Conserved Functions of Orthohepadnavirus X Proteins to Inhibit Type-I Interferon Signaling

Orthohepadnavirus causes chronic hepatitis in a broad range of mammals, including primates, cats, woodchucks, and bats. Hepatitis B virus (HBV) X protein inhibits type-I interferon (IFN) signaling, thereby promoting HBV escape from the human innate immune system and establishing persistent infection. However, whether X proteins of Orthohepadnavirus viruses in other species display a similar inhibitory activity remains unknown. Here, we investigated the anti-IFN activity of 17 Orthohepadnavirus X proteins derived from various hosts. We observed conserved activity of Orthohepadnavirus X proteins in inhibiting TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-mediated IFN-β signaling pathway through TRIF degradation. X proteins from domestic cat hepadnavirus (DCH), a novel member of Orthohepadnavirus, inhibited mitochondrial antiviral signaling protein (MAVS)-mediated IFNβ signaling pathway comparable with HBV X. These results indicate that inhibition of IFN signaling is conserved in Orthohepadnavirus X proteins.


Introduction
Hepatitis B virus (HBV) (family: Hepadnaviridae, genus: Orthohepadnavirus) affects >248 million individuals worldwide, and is the major cause of chronic liver disease and liver cancer in humans [1].Viruses in the Orthohepadnavirus genus infect mammals, including primates, cats, woodchucks, and bats [2].Orthohepadnavira exhibit a partially doublestranded DNA genome, ranging between 3.0 and 3.4 kb, encoding four viral (i.e., core, polymerase, surface, and X) proteins [2].HBV is a "stealth virus" because it triggers a minimal immune response, particularly the type-I interferon (IFN) response, during the initial stages of infection [3].The failure to induce an innate immune response in infected hepatocytes can lead to incomplete clearance of infected hepatocytes and cause chronic infection [4].
In 2018, domestic cat hepadnavirus (DCH), a novel member of the genus Orthohepadnavirus, was identified from a domestic cat in Australia [27].Since then, the prevalence of DCH in cats has been investigated in Italy [28], Thailand [29], Malaysia [30], the United Kingdom [31], Japan [32], the USA [33], Hong Kong [34], Taiwan [35], and Türkiye [36].The prevalence of DCH infection ranged from 0.2% in the USA to 18.5% in Thailand [33,37].DCH infection is associated with chronic hepatitis and hepatocellular carcinoma in cats, with increased levels of alanine transaminase (ALT) and aspartate transaminase (AST) [35,38], suggesting similarity with HBV infection in humans.We reported that DCH shares the sodium/bile acid cotransporter (NTCP) with HBV as a cellular entry receptor [39].However, the functional conservation of DCH X proteins is unclear.We aimed to investigate the functional conservation of Orthohepadnavirus X protein from other animal species in inhibiting the IFN-β signaling pathway to better understand viral immune evasion strategies.
In this study, we investigated the function of 17 Orthohepadnavirus X proteins on the IFN-β signaling pathway.We found that DCH X and other Orthohepadnavirus X proteins tested inhibited TRIF-mediated IFN-β signaling.DCH X protein required 20 amino acids at the C-terminus for efficient expression and function, suggesting that this domain is required for protein stability and is conserved between HBV and DCH X proteins.Thus, the inhibitory function of TRIF-mediated IFN-β signaling pathway is conserved in Orthohepadnavirus X protein.

