.\n• Authentic Virus Data: Data from authentic virus experiments, currently in supplements S9E and F, should be moved to the main figures and discussed earlier in the manuscript to highlight their importance.\n• Figure 6A: The inclusion of neutralization controls using antibodies or sera is recommended to validate the findings.\n• Immunofluorescence Images: Ensure scale bars and magnification information are provided for all IF images to enhance clarity and reproducibility.\n• Figure 6A: The use of authentic virus, rather than VLPs, would provide more relevant insights into viral behavior.\n• Figure 6D, E: Include controls and present infection data, including IF images, using authentic virus to extend the study beyond mere binding analysis.\n• Figure 6F: The authors should simplify the visualization of the data, enhance the x-axis legends, increase the number of animals in the study, and measure RNA levels in the liver to provide a more detailed and informative analysis.", "reviewer_2": "HEV has multiple genotypes: HEV1 and HEV2 infect only humans, while HEV3 and HEV4 infect a wide range of animal species including pigs other than humans. The basis of this difference in HEV’s host range is not well understood. The study by Tang et al. identifies two amino acid residues (N490 and M492) on the HEV4 capsid critical for binding to an HEV3- and HEV4-specific monoclonal antibody 6H8 and infecting pig hepatocytes. Mutations in these residues in HEV4 abolished its ability to bind 6H8 and infect pig cells but had no effect on its ability to infect human cells. Conversely, substitution of these residues for those in HEV1 rendered HEV1 capable of infecting pig cells. However, HEV1 bearing N490 and M492 failed to establish infection in minipigs, indicating additional viral factors are also important for infection in pigs.\nThis is an important study that advances our understanding of the viral determinants of HEV’s host range. The manuscript is well-written. The data are of high quality and compelling. There are some minor issues that are listed below and should be addressed.\n-Line 157 and Fig. 1G: Fig. 1G can be removed since no virions were detected in the cells.\n-Fig. 3E: The amino acid alignment includes only one strain in each HEV genotypes. It would be good to include more HEV sequences to show the conservation of these residues.\n-Line 263-264: Serine (S) should be Thr (T), and tyrosine (Y) should be Ser (S).\n-Line 277: This sentence doesn’t read well. How about “The structure…displayed greater similarity in the 480- and 530-loop to E2s_4 than E2s_1”?\n-Fig. 5A: Change RMSF to RMSD on the y-axis.\n-Line 358: Change formed to forming.\n-Line 382: Change the cell lysate to the lysate of cells.\n-Line 399: Change HEV_4 to HEV_1.", "reviewer_3": "This study provides insights into the molecular determinants of Hepatitis E virus (HEV) host tropism, particularly focusing on the zoonotic group HEV. The authors systematically identify key residues (N490 and M492) within the E2 domain of the capsid protein that contribute to HEV’s specific binding to porcine hepatocytes. Various methodologies, including structure-based mutagenesis, molecular dynamics simulations, and virus-cell attachment assays, strengthen the conclusions drawn from their findings. Identifying the 6H8 antibody epitope, its structural characterization through crystallography and cryo-EM, and the exploration of mutant viruses in both in vitro and in vivo models are comprehensive.\n\nHowever, there are areas for improvement.\n1. Figure 3D: The labeling of amino acid residue appears quite crowded. The stick model, label text, and unnecessary shadows make it difficult for the reader to clearly distinguish the positions of key amino acids. A clearer layout or reducing label overlap would greatly improve the readability.\n2. Figure 4: Several interactions lack distance annotations, which diminishes the clarity of the structural relationships between the residues.\n3. Figure 5D: The use of PDB file Chain IDs as identifiers is understandable, but the two symmetric protomers of the E2s dimer do not appear to behave consistently, especially for E2s_1 V492M and E2s_4 N490G. This inconsistency is intriguing —does it suggest structural or functional differences between these protomers that could be relevant to the virus’s mechanism or stability? It would be beneficial to provide a discussion of these discrepancies.\n4. Figure 6E: There appears to be an absence of error bars.\n7. Crystal Structure Model: The crystal structures, particularly E2s_4_N490G/M492V and E2s_1, may require further refinement. Notably, the Ramachandran plots for these two structures show a higher percentage of disallowed regions compared to the others (0.7% vs. 0%) , which is unexpected. This could indicate either some special structural features arising from the crystallization conditions or simply an incomplete refinement process. It would be helpful for the authors to clarify whether these discrepancies stem from the protein crystallization energy state (e.g. packing interactions), or if further refinement was needed. Additionally, the authors’ provided crystal structure PDB validation report is not as curated (NOT for manuscript review) as the cryo-EM structure validation (which was ready for manuscript review).\n\nThe results in this manuscript enhance the understanding of HEV’s cross-species transmission potential, particularly regarding its zoonotic transmission. If the above deficiencies are addressed or clearly explained, I recommend this manuscript to be published in Nature Communications"}, "author_response": "1 Response to Reviewer Comments on the manuscript NCOMMS-25-13653-T:\n2\n4 significantly improved our manuscript. To facilitate the navigation of this document,\n5 we have copied the reviewer’s comments verbatim in blue and typed out our responses\n6 in black. Thanks to the valuable suggestions, the manuscript now offers more insightful\n7 information for readers.\n8\n12 investigates the molecular mechanisms underlying the host specificity of Hepatitis E\n13 virus (HEV) by focusing on the viral capsid protein E2 and its interaction with host\n14 cells. The study identifies key residues in the E2 protein that influence host specificity\n15 and virus-cell attachment, using a combination of structure-based mutagenesis,\n16 molecular dynamics simulations, and virus-cell attachment assays.\n18 recognition of our work. We truly appreciate these constructive feedback, which greatly\n19 helped improve the clarity and quality of our manuscript. We have addressed all the\n20 reviewer’s comments and revised the manuscript accordingly. All changes in the\n21 manuscript are highlighted in yellow.\n22\n24 1. The study’s findings on the role of residues N490 and M492 in HEV host tropism are\n25 not sufficiently novel. Previous studies have already highlighted the importance of the\n27 advance this understanding.\n29 role in HEV host specificity. Indeed, previous studies have demonstrated that the E2s\n1\n30 domain plays an important role in host specificity and swapping the E2s domain\n31 between HEV-1 (human-restricted) and HEV-3 (zoonotic) alters infectivity in porcine\n32 kidney cells in vitro (He et al, 2008, J Gen Virol; Nguyen et al, 2014, J Virol). However,\n33 this approach left two critical gaps:\n34 (1) Whether alteration of the E2s domain could disrupt the host's tropism of zoonotic\n36 (2) The functional mechanism of HEV host specificity remained unresolved. Prior work\n37 did not identify specific residues governing tropism, nor did it explain why E2s domain\n38 swaps only partially modulated infectivity.\n40 (1) We confirmed the critical role of E2s domain in determining HEV host specificity\n41 both in vitro and in vivo.\n42 (2) We identified of tropism-determining residues: through systematic mutagenesis, we\n43 pinpointed N490 and M492 as the specific residues within E2s that dictate HEV-4’s\n44 ability to infect swine. Mutating these sites abolished HEV-4 infectivity in both porcine\n45 cells (in vitro) and Bama miniature swine (in vivo), while reciprocal mutations in HEV-\n46 1 restored its ability to infect porcine cells in vitro (Fig. 7C-J). This residue-level\n47 resolution provides a mechanistic leap beyond domain-swap approaches.\n48 (3) We performed in vivo tropism reprogramming: we showed that HEV-1 with\n50 vitro infectivity (Fig. 7C-J). This disconnect reveals that additional viral factors (e.g.,\n52 receptors) constrain cross-species transmission in vivo.\n54 functionally mapped determinant of HEV host range. We have revised the manuscript\n55 on Page 6, line 110-113, Page 20, Page 17, line 422-431, Page 18, line 432-434, Page\n56 20, line 501-503, line 513-514, Page 21, line 516-529, Page 30, line 782-795, Page 31,\n57 line 796-814, to emphasize our findings.\n58\n2\n59 2. The use of primary porcine hepatocytes (PPH) as a model for HEV study is\n60 problematic due to the lack of standardization and potential variability in cell isolation\n61 and culture conditions. This raises concerns about the reproducibility and reliability of\n62 the results.\n64 consideration. Primary cells provide an widely used model for studying HEV interplay\n65 with host, including host response and host tropism. Since pigs represent the natural\n66 host of HEV, the isolation of primary porcine hepatocytes (PPH) have been reported in\n67 many previous studies (Chen, et al, 2019, Hepatology; Angelini et al, 2021, Gut; Todt\n68 et al, 2020, PNAS; Li et al, 2024, Biomed Pharmacother; Nyberg et al, 2005, Liver\n70 studies, which have been used as a model of HEV study in previous studies (Todt et al,\n71 2020, PNAS; Schlienkamp et al, 2025, Emerg Microbes Infect).\n75 cross-verified. Therefore, we believe that our core findings on the PPH models is\n76 reasonable.\n77\n78 3. The molecular dynamics simulations, while extensive, do not provide conclusive\n79 evidence for the proposed mechanisms. The simulations should be complemented with\n80 additional experimental validation to strengthen the claims.\n82 validation for computational findings. Our main conclusion from the MD simulations\n83 is that the N490-Y532 hydrogen bond critically stabilizes the 6H8 epitope conformation;\n84 disruption of this bond, as seen in the G490 mutants, increases local flexibility and\n85 abrogates 6H8 binding.\n87 computational data in our study. Specifically, binding assays (Fig. 4F-G) showed that\n3\n89 completely lose 6H8 binding, while variants retaining N490 (E2-4, E2-1-G490N, E2-\n90 1-G490N/V492M, E2-4-M492V) preserve antibody recognition. Moreover, crystal\n91 structures (Fig. 4H-I) corroborated the structural basis of this observation: the critical\n93 provides both computational and experimental evidence that consistently support the\n94 mechanism we propose.\n95\n96 4. The inoculums used in Figure 1 have not been characterized, as genome copy\n97 numbers are used instead of infection units or FFU. This approach is rather unspecific\n98 for determining HEV infection events, especially given that the authors appear capable\n99 of directly visualizing HEV via immunofluorescence.\n101 suggestion, we performed additional experiment to detect the infectivity of HEV on\n103 showed that the infectivity of HEV-3 and HEV-4 were observed both on HepG2 and\n106 on HepG2 and PPH (Fig. 1C, Fig. S3, and Fig.S4) and provided corresponding\n107 descriptions at page 7, lines 143-149, Page 30, line 782-794, and Page 31, line 805-814.\n109 revised in Fig. 1A and 1B.\n4\n110\n112 immunofluorescence assay. Green fluorescence indicates HEV ORF2 protein\n113 expression, and blue fluorescence indicates 4',6-diamidino-2-phenylindole (DAPI)\n114 nuclear staining. Scale bar, 25 μm.\n115\n116 5. The in vivo experiments using Bama miniature swine and cynomolgus monkeys do\n117 not convincingly demonstrate the role of residues N490 and M492 in HEV host tropism.\n119 binding affinity to porcine hepatocytes, suggests that other factors are involved in host\n120 specificity. This undermines the central hypothesis of the study.\n122 complexity of HEV host tropism. This study demonstrated that the residues N490 and\n124 additional factors may modulate cross-species transmission, our data unequivocally\n125 demonstrate that N490 and M492 serve as non-redundant molecular switches. We\n126 provided loss-of-function proof by mutating N490/M492 in HEV-4 (a swine-adapted\n127 strain), which abolished infectivity of HEV in both porcine hepatocytes (in vitro) and\n129 introducing N490/M492 mutations into HEV-1 (human-restricted), which restored\n130 binding and infectivity in porcine hepatocytes in vitro. This residue-level resolution\n5\n131 achieved through systematic mutagenesis across genotypes (HEV-1/4) provides the\n132 direct evidence that N490/M492 are gatekeepers for swine entry.