Genetic Characteristics of Orthohepadnavirus X Proteins
We aligned 17 Orthohepadnavirus X proteins, including four strains of human HBV (genotype A, D, G, and H), four strains of DCH (KT-116, Rara, Sydney, and TR-SV15), three strains of bat HBV (pomona bat, horseshoe bat, and tent-making bat), one strain of woodchuck hepatitis virus, and one strain of HBV from the domestic donkey, Asian gray shrew, capuchin monkey, orangutan, and woolly monkey (Figure 1A).Although X proteins derived from the four DCH strains consist of 145 amino acids (aa), HBV X protein comprises 154 aa.The X protein from DCH (Rara) was distant from the X proteins of other DCH strains (Figure 1B).X proteins from DCH (Rara) and DCH (Sydney: a reference strain of DCH) differed by 14 residues [35].We observed that the X protein of tentmaking bat HBV was more distant from the X proteins of pomona bat HBV and horseshoe bat HBV.X proteins from bat HBV were not closely related, but were genetically similar to HBV strains from other animal species.This suggests viral transmission between bats and other animals (Figure 1B).In addition, the X protein of orangutan HBV is genetically closer to the X protein of HBV than the X proteins of capuchin monkey HBV or woolly monkey HBV.This result suggests that the X protein of ape HBV is more similar to the X proteins of human HBVs than the X proteins of New World monkey HBVs.
To support phylogenetic tree information, we observed the sequence identity of X proteins (Figure S1A).Four strains of HBV X proteins showed a percentage identity (PID) of 100%, the same as four strains of DCH X proteins, while the PID between both groups was 39.87%.Consistent with the result of the phylogenetic tree, we found that the X protein of orangutan HBV has an 87.73% identity compared with the X proteins of HBV, which is higher than the X proteins of capuchin monkey HBV and woolly monkey HBV.Moreover, we investigated the sequence similarity of Orthohepadnavirus X proteins (Figure S1B) to identify homologous sequences.DCH X proteins showed a percent similarity with HBV X protein (42.48%) more than other Orthohepadnavirus X proteins.This result suggests that HBV and DCH X proteins are closely related.

Both HBV (A) and DCH (KT-116) X Proteins Inhibited IFN-β Signaling Mediated by TRIF, MAVS, and IRF3
Considering the genetic diversity of Orthohepadnavirus X proteins across species and strains, we sought to investigate whether the inhibitory effect of HBx on IFN-β induction is conserved in other Orthohepadnavirus X proteins.First, we measured the expression level of HA-tagged Orthohepadnavirus X proteins in Lenti-X 293T cells with Western blotting.We found comparable expression levels of X proteins in transfected Lenti-X 293T cells (Figure 2).Before testing the inhibitory effect of Orthohepadnavirus X proteins, we confirmed that co-transfection with the following plasmids: Next, we examined the inhibitory effect of HBV (genotype A) and DCH (KT-116) X proteins on IFN signaling pathways by co-transfection of Lenti-X 293T cells with plasmids expressing X proteins.Consistent with other findings [26], HBx inhibited the induction of Firefly luciferase in cells co-transfected with the following plasmids: IFN-β Luc and TRIF (Figure 3A), IgK-IFN Luc and MAVS (Figure 3B), and ISRE Luc and IRF3 (Figure 3D), but Next, we examined the inhibitory effect of HBV (genotype A) and DCH (KT-116) X proteins on IFN signaling pathways by co-transfection of Lenti-X 293T cells with plasmids expressing X proteins.Consistent with other findings [26], HBx inhibited the induction of Firefly luciferase in cells co-transfected with the following plasmids: IFN-β Luc and TRIF (Figure 3A), IgK-IFN Luc and MAVS (Figure 3B), and ISRE Luc and IRF3 (Figure 3D), but not IgK-IFN Luc and IKKe (Figure 3C).DCH (KT-116) X protein showed similar inhibitory effects with HBV (genotype A) X protein, suggesting functional conservation between HBV (genotype A) and DCH (KT-116) X proteins.