\n134 despite restored hepatocyte binding and infectivity. Our data show that N490/M492 are\n135 necessary for the initial entry step, but post-entry factors (e.g., innate immune evasion\n136 or replication machinery) may limit in vivo fitness. This aligns with known HEV\n137 biology: many other viral factors such as ORF1, 5’UTR, codon usage, and adaptive\n138 evolution, may be involved in determinizing HEV host tropism (Feagins et al, 2011,\n140 al, 2012, Infect Genet Evol; Nguyen et al, 2014, J Virol; Sun et al, 2020, Infect Genet\n144 this study demonstrated that two-residue edits yield binary on/off phenotypes in cellular\n145 models. To contextualize these mechanisms, we have revised the title to emphasize the\n146 pivotal role of the two residues: “The Residues 490 and 492 on E2s Domain of the\n149 findings and limitations in the revised manuscript on Page 1, line 1-2, Page 5, line 93-\n150 95, Page 6, line 96-98, Page 20, line 501-503, Page 21, line 517-529, Page 23, line 594-\n151 599, and Page 24, line 600-602.\n152\n153 6. The manuscript overinterprets the significance of the identified residues in\n154 determining HEV host tropism. The data do not support the conclusion that N490 and\n157 study.\n159 interpretation of our data. We acknowledge that the original manuscript contained\n6\n160 overstatements about the role of N490 and M492 residues in determining HEV-4 host\n161 tropism. To address this concern, we have implemented the following modifications:\n162 (1) We have revised the title to emphasize the pivotal role of the two residues more\n163 rigorously: “The Residues 490 and 492 on E2s Domain of the Capsid Protein Play\n164 a Pivotal but Insufficient Role in Determining the Host Tropism of Hepatitis E\n166 (2) Content Refinements: N490 and M492 play a pivotal but insufficient role in\n167 determining HEV host tropism, as mutation of these two residues disrupts both the\n168 binding and infection of primary porcine hepatocytes in vitro and in vivo infection in\n170 adaptive evolution, and host determinants, including host cellular factors and host\n171 immune status, were also involved in determining HEV-4’s specific tropism for porcine\n172 hepatocytes. We have further elaborated on the relevant descriptions in the\n173 “Introduction” section on Page 5, line 93-95, Page 6, line 96-98, and “Discussion”\n174 section on Page 20, line 501-503, Page 21, line 516-529.\n175 (3) We also revised and provided a balanced conclusion for our findings: While\n177 modulated by uncharacterized viral and host co-factors.\n179 while acknowledging the systemic complexity of tropism—a nuance we regret was\n180 initially overstated.\n181\n182 7. The study does not provide sufficient mechanistic insights into how the identified\n183 residues influence HEV host specificity. The proposed role of the N490-Y532 hydrogen\n184 bond in stabilizing the epitope conformation is speculative and requires further\n185 experimental validation.\n187 prior responses, we believe that our study is the first to resolve residue-level\n188 determinants of HEV host tropism through an integrated structural and functional\n7\n189 approach. Below we want to address the mechanistic insights with enhanced clarity:\n191 mAb 6H8 was associated with HEV-4's specific tropism for porcine hepatocytes. The\n192 binding assays of mutants with mAb 6H8 (Fig. 4F-G) showed that the G490-containing\n193 mutants completely lose 6H8 binding, while variants retaining N490 preserve antibody\n194 recognition. We also determined high-resolution (<2 Å) crystal structures of E2s-1,\n197 hydrogen bond is present in E2s-1 but absent in E2s-4. Given that crystal structures\n198 represent time- and space-averaged states and are limited in capturing dynamic\n199 conformational changes, we further performed molecular dynamics (MD) simulations\n200 to elucidate potential molecular mechanisms. Our MD simulations across various E2s-\n201 1 and E2s-4 wildtype and mutants consistently demonstrated a strong correlation\n202 between the presence of the N490-Y532 hydrogen bond and epitope stability (Fig. 5C).\n204 determinant in stabilizing the 6H8-recognized epitope, thereby influencing host tropism.\n205\n206 8. The manuscript does not adequately discuss the limitations of the study, including\n207 the potential variability in primary cell cultures, the limitations of molecular dynamics\n208 simulations, and the need for additional in vivo validation. The manuscript would\n209 benefit from a more thorough review of the existing literature to better contextualize\n210 the findings.\n212 related description regarding the limitations of this study in the “Discussion” section on\n214 description of the factors that determined the HEV host tropism on Page 5, Line 93-95,\n216\n217 9. The title “Molecular Determinants of Hepatitis E Virus Host Tropism” is rather\n8\n218 unspecific and overstated and should be more precise.\n222 revised manuscript.\n223\n224 10. For some figures, it is unclear how often an experiment was conducted. It is\n225 essential to report in every figure legend how many technical or biological replicates\n226 were included. The authors should clearly state this as it is important to evaluate the\n227 robustness of the results. Additionally, it is often unclear how many cells were imaged\n228 or quantified or if images were taken from random areas. Information on this is also\n229 important for scientific soundness.\n231 have been added in the corresponding figure legends. In particular, about 2000 cells\n232 were quantified in Fig. 6B and Fig. 6C. In the Fig. 7G, about 69 imaged area were\n233 quantified.\n234\n236 11. Line 45: A more widely used term for the HEV capsid protein is P domain rather\n237 than E2.\n239\n240 12. Line 52 and later in the manuscript: Change HEV_1 and HEV_4 to HEV-1 and\n244 figures. We have also revised the E2_1, E2_2, E2_3, and E2_4 to E2-1, E2-2, E2-3, and\n246 p239-3, and p239-4, respectively.\n9\n249 13. Figure 1: It is recommended to use primary human hepatocytes (PHH) as a more\n250 appropriate control for comparison to primary porcine hepatocytes (PPH). In panels A\n251 and B, inclusion of HEV-3 would provide a more comprehensive comparison.\n253 potency of HEV on PHH. Therefore, we have analyzed the binding of HEV on PHH as\n254 well as the binding and penetration of VLP on PHH and added the related results in the\n255 revised Fig. S2 and Fig. S5. We have also added the related description in the revised\n256 manuscript on Page 7, line 136, line 142-143, Page 25, line 655, Page 26, line 656-670,\n258 of the binding of HEV-3 on PPH and HepG2 in the revised Fig. S1 and added the related\n259 description in the revised manuscript on Page 7, line 136-137, line 139-140, line 142,\n260 line 145.\n261\n262\n264 hepatocytes (PHH). HEV-1: genotype 1 HEV; HEV-3: genotype 3 HEV; HEV-4:\n265 genotype 4 HEV. Binding of HEV to PHH was blocked by cross-genotype neutralizing\n266 monoclonal antibody 8G12. Data represent two biological replicates.