X Proteins Derived from a Range of Orthohepadnavirus sp. Inhibited TRIF-Mediated IFN Signaling
We observed a conserved inhibitory effect of HBV (genotype A) and DCH (KT-116) X proteins on IFN signaling (Figure 3A,B,D).Notably, HBV (genotype A) and DCH (KT-116) X proteins potently inhibited TRIF-mediated IFN-β signaling (Figure 3A).To investigate whether the inhibitory effect of X proteins on TRIF-mediated IFN-β signaling is conserved across Orthohepadnavirus sp., Lenti-X 293T cells were co-transfected with plasmids expressing the 17 Orthohepadnavirus X proteins with IFN-β Luc and TRIF plasmids (Figure

X Proteins Derived from a Range of Orthohepadnavirus sp. Inhibited TRIF-Mediated IFN Signaling
We observed a conserved inhibitory effect of HBV (genotype A) and DCH (KT-116) X proteins on IFN signaling (Figure 3A,B,D).Notably, HBV (genotype A) and DCH (KT-116) X proteins potently inhibited TRIF-mediated IFN-β signaling (Figure 3A).To investigate whether the inhibitory effect of X proteins on TRIF-mediated IFN-β signaling is conserved across Orthohepadnavirus sp., Lenti-X 293T cells were co-transfected with plasmids expressing the 17 Orthohepadnavirus X proteins with IFN-β Luc and TRIF plasmids (Figure 4).We found that all Orthohepadnavirus X proteins significantly inhibited TRIF-mediated IFN-β signaling (Figure 4A,B), suggesting that the inhibitory effect on TRIF-mediated IFN signaling is conserved in X proteins across Orthohepadnavirus sp.To further test the inhibitory effect of Orthohepadnavirus X proteins, we co-transfected Lenti-X 293T cells with plasmids expressing IFN-β Luc, MAVS, and HBV strains (genotype A, D, G, and H) or DCH strains (KT-116, Rara, Sydney, and TR-SV15) X proteins.Although the inhibitory effect is smaller than on TRIF-mediated IFN-β signaling (Figure S3), Orthohepadnavirus X proteins target MAVS-mediated IFN-β signaling.
Next, we examined the subcellular localization of X proteins in Lenti-X 293T cells.Wildtype (WT) X proteins of HBV (genotype A) and DCH (KT-116) were primarily localized in the nucleus, but were also detected in the cytoplasm (Figure S4), which is consistent with another study [44].Mutant X proteins with deletions had similar localization, suggesting that the deletion of N-and C-terminal amino acids of HBV (genotype A) and DCH (KT-116) X proteins minimally affected protein localization in Lenti-X 293T cells.
Consistent with the result of Western blotting, we observed both del(52-148) and del(45-140) X protein mutants of both HBV (genotype A) and DCH (KT-116) failed to inhibit TRIF-mediated IFN-β signaling (Figure 5D,E).These results demonstrated that the transactivation domain at the C-terminus has an important role in stabilizing the expression and function of Orthohepadnavirus X proteins.
To elucidate the mechanism of the inhibitory effect on the TRIF-mediated IFN-β signaling pathway, we co-transfected Lenti-X 293T cells with plasmids expressing TRIF and X proteins from a broad range of species.We found that the expression of X proteins induced 13-100% degradation of TRIF (Figure 6E).Although DCH (Rara) X protein degraded TRIF completely, TMBHBV X protein decreased TRIF expression by ~13% in Lenti-X 293T cells.These results suggest that X protein inhibits TRIF-mediated IFN-β signaling by degrading TRIF.significantly lower expression levels of del(52-148) and del(45-140) mutants of HBV (genotype A) X protein in Lenti-X 293T cells (Figure 5C).Although the similarity of the transactivation domains between HBV (genotype A) and DCH (KT-116) X proteins is low, del(52-148) and del(45-140) X protein mutants of DCH (KT-116) X protein shared phenotype with HBV (genotype A) del(52-148) and del(45-140) mutants.
Next, we examined the subcellular localization of X proteins in Lenti-X 293T cells.Wild-type (WT) X proteins of HBV (genotype A) and DCH (KT-116) were primarily localized in the nucleus, but were also detected in the cytoplasm (Figure S4), which is consistent with another study [44].Mutant X proteins with deletions had similar localization, suggesting that the deletion of N-and C-terminal amino acids of HBV (genotype A) and DCH (KT-116) X proteins minimally affected protein localization in Lenti-X 293T cells.
Consistent with the result of Western blotting, we observed both del(52-148) and del(45-140) X protein mutants of both HBV (genotype A) and DCH (KT-116) failed to inhibit TRIF-mediated IFN-β signaling (Figure 5D,E).These results demonstrated that the transactivation domain at the C-terminus has an important role in stabilizing the expression and function of Orthohepadnavirus X proteins.
To elucidate the mechanism of the inhibitory effect on the TRIF-mediated IFN-β signaling pathway, we co-transfected Lenti-X 293T cells with plasmids expressing TRIF and X proteins from a broad range of species.We found that the expression of X proteins induced 13-100% degradation of TRIF (Figure 6E).Although DCH (Rara) X protein degraded TRIF completely, TMBHBV X protein decreased TRIF expression by ~13% in Lenti-X 293T cells.These results suggest that X protein inhibits TRIF-mediated IFN-β signaling by degrading TRIF.

Variation in the C-Terminus Transactivation Domain of DCH X Protein Determines the Inhibitory Effect on ISRE-Mediated IFN-β Signaling
To evaluate the effect of Orthohepadnavirus X proteins in inhibiting ISRE-mediated IFN-β signaling, we used 293T-ISRE-luc2 cells.We confirmed that 293T-ISRE-luc2 cells could be stimulated with recombinant human IFN-β, leading to a significant induction of luciferase (Figure S5).Next, we transfected 293T-ISRE-luc2 cells with plasmids expressing Orthohepadnavirus X proteins to test the inhibitory effect of Orthohepadnavirus X proteins on ISRE-mediated IFN-β signaling.Most X proteins, except DCH (TR-SV15) X, suppressed ISRE-mediated IFN-β signaling (Figure 7A,B).Alignment of the X proteins of the four DCH strains showed that in DCH (TR-SV15) X protein, five residues (positions 102, 106, 137, 140, and 144) differed in the C-terminus transactivation domain compared with the other three strains (Figure 7C).This indicates that these five amino acids might be responsible for the impaired inhibition of ISRE-mediated IFN-β signaling.

Variation in the C-Terminus Transactivation Domain of DCH X Protein Determines the Inhibitory Effect on ISRE-Mediated IFN-β Signaling
To evaluate the effect of Orthohepadnavirus X proteins in inhibiting ISRE-mediated IFN-β signaling, we used 293T-ISRE-luc2 cells.We confirmed that 293T-ISRE-luc2 cells could be stimulated with recombinant human IFN-β, leading to a significant induction of luciferase (Figure S5).Next, we transfected 293T-ISRE-luc2 cells with plasmids expressing Orthohepadnavirus X proteins to test the inhibitory effect of Orthohepadnavirus X proteins on ISRE-mediated IFN-β signaling.Most X proteins, except DCH (TR-SV15) X, suppressed ISRE-mediated IFN-β signaling (Figure 7A,B).Alignment of the X proteins of the four DCH strains showed that in DCH (TR-SV15) X protein, five residues (positions 102, 106, 137, 140, and 144) differed in the C-terminus transactivation domain compared with the other three strains (Figure 7C).This indicates that these five amino acids might be responsible for the impaired inhibition of ISRE-mediated IFN-β signaling.