\n267\n10\n268\n270 and genotype 4 p239 (p239-4) to primary human hepatocytes (PHH). Green\n271 fluorescence indicates positive for p239 proteins; blue fluorescence indicates positive\n272 for 4',6-diamidino-2-phenylindole (DAPI) nuclear staining. Cross-genotype\n273 neutralizing monoclonal antibody (mAb) 8G12 was used to block the binding and\n274 penetration of p239 proteins. 1h+8G12 and 4h+8G12 indicate the binding and\n275 penetration of p239 proteins in the presence of mAb 8G12, respectively. The binding\n276 and penetration of p239-1 and p239-4 were blocked by mAb 8G12. NC (negative\n277 control) represents untreated cells. Scale bar, 25 μm.\n278\n279\n281 primary porcine hepatocytes (PPH). Magenta column represents HEV-3 binding\n282 capacity to HepG2 cells, and cyan column represents HEV-3 binding capacity to PPH.\n284 of infection.\n11\n285 14. Figure 1C, D: Incorporating negative controls (untreated samples) and additional\n286 controls such as serum or other blocking agents would enhance the interpretability of\n287 the results.\n289 not treated with p239 proteins and a cross-genotype monoclonal antibody 8G12 were\n290 used as the blocking agents in the immunofluorescence assay of p239 wildtype. As\n291 excepted, the binding and penetration of both p239-1 and p239-4 on HepG2 and\n292 primary porcine hepatocytes could be blocked by a previously reported HEV cross-\n293 genotype neutralizing antibody 8G12. We have added the related results as Fig. S6 in\n294 supplement material and revised the description on Page 8, line 157-158, and Page 30,\n295 line 778-779.\n296\n297\n299 and genotype 4 p239 (p239-4) on HepG2 cells (A) and primary porcine hepatocytes\n12\n300 (PPH) (B). Green fluorescence indicates positive for p239 protein; blue fluorescence\n301 indicates positive for 4',6-diamidino-2-phenylindole (DAPI) nuclear staining. Cross-\n302 genotype neutralizing monoclonal antibody (mAb) 8G12 was to block the binding and\n303 penetration of p239 proteins on both cells. 1h+8G12 and 4h+8G12 indicate the binding\n304 and penetration of p239 proteins in the presence of mAb 8G12, respectively. The\n305 binding and penetration of p239-1 and p239-4 on both cell types were blocked by mAb\n306 8G12. NC (negative control) represents untreated cells. Scale bar, 25 μm.\n307\n308 15. Figure 1E-F: The absence of blocking controls in these panels is noted. Including\n309 such controls would strengthen the conclusions drawn from these experiments.\n311 was used as blocking controls and the corresponding results were presented in Fig. 1F-\n313\n314 16. Figure 2G: It is suggested to add an ELISA-based readout in addition to the PCR-\n315 based readout to provide a more robust analysis.\n317 the capture abilities of mAb 6H8 were analyzed using ELISA-based immune capture\n318 assay. The results of ELISA-based immune capture assay demonstrated that mAb 6H8\n319 presented capture ability for both HEV-3 and HEV-4, but not for HEV-1.\n322\n13\n324 immune capture experiment with ELISA for HEV antigen quantitation. The dash line\n325 indicates the cut-off value. Data represent two biological replicates.\n326\n327 17. Line 188: Correct the reference from Fig 2G to Fig 2H.\n329 added in Fig. 2H, we have changed to Fig. 2I in the revised manuscript.\n330\n331 18. Figure 2H, I: Presenting neutralization data on authentic virus across different HEV\n332 genotypes would be beneficial. An immunofluorescence assay (IFA)-based readout,\n333 accompanied by respective IF images, would provide visual confirmation of the results.\n335 different genotypes HEV were assessed only in HepG2. According to the reviewer’s\n336 suggestion, we further performed the neutralization assay of mAb 6H8 against HEV-3\n337 and HEV-4 in PPH. The results showed that 6H8 showed similar neutralizing activity\n338 against HEV-3 and HEV-4 on PPH compared with those on HepG2. We have added the\n339 corresponding results in the revised Fig. 2J and Fig. 2L (see attached below), and\n340 revised the description at Page 9, line 204-206, page 10, line 208-209.\n342 genotypes HEV infection were currently absent (Todt et al, 2020, PNAS; Brüggemann\n343 et al, 2025, Trends Microbiol). Most HEV study based on immunofluorescence assay\n344 were rely on a specific HEV-3 strain (Kernow), which were derived from an\n345 immunosuppressed patient with chronic HEV infection (Todt et al, 2020, PNAS; Liu et\n346 al, 2019, Viruses; Yin et al, 2016, J Virol; Schlienkamp et al, 2025, Emerg Microbes\n348 6H8 against HEV-1, HEV-3, and HEV-4 using a single-concentration IFA-based assay,\n349 with mAb A286 serving as the reference control. The results demonstrated that mAb\n350 6H8 exhibited potent neutralizing activity exclusively against HEV-3, and HEV-4 on\n352 revised manuscript at Page 10, line 209-211, and Page 31, line 805-814, and Fig. S3\n14\n353 and Fig. S4 (see attached below).\n354\n355\n357 of HEV in primary porcine hepatocytes (PPH). Data represent two biological replicates.\n358 (K) Neutralizing activity of mAb 8G12 for different genotypes of HEV on HepG2. Data\n359 represent two biological replicates. (L) Neutralizing activity of mAb 8G12 against\n360 different genotypes of HEV in PPH. Data represent two biological replicates. mAb\n361 8G12 with cross-genotype neutralizing capability for HEV was used as a control for the\n362 neutralizing assay.\n363\n365 assessed by immunofluorescence assay. Green fluorescence indicates positive for\n15\n367 phenylindole (DAPI) nuclear staining. +6H8 and +A286 represent HEV infectivity in\n368 the presence of mAb 6H8 and mAb A286, respectively. All wild-type and mutant\n369 viruses were neutralized by mAb A286. HEV-3, HEV-4, and HEV-1-G490N/V492M\n370 were neutralized by mAb 6H8. Scale bar, 50 μm.\n371\n372\n374 porcine hepatocytes (PPH) assessed by immunofluorescence assay. Green\n375 fluorescence indicates positive for HEV ORF2 protein and blue indicates positive for\n376 4',6-diamidino-2-phenylindole (DAPI) nuclear staining. +6H8 and +A286 represent\n378 with demonstrable infectivity on PPH are shown. HEV-3, HEV-4, and HEV-1-\n380\n381 19. Supplementary Figures: Move Fig S5 to the main figures to ensure critical data is\n382 prominently displayed.\n384 manuscript), the mutation at residues 488 and 532 completely abolished the binding of\n16\n385 p239-4 on PPH, as well as the binding of p239-1 and p239-4 on HepG2. These results\n386 indicated that these two residues may play an important role in the receptor recognition\n387 on HepG2 and PPH. In current, the genuine functional receptor for HEV remain\n388 unknown (Wißing et al, 2021, Trends Microbiol; Wang et al, 2021, Curr Opin Microbiol;\n390 mechanism determining the HEV host range and cross-species infection. Thus, we think\n391 that the original Fig. S5 (Fig. S12 in the revised manuscript) is more suitable to be\n392 placed in the supplement material.\n393\n394 20. Alanine Scanning: The study would benefit from alanine scanning of residues 488,\n395 489, 490, and 532 on authentic virus and assess neutralization capacity. Additionally,\n396 investigate attachment not only in HepG2 cells but also in PHH to provide a more\n397 comprehensive analysis.\n399 completely abolished the binding of HEV VLP p239 to both primary porcine\n400 hepatocytes (PPH) and HepG2, indicating that residues 488 and 532 may be implicated\n401 in HEV receptor recognition. The primary objective of this study was to elucidate the\n402 molecular mechanisms underlying HEV cross-species infection, the roles of residues\n403 488, 489, 490, and 532 on both PPH and HepG2 were not comprehensively investigated\n404 in this study, and alanine-scanning mutant viruses targeting residues 488, 489, 490, and\n405 532 were not generated, and their neutralization capacity were not assay. Due to the\n406 limit of primary human hepatocytes (PHH), we investigated the attachment of only\n407 p239 wildtype but not p239 mutants on PHH.\n408\n409 21. Authentic Virus Data: Data from authentic virus experiments, currently in\n410 supplements S9E and F, should be moved to the main figures and discussed earlier in\n411 the manuscript to highlight their importance.\n17\n414 the revised manuscript. We have revised the description on Page 17, line 407-410.\n415\n416 22. Figure 6A: The inclusion of neutralization controls using antibodies or sera is\n417 recommended to validate the findings.\n419 used as the neutralization control to validated the findings. We have revised the\n420 description on Page 30, line 778-779. We added the related results performed on PPH\n421 and HepG2 in Fig. S16 and Fig. S17, respectively.\n422\n423 23. Immunofluorescence Images: Ensure scale bars and magnification information are\n424 provided for all IF images to enhance clarity and reproducibility.\n426 have provided scale bars in the figures with immunofluorescence images, including Fig.\n427 1C-1E, Fig. 6A, Fig. 7H, Fig. S3. Fig. S4, Fig. S6, Fig. S12, Fig. S16, and Fig. S17.\n429 legends on Page 42, line 1169, 1172, 1174, Page 45, line 1268, Page 46, line 1293.\n430\n431 24. Figure 6A: The use of authentic virus, rather than VLPs, would provide more\n432 relevant insights into viral behavior.\n435 and primary porcine hepatocytes (PPH), respectively, and then incubated at 37°C for 4\n436 hours. The cells were then analyzed through immunofluorescence assay and\n437 fluorescence images were acquired with Leica SP8 (Leica, Wetzlar, Germany). The\n438 results showed that faint fluorescence signals were observed (see Appendix Fig. 1\n439 attached below). Due to the lack of efficient HEV cell culture systems to different\n440 genotypes, it is difficult for us to provide relevant insights for all wildtype and mutant\n441 viruses at both 1h-incubation and 4h-incubation.\n18\n442\n444 hepatocytes (PPH) detected by immunofluorescence assay. Scale bar: 50 μm. NC\n445 (negative control) represents untreated cells.\n446\n447 25. Figure 6D, E: Include controls and present infection data, including IF images, using\n448 authentic virus to extend the study beyond mere binding analysis.\n450 have assessed the infectivity of mutant and wild-type viruses in PPH and HepG2 cells\n451 using ELISA and immunofluorescence assay, with mAb A286 serving as a neutralizing\n452 control. The results showed that HEV-1, HEV-4-N490G/M492V, HEV-4 and HEV-1-\n456 in the revised manuscript on page (Fig.7E-7H, Fig. S3 and Fig. S4). We also added the\n457 description on Page 16, line 385, Page 17, line 422-431, Page 28, line 432-434, Page\n458 30, line 782-795, and Page 31, line 796-814.\n19\n459\n462 replicates. (F) Ratio of HEV levels in the supernatant of PPH and HepG2 cells after\n464 assay after HEV infection. Approximately 69 imaging areas were quantified. Data\n465 represent three biological replicates (H) Infection capability of mutant viruses in\n467 indicates HEV ORF2 protein expression and blue fluorescence indicates positive for\n468 4',6-diamidino-2-phenylindole (DAPI) nuclear staining. Scale bar, 25 μm.\n469\n470 26. Figure 6F: The authors should simplify the visualization of the data, enhance the x-\n471 axis legends, increase the number of animals in the study, and measure RNA levels in\n472 the liver to provide a more detailed and informative analysis.\n474 et al, 2011, Virus Res; Gu et al, 2015, Cell Res; Tang et al, 2015, Sci Rep). During the\n475 animal experiments, the animals were monitored for viral shedding and the liver tissues\n20\n476 from these animals were not collected. Thus, we were unable to measure RNA levels\n477 in the liver. As recommended by the reviewer, we have simplified the visualization of\n478 the data concerning animal experiments (please referred to the revised Fig. 7I and Fig.\n479 7J) and revised the corresponding figure legends on Page 46, line 1296, and Page 47,\n480 line 1297, line 1299-1300.\n481\n21\n486 basis of this difference in HEV’s host range is not well understood. The study by Tang\n487 et al. identifies two amino acid residues (N490 and M492) on the HEV4 capsid critical\n488 for binding to an HEV3- and HEV4-specific monoclonal antibody 6H8 and infecting\n489 pig hepatocytes. Mutations in these residues in HEV4 abolished its ability to bind 6H8\n490 and infect pig cells but had no effect on its ability to infect human cells. Conversely,\n491 substitution of these residues for those in HEV1 rendered HEV1 capable of infecting\n492 pig cells. However, HEV1 bearing N490 and M492 failed to establish infection in\n493 minipigs, indicating additional viral factors are also important for infection in pigs.\n496 compelling. There are some minor issues that are listed below and should be addressed.\n498 addressed all the reviewer’s comments and revised the manuscript accordingly. All\n499 changes in the manuscript are highlighted in yellow.\n500\n501 1. Line 157 and Fig. 1G: Fig. 1G can be removed since no virions were detected in the\n502 cells.\n504\n505 2. Fig. 3E: The amino acid alignment includes only one strain in each HEV genotypes.\n507 residues.\n510 and brevity of Fig. 3, we present the representative E2s sequences from HEV1-4 in the\n22\n511 main figure, and show the sequence logo plots, generated using WebLogo, in Fig. S11.\n513 acids at each position. The methods for multiple sequence alignment and logo\n514 generation are described in detail in the Methods section (Page 34, line 890-897).\n515\n516\n518 of HEV genotypes 1-4. (A) Sequence logo representing the E2s region of genotype 1\n520 logo representing the E2s region of HEV genotype 3 (n = 230) and genotype 4 (n = 6)\n23\n521 strains. The height of each letter at a given position represents the relative frequency of\n522 the corresponding amino acid, and the overall height of the stack indicates the degree\n523 of conservation at that position. Red arrowheads indicate critical amino acid residues\n524 at positions 490 and 492.\n525\n526 3. Line 263-264: Serine (S) should be Thr (T), and tyrosine (Y) should be Ser (S).\n528 tyrosine (Y) to Ser (S), respectively, in the revised manuscript.\n529\n530 4. Line 277: This sentence doesn’t read well. How about “The structure…displayed\n531 greater similarity in the 480- and 530-loop to E2s_4 than E2s_1”?\n533 original sentence did not express our opinion well. Our original intention was to express\n534 that the conformations of the 480- and 530-loop regions in E2s-1-G490N/V492M are\n535 closer to those in E2s-4 than to E2s-1, which is reflected by lower RMSD values. We\n536 agree that the sentence could be made clearer. Following your advice, we have revised\n537 the sentence as follows to improve readability and explicitly highlight the comparison:\n538 “The structural conformation of the 480- and 530-loop regions (6H8 epitope region) in\n539 the structure of E2s-1-G490N/V492M is more similar to that in E2s-4 than to E2s-1, as\n540 evaluated by root mean square deviations (RMSDs) of 0.227 and 0.458 Å.” (Page 13,\n541 lines 298-300)\n542\n543 5. Fig. 5A: Change RMSF to RMSD on the y-axis.\n545 carefully reviewed Fig. 5A and would like to clarify that our intention in this figure was\n546 to represent residue-specific flexibility. Therefore, we used root mean square\n547 fluctuation (RMSF), which reflects the per-residue variations throughout the MD\n548 simulation, rather than root mean square deviation (RMSD), which describes the\n549 overall structural deviation. We believe RMSF is more appropriate for this purpose. To\n24\n550 prevent potential confusion, we added a brief introduction of the calculation of RMSF\n551 in the revised manuscript for clarity: “To quantitatively compare the flexibility of\n552 different structures, we calculated the root mean square fluctuation (RMSF) for each\n553 residue based on the MD simulation trajectories.” (Page 14, line 325-327)\n554\n555 6. Line 358: Change formed to forming.\n557\n558 7. Line 382: Change the cell lysate to the lysate of cells.\n560\n561 8. Line 399: Change HEV_4 to HEV_1.\n25\n565 host tropism, particularly focusing on the zoonotic group HEV. The authors\n566 systematically identify key residues (N490 and M492) within the E2 domain of the\n567 capsid protein that contribute to HEV’s specific binding to porcine hepatocytes. Various\n568 methodologies, including structure-based mutagenesis, molecular dynamics\n569 simulations, and virus-cell attachment assays, strengthen the conclusions drawn from\n570 their findings. Identifying the 6H8 antibody epitope, its structural characterization\n571 through crystallography and cryo-EM, and the exploration of mutant viruses in both in\n572 vitro and in vivo models are comprehensive.\n574 recognition of our work. We truly appreciate these constructive feedbacks, which\n575 greatly helped improve the clarity and quality of our manuscript. We have addressed all\n576 the reviewer’s comments and revised the manuscript accordingly. All changes in the\n577 manuscript are highlighted in yellow.\n578\n580 1. Figure 3D: The labeling of amino acid residue appears quite crowded. The stick\n581 model, label text, and unnecessary shadows make it difficult for the reader to clearly\n582 distinguish the positions of key amino acids. A clearer layout or reducing label overlap\n583 would greatly improve the readability.\n585\n26\n586\n588 blue, positive; and gray, neutral), with key residues for interaction from 6H8 CDR loops\n589 represented as sticks.\n590\n591 2. Figure 4: Several interactions lack distance annotations, which diminishes the clarity\n592 of the structural relationships between the residues.\n594 4.\n595\n596 3. Figure 5D: The use of PDB file Chain IDs as identifiers is understandable, but the\n597 two symmetric protomers of the E2s dimer do not appear to behave consistently,\n598 especially for E2s_1 V492M and E2s_4 N490G. This inconsistency is intriguing—does\n599 it suggest structural or functional differences between these protomers that could be\n600 relevant to the virus’s mechanism or stability? It would be beneficial to provide a\n601 discussion of these discrepancies.\n603 asymmetry between the two protomers of the E2s dimer in our MD simulations\n604 (original Fig. 5D), and thank you for raising this important point. While the asymmetry\n605 is indeed visually noticeable, we believe it does not reflect inherent structural or\n606 functional differences between the protomers. Rather, this variability arises from the\n607 stochastic nature of MD simulations. Although the two chains are structurally identical\n27\n608 in the starting model, minor differences in initial atomic velocities and local thermal\n609 fluctuations can lead to asymmetric conformational behavior over the course of the\n610 simulation, particularly in flexible regions such as the 6H8 epitope.\n612 independent simulation replicates for each variant under identical conditions. In all\n613 cases, the degree of epitope deflection varied between the two chains. This supports the\n614 conclusion that the observed asymmetry is a result of simulation randomness rather\n615 than intrinsic protomer-specific properties.\n617 given simulation, they exhibit the same overall trend across trajectories: variants\n618 containing the N490G mutation consistently show greater epitope destabilization than\n619 those retaining N490. We now include angular distribution frequency plots to better\n620 illustrate this phenomenon in the revised Fig. 5D.\n28\n621\n623 mutants in three replicate 1000-ns simulations. Each panel represents a distinct\n624 mutation, with six curves corresponding to three independent simulation runs (run1,\n625 run2, run3) for each chain (A and B) of the E2s dimer.\n626\n29\n627 4. Figure 6E: There appears to be an absence of error bars.\n630 manuscript, we change the date presentation by separately calculation the ratio of\n631 virions bound to PPH and HepG2, enabling to display the error bars. We have update\n632 the Fig. 7C with error bars in the revised manuscript.\n633\n634 5. Crystal Structure Model: The crystal structures, particularly E2s_4_N490G/M492V\n635 and E2s_1, may require further refinement. Notably, the Ramachandran plots for these\n636 two structures show a higher percentage of disallowed regions compared to the others\n637 (0.7% vs. 0%), which is unexpected. This could indicate either some special structural\n638 features arising from the crystallization conditions or simply an incomplete refinement\n639 process. It would be helpful for the authors to clarify whether these discrepancies stem\n640 from the protein crystallization energy state (e.g. packing interactions), or if further\n641 refinement was needed. Additionally, the authors’ provided crystal structure PDB\n642 validation report is not as curated (NOT for manuscript review) as the cryo-EM\n643 structure validation (which was ready for manuscript review).\n645 structures and the insightful questions raised regarding structural refinement. The two\n646 structures mentioned by the reviewer (E2s-4-N490G/M492V at 0.95 Å, E2s-1 at 1.40\n647 Å) do exhibit slightly higher Ramachandran disallowed percentages (0.7% vs. 0% in\n648 other structures). However, this corresponds to only one residue per structure (see\n650 protocols and all steps (including TLS parameterization and iterative model rebuilding)\n651 followed standard high-resolution refinement workflows. We have also validated\n652 model-to-density fit and found that the disallowed residue showed unambiguous\n653 electron density, confirming their accurate placement (see Appendix Fig. 2 and 3\n654 attached below). We believed that, at such high (atomic) resolution, subtle\n655 conformational strains—potentially induced by crystal packing interactions (e.g.,\n30\n656 intermolecular H-bonds)—may locally distort backbone geometry without indicating\n657 refinement errors.\n658\n660 disallowed residue of E2s-4-N490G/M492V in the Coot window\n661\n662\n664 disallowed residue of E2s-1 in the Coot window\n665\n31\n667 validation reports. Revised validation reports with \"for manuscript review\" status for\n668 all crystal structures have now been uploaded to the submission system.\n669\n670 6. The results in this manuscript enhance the understanding of HEV’s cross-species\n671 transmission potential, particularly regarding its zoonotic transmission. If the above\n672 deficiencies are addressed or clearly explained, I recommend this manuscript to be\n673 published in Nature Communications\n675 sincerely appreciate the encouraged comments for our study. According to the\n676 reviewers’ suggestions, we have included additional data to address all the reviewers’\n677 comments in the revised manuscript.\n32"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed many of the issued raised by the reviewers. Changing the title to \"The Residues 490 and 492 on E2s Domain of the Capsid Protein Play a Pivotal but Insufficient Role in Determining the Host Tropism of Hepatitis E Virus\" now discribes the findings more accuratly, nevertheless, is somewhat long and not elegant. Think about rephrasing.\n\nIt was very difficult to assess changes made to the manuscript, as new parts where only hghlighted in yellow instead of tracking all changes including deletions and so on. Keep in mind for new submissions.\n\nMy major concerns still open are concerning the FFU measurements of the authors. Did you calculate the focus forming units per mL based on titrating the virus stocks? MOIs should refelct the number of infectious/intact particles per cell and mL. From literature we know that the ratio of RNA copies to infectious particles is roughly 1:1,000 (doi: 10.1073/pnas.1912307117). MOIs of 64 (Fig 1) or even 1,000 (p29 of revised manuscript) are extremly high and never have been reporte for HEV before. Please make sure not to use RNA copies, but rather titrate the virus and assess FFU. Please also provide images of complete wells of your titration plate, usually 96well plate (compare e.g. doi: 10.1073/pnas.2202653119).\n\nThe sequence logos now presented in Fig S11 are somewhat confusing. The label for the y-axes are missing. Ususally the height of the entire stack of residues is the information measured in bits. What do the authors mean by \"degree of conservation\"? How can conservation be the same over all residues here? Please explain and addapt.", "reviewer_2": "The revised manuscript has satisfactorily addressed my concerns.", "reviewer_3": "The authors revised the manuscript and responded one by one to all of my suggestions and concerns on structure, which was persuasive, although I still had doubts about the reliability of the “random” MD results and the extent to which they could be accepted to support the conclusions of the manuscript. I prefer to leave other virology and functional mechanisms to the other two reviewers to make judgments."}, "author_response": "Response to Reviewer Comments on the manuscript NCOMMS-25-13653A:\nWe thank the reviewers for the positive comments. We have addressed all the reviewer’s\ncomments and revised the manuscript accordingly. To facilitate the navigation of this\ndocument, we have copied the reviewer’s comments verbatim in blue and typed out our\nresponses in black.\n\nReviewer #1 (Remarks to the Author):\n1. The authors addressed many of the issued raised by the reviewers. Changing the title\nto \"The Residues 490 and 492 on E2s Domain of the Capsid Protein Play a Pivotal but\nInsufficient Role in Determining the Host Tropism of Hepatitis E Virus\" now discribes\nthe findings more accuratly, nevertheless, is somewhat long and not elegant. Think\nabout rephrasing.\nResponse: We thank the reviewer for this insightful comment. In response to the\nreviewer's suggestion, we have revised the title to “The Crucial but Insufficient Role\nof E2s Domain’s Residues 490 and 492 in Determining the Host Tropism of\nHepatitis E Virus”. We have revised the manuscript accordingly on page 1, lines 1-4.\n\n2. It was very difficult to assess changes made to the manuscript, as new parts where\nonly hghlighted in yellow instead of tracking all changes including deletions and so on.\nKeep in mind for new submissions.\nResponse: We thank the reviewer for this comment. In response to this feedback, we\nhave enabled Track Changes to ensure all revisions are clearly visible throughout the\nrevised manuscript. Since track change mode of Word cannot highlight the modified\ncontent when generating a PDF, all changes were also highlighted in yellow.\n\n3. My major concerns still open are concerning the FFU measurements of the authors.\nDid you calculate the focus forming units per mL based on titrating the virus stocks?\nMOIs should refelct the number of infectious/intact particles per cell and mL. From\nliterature we know that the ratio of RNA copies to infectious particles is roughly 1:1,000\n(doi: 10.1073/pnas.1912307117). MOIs of 64 (Fig 1) or even 1,000 (p29 of revised\nmanuscript) are extremly high and never have been reporte for HEV before. Please\nmake sure not to use RNA copies, but rather titrate the virus and assess FFU. Please\nalso provide images of complete wells of your titration plate, usually 96well plate\n(compare e.g. doi: 10.1073/pnas.2202653119).\nResponse:\nWe thank the reviewer for these important questions regarding our viral quantification\nmethodology. In this study, viral infectivity was assessed by enumerating positive cells\nthrough immunofluorescence assay (IFA) as previously reported (Liu et al, 2019,\nViruses). We considered this enumeration method to be fundamentally analogous to the\nfocus-forming unit (FFU) assay, as both approaches rely on immunofluorescence\nstaining of infected cells to assess viral infectivity.\nIn this study, MOI refers to copies per cell rather than infectious particles per cell. For\nviral binding and infection assays, HEV virions were applied at 64 copies per cell or\n1000 copies per cell. The use of copy numbers or genome equivalents (GE) as viral\ninput measures has been widely employed in previous studies (Knegendorf et al, 2018,\nHepatol Commun; Yin et al, 2016, J Virol; Yin et al, 2018, PNAS; Liu et al, 2019,\nViruses). For instance, 25 GE per cell (Knegendorf et al, 2018, Hepatol Commun) or up\nto 10,000 GE per cell of HEV virions (Yin et al, 2016, J Virol; Yin et al, 2018, PNAS)\nwere utilized. Furthermore, Liu et al. (2019, Viruses) demonstrated that 40-1000 GE\nper cell were required to achieve reliable IFA detection results. Notably, these studies\nwere primarily based on the Kernow C1/p6 strain, which was isolated from a\nchronically HEV-infected patient (Shukla et al, 2011, PNAS). Many studies have\ndemonstrated that the cell culture-adapted Kernow C1/p6 exhibits significantly higher\ninfectivity and replication capacity compared to the primary isolate Sar55 (Shukla et al,\n2011, PNAS; Knegendorf et al, 2018, Hepatol Commun). Given that the primary isolates\nof the genotype 1 and 4 strains (Sar55 and D32) used in this study inherently\ndemonstrate lower replication competence, the higher viral input is scientifically\njustified.\nThis study investigated the differences in binding and infectivity among different\ngenotypes of HEV in human and porcine hepatocytes. The results demonstrated that, at\nequivalent copy-based viral input, the binding and infection efficiencies of different\ngenotypes were comparable in human hepatocytes (Fig. 1A-E, Fig. 7C–H, Fig. S2,\nand Fig. S3). Similar levels of HEV antigen in supernatants and comparable numbers\nof IFA-positive cells were observed following HEV infection of the human hepatogenic\ncell line HepG2, indicating that the number of infectious particles was similar across\ndifferent genotypes of HEV. We have revised the description accordingly in the revised\nmanuscript on Page 29 line 770.\n\n4. The sequence logos now presented in Fig S11 are somewhat confusing. The label for\nthe y-axes are missing. Ususally the height of the entire stack of residues is the\ninformation measured in bits. What do the authors mean by \"degree of conservation\"?\nHow can conservation be the same over all residues here? Please explain and addapt.\nResponse: We thank the reviewer for pointing this out and apologize for the ambiguity\nin our original presentation. In the original Fig. S11, the y-axis represented raw residue\nprobabilities rather than information content (in bits), which may lead to confusion in\ninterpreting sequence conservation. Following the reviewer’s suggestion, we have\nrevised Fig. S11 to adopt the conventional representation, where the y-axis denotes\ninformation content in bits. Please refer to the updated Fig. S11, which is attached below.\nRevised Fig. S11. Sequence logo representation of the E2s region (residues 459–\n606) of HEV genotypes 1–4. (A) Sequence logo representing the E2s region of HEV\ngenotype 1 (n = 22) and genotype 2 (n = 1) strains. (B) Sequence logo representing the\nE2s region of HEV genotype 3 (n = 230) and genotype 4 (n = 6) strains. The height of\neach letter at a given position represents the information content (in bits) of the\ncorresponding amino acid at that site. Red arrowheads indicate the critical amino acid\nresidues at positions 490 and 492.\n\nReviewer #2 (Remarks to the Author):\nThe revised manuscript has satisfactorily addressed my concerns.\nResponse: We sincerely thank the reviewer for this positive comments.\n\nReviewer #3 (Remarks to the Author):\nThe authors revised the manuscript and responded one by one to all of my suggestions\nand concerns on structure, which was persuasive, although I still had doubts about the\nreliability of the “random” MD results and the extent to which they could be accepted\nto support the conclusions of the manuscript. I prefer to leave other virology and\nfunctional mechanisms to the other two reviewers to make judgments.\nResponse: We thank the reviewer for this follow-up feedback. We would like to\nreiterate that in this study, molecular dynamics (MD) was only used to assist us in our\nstructural and biochemical findings, specifically regarding the role of the unique N490\nin HEV-4 in stabilization of the epitope recognized by the zoonotic HEV-specific mAb\n6H8. To address this, we performed three independent replicate MD simulations for\neach variant. Across all replicates, we observed highly consistent trends: variants\ncontaining G490 consistently exhibited increased epitope destabilization, while those\ncontaining N490 maintained structural stability. These results suggested that the\nobserved effects are robust and not artifacts of random variation in the simulations.\nTherefore, we believe that the reproducibility and consistency of our findings across\nmultiple independent runs provide strong support for the reliability of our conclusions.\nWe acknowledge that stochastic fluctuations are inherent features of atomistic MD,\nreflecting the natural conformational variability of biomolecules. Nonetheless, we\nbelieve that our MD results corroborate our structural and biochemical findings, rather\nthan drawing key conclusions solely based on MD analysis."}, {"round": 3, "reviewer_comments": {"reviewer_1": "Thanky you for addressing my concerns and correcting FFU. I hope the authors feel that their manuscript improved by this substantial revision.\nCongratulations!"}, "author_response": ""}]

Decision letter