Discussion
In this study, we demonstrated that Orthohepadnavirus X protein inhibits TRIF-mediated IFN-β signaling, mainly through TRIF degradation.This inhibitory activity is conserved across Orthohepadnavirus X proteins.The C-terminus transactivation domains have an important role in stabilizing the expression and function of Orthohepadnavirus X protein to inhibit TRIF-mediated IFN-β signaling.
In 1991, the domains of HBx were characterized; the domain between residues 103 and 117 of the C-terminus transactivation domain was found to be important for a fully functional HBx [45].Here, we aligned 17 Orthohepadnavirus X proteins and found high genetic diversity at the C-terminus transactivation domains (Figure 1A).The phylogenetic tree showed that DCH X proteins are more distant from HBx than X proteins from other species (Figure 1B).Nevertheless, DCH X proteins shared a function with HBx to inhibit IFN-β signaling (Figure 3A,B,D).We recently reported that DCH shares the cell entry molecule, sodium/bile acid cotransporter, with HBV [39].However, DCH preS1 is genetically closer to HBV preS1 than woodchuck hepatitis virus or Arctic ground squirrel HBV [39].Understanding the similarities and differences between HBV and DCH is critical for using DCH as a model for HBV research.
HBx was reported to induce ubiquitin-mediated protein degradation [46] and TRIF degradation through a proteasomal pathway in hepatoma cells [26].In this study, Orthohepadnavirus X proteins from broad range of species exhibited a conserved mechanism for TRIF degradation to perturb innate immunity.Our results increase our understanding of Orthohepadnavirus X protein-mediated suppression of TLR signaling.Consistently, HBV was reported to utilize X protein to avoid the innate immune system, establishing chronic infection in the host [14,24,47,48].
The 3D structure of Orthohepadnavirus X protein is unsolved.However, it has two functional domains, including an N-terminal negative regulatory and a C-terminal transactivation domain [20].The N-terminal region (aa 1-50) contains a highly conserved region (aa 1-20) and a Ser/Pro-rich region (aa 21-50) that is essential for negative regulatory effects and association of the regulatory domain [21].C-terminal residues 58-119 are associated with signal transduction to the nucleus [49], residues 120-140 play an important role in nuclear transactivation, and the last 20 amino acids (aa 134-154) are involved in protein stability [22,23].Consistently, we found that the deletion of residues 120-140 impaired the stability of the X protein (Figure 5C) and the degradation activity of TRIF (Figure 6D), leading to failed inhibition of IFN signaling (Figure 5D,E).Although the amino acid length of DCH X proteins is nine amino acids shorter than HBx, and there is high genetic variation at position 120-140 (Figure 5A), our results suggest that the C-terminal domains of DCH X proteins have conserved function with HBx (regarding protein stability and inhibition of IFN signaling).
The localization of HBx was shown to depend on its expression level: low expression leads to nuclear localization [44]; high expression leads to cytoplasmic localization and abnormal mitochondrial distribution [44].In this study, Orthohepadnavirus X proteins from various species showed conserved localization primarily in the nucleus, but also localized to the cytoplasm (Figure S4A).Deletion at the N-or C-terminal domain marginally affected the localization of HBV and DCH X proteins (Figure S4B).These observations suggest that the C-terminal domain is important for the inhibitory effect of the X proteins, but does not determine their cellular localization.
X proteins derived from HBV and DCH showed an inhibitory activity on ISREmediated IFN-β signaling, except DCH (TR-SV15) (Figure 7A,B).Alignment of X proteins derived from the four DCH strains showed that DCH (TR-SV15) had five different amino acids in the C-terminus transactivation domain (Figure 7C).Analysis using chimeric X proteins between DCH (Sydney) and DCH (TR-SV15) proteins suggested that genetic variations in the C-terminus of DCH X protein determine its inhibitory effect on ISRE-mediated IFN-β signaling (Figure 7E,F).Further analyses are required to identify specific residue(s) that determine inhibitory activity.
Several host and viral factors, such as host age and gender, viral load, and viral genotype, can influence the IFN response during infection and IFN treatment [50].Among viral factors, the X protein is one of the important factors in the IFN response, because it not only inhibits TLR3-TRIF and RIG-I signaling, but also suppresses IFN induction by suppressing the transcription of tripartite motif 22 in a mouse model, primary human hepatocytes, and human liver tissues [51].The mechanism for the loss of inhibitory effect of DCH (TR-SV15) on ISRE-mediated IFN-β signaling is unclear.Therefore, we must elucidate the determinant(s) that can affect the IFN response in DCH-infected cats.Our results provide clues to improve the efficiency of IFN therapy in cats with chronic hepatitis.
A limitation of this study is that we used a luciferase reporter system to probe the interaction between Orthohepadnavirus X protein and human-derived molecules involved in IFN signaling.We must investigate whether (1) Orthohepadnavirus X proteins a show similar effect on molecules derived from other species, and (2) our results can be reproduced in primary cells.Lastly, we must investigate the immunopathogenesis induced by Orthohepadnavirus X proteins, especially DCH.These findings will contribute to developing therapeutic guidelines to counteract DCH infection effectively.
In conclusion, our results revealed that DCH X proteins and other Orthohepadnavirus X proteins inhibit TRIF-mediated IFN-β signaling by degrading TRIF, suggesting that this mechanism is a conserved function of Orthohepadnavirus X proteins to perturb the host's innate immune response.Our findings show that the C-terminus domains of HBV and DCH X proteins are important for protein stability and inhibitory function.The results of this study deepen our understanding of the function of Orthohepadnavirus X proteins in inhibiting the IFN-β signaling pathway.Further investigations are required to understand the evasion strategy of viruses belonging to Orthohepadnavirus.cDNA sequences of 17 Orthohepadnavirus X proteins with an N-terminal HA-tag were synthesized with codon optimization to human cells (Twist Bioscience, San Francisco, CA, USA).Synthesized DNA sequences are summarized in Supplementary Table S1.Inserts encoding cDNA were cloned into the pCAGGS vector [57], predigested with EcoRI-HF (New England Biolabs [NEB], Ipswich, MA, USA, Cat# R3101M) and NheI-HF (NEB, Cat# R3131M) using In-Fusion Assembly Master Mix (TaKaRa, Kusatsu, Japan, Cat# Z8947N).Plasmids were amplified using NEB 5-alpha F Iq Competent E. coli (High Efficiency) (NEB, Cat# C2992H) and extracted with PureYield Plasmid Miniprep System (Promega, Cat# A1222).Sequences of all plasmids were verified using SupreDye v3.1 Cycle Sequencing Kit (M&S TechnoSystems, Osaka, Japan, Cat# 063001) with Spectrum Compact CE System (Promega).

Construction of Plasmids Encoding Orthohepadnavirus X Proteins with Deletions
To construct pCAGGS vectors of Orthohepadnavirus X proteins with deletions, mutagenesis was performed with overlapping PCR using PrimeSTAR GXL DNA polymerase (TaKaRa, Cat# R050A).Primers are listed in Supplementary Table S2.The PCR protocol consisted of 35 cycles at 98 • C for 10 s, 60 • C for 15 s, and 68 • C for 1 min, followed by 68 • C for 7 min.Amplified PCR fragments encoding the deletion were cloned into the pCAGGS vector, as described in Section 4.1.Plasmids were verified by sequencing.

Construction of Plasmids Encoding Myc-Tagged TRIF
To construct the pCAGGS vector encoding Myc-tagged human TRIF, the insert encoding human TRIF was PCR amplified from the pEF-Bos TRIF Flag plasmid using PrimeSTAR GXL DNA polymerase.The primers are listed in Supplementary Table S3.The PCR protocol consisted of 35 cycles at 98 • C for 10 s, 60 • C for 15 s, and 68 • C for 1 min, followed by 68 • C for 7 min.Amplified PCR fragments encoding human TRIF were cloned into the pCAGGS vector, as described in Section 4.1.Plasmids were verified by sequencing.

Construction of Plasmids Encoding Chimeric DCH X Proteins
To construct pCAGGS vectors encoding chimeric X proteins, mutagenesis was performed with overlapping PCR using PrimeSTAR GXL DNA polymerase.Primers are listed in Supplementary Table S4.The PCR protocol consisted of 35 cycles of 98 • C for 10 s, 60 • C for 15 s, and 68 • C for 1 min, followed by 68 • C for 7 min.Amplified PCR fragments encoding DCH (Sydney) and DCH (TR-SV15) X proteins were mixed and cloned into the pCAGGS vector using NEBuilder HiFi DNA Assembly Master Mix (NEB, Cat# E2621F).Plasmids were amplified as described in Section 4.1.Plasmids were verified by sequencing.
Single-cell cloning was then performed.After cell growth, we evaluated the induction of luciferase activity upon treatment with recombinant human IFN-β (PeproTech, Cranbury, NJ, USA, Cat# 300-02BC) in each clone.

Luciferase Reporter Assay 4.8.1. IFN-β Luciferase Reporter Assay
Lenti-X 293T cells were seeded in a 96-well plate (Fujifilm, Osaka, Japan, Cat# 635-28511) at 3 × 10 4 cells per well, cultured overnight, and transfected with 2.5 ng pIFN-β-Luc plasmid, 45 ng pRL-TK, 2.5 ng TRIF or MAVS plasmid, and 50 ng pCAGGS plasmid, encoding HA-tagged X protein or pCAGGS empty plasmid, using TransIT-LT1 Transfection Reagent in Opti-MEM.After 24 h, the cells were assayed for luciferase activity with Dual-Glo Luciferase Assay System (Promega, Cat# E2920).Firefly luciferase activity was normalized based on Renilla luciferase activity.Percent relative activity was calculated by comparing normalized luciferase data of Orthohepadnavirus X proteins plasmid transfected cells and empty plasmid transfected cells.The assays were repeated at least three times.The data shows mean values ± SD from one representative experiment.

IgK-IFN and ISRE Luciferase Reporter Assay
Lenti-X 293T cells were seeded in a 96-well at 3 × 10 4 cells per well.After overnight incubation, cells were transfected with 5 ng IgK-IFN-Luc plasmid or ISRE Luc plasmid, 40 ng pRL-TK, 5 ng MAVS or IKKe or IRF-3 plasmid, and 50 ng plasmids encoding Orthohepadnavirus X proteins or empty plasmid.At 24 h after transfection, cells were assayed for luciferase activity with a Dual-Glo Luciferase Assay System, as described above.

IFN-β Bioassay in 293T-ISRE-luc2 Cells
The 293T-ISRE-luc2 cells were seeded in a 96-well at 3 × 10 4 cells per well, cultured overnight, and transfected with 50 ng pRL-TK and 50 ng pCAGGS plasmid encoding Orthohepadnavirus X proteins, chimeric DCH X protein, or empty plasmid.At 24 h after transfection, the cells were treated with 50 or 10 U/mL recombinant human IFN-β.At 48 h after transfection, the cells were assayed for luciferase activity with Dual-Glo Luciferase Assay System.

TRIF-Degradation Assay
To investigate the effect of protein X-mediated degradation of TRIF, Lenti-X 293T cells were seeded in a 24-well plate (Fujifilm, Cat# 630-28441) at 1.25 × 10 5 cells per well.The cells were cultured overnight and co-transfected with 250 ng of pCAGGS plasmid encoding HA-tagged protein X and 250 ng of pCAGGS plasmid encoding Myc-tagged human TRIF.Cellular lysates were prepared as described above.The expression of Myc-tagged TRIF was measured using an anti-Myc (9B11) mouse monoclonal antibody (CST, Cat# 2276S, ×100) and Anti-Mouse Detection Module.The amount of input protein was measured using Total Protein Detection Module, as described above.

Alignment of Orthohepadnavirus Protein X and Phylogenetic Analysis
The amino acid sequences of protein X from 17 Orthohepadnavirus sp. were aligned using the MUSCLE algorithm in MEGA X (MEGA Software) version 11.0.13.A phylogenetic tree was constructed using the alignment of amino acid sequences from public databases, and evolutionary analysis was conducted using the maximum likelihood method and neighbor-joining method based on the Jones-Taylor-Thornton matrix-based model with 1000 bootstrap replicates.

Statistical Analysis
The results are presented as the mean and standard deviation of four measurements from one assay, representing at least two or three independent experiments.Differences in relative values between Orthohepadnavirus X proteins and empty plasmid were examined by one-way ANOVA followed by Dunnett's multiple comparison test.A p ≤ 0.05 was considered statistically significant.Analysis was performed using Prism 10 software v10.1.2for Windows (GraphPad Software, Boston, MA, USA).

Figure 1 .
Figure1.The alignment and phylogenetic tree of Orthohepadnavirus X proteins.(A) Amino acid alignment of Orthohepadnavirus X proteins obtained from public databases.(B) The phylogenetic tree was constructed using the MEGA software, and the evolutionary analysis was conducted using the maximum likelihood and neighbor-joining methods based on the Jones-Taylor-Thornton matrixbased model with 1000 bootstrap replicates.

Figure 4 .
Figure 4. Inhibitory effect of Orthohepadnavirus X proteins on TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-mediated IFN-β signaling.(A) Raw data of the luciferase reporter assay.The RLU of Firefly luciferase was divided by the RLU of Renilla luciferase.(B) Relative value of IFNβ luciferase reporter assay.Differences between cells transfected with plasmids expressing Orthohepadnavirus X protein or an empty plasmid were examined by one-way ANOVA followed by Dunnett s multiple comparison test.

Figure 4 .
Figure 4. Inhibitory effect of Orthohepadnavirus X proteins on TIR-domain-containing adaptor protein inducing IFN-β (TRIF)-mediated IFN-β signaling.(A) Raw data of the luciferase reporter assay.The RLU of Firefly luciferase was divided by the RLU of Renilla luciferase.(B) Relative value of IFN-β luciferase reporter assay.Differences between cells transfected with plasmids expressing Orthohepadnavirus X protein or an empty plasmid were examined by one-way ANOVA followed by Dunnett's multiple comparison test.

Figure 5 .
Figure 5.The C-terminus transactivation domain of X protein plays an important role in protein stability and function.(A) Amino acid alignment of HBV (genotype A) and DCH (KT-116) X proteins.(B) Deletion mutants of X proteins.(C) HBV (genotype A) and DCH (KT-116)-derived mutant X protein expression levels in Lenti-X 293T cells.The expected HA-tagged WT and mutant X protein sizes were approximately 16 kDa and ranged between 5.40 and 14.04 kDa, respectively.(D) Raw data from the luciferase reporter assay.The RLU of Firefly luciferase was divided by the RLU of Renilla luciferase.(E) Relative value of the IFN-β luciferase reporter assay.Differences between cells transfected with Orthohepadnavirus X protein plasmids or an empty plasmid were examined by oneway ANOVA followed by Dunnett s multiple comparison test.

Figure 5 .
Figure 5.The C-terminus transactivation domain of X protein plays an important role in protein stability and function.(A) Amino acid alignment of HBV (genotype A) and DCH (KT-116) X proteins.(B) Deletion mutants of X proteins.(C) HBV (genotype A) and DCH (KT-116)-derived mutant X protein expression levels in Lenti-X 293T cells.The expected HA-tagged WT and mutant X protein sizes were approximately 16 kDa and ranged between 5.40 and 14.04 kDa, respectively.(D) Raw data from the luciferase reporter assay.The RLU of Firefly luciferase was divided by the RLU of Renilla luciferase.(E) Relative value of the IFN-β luciferase reporter assay.Differences between cells transfected with Orthohepadnavirus X protein plasmids or an empty plasmid were examined by one-way ANOVA followed by Dunnett's multiple comparison test.

Figure 6 .
Figure 6.Orthohepadnavirus X protein degrades TIR-domain-containing adaptor protein inducing IFN-β (TRIF).(A,C) HA-tagged Orthohepadnavirus X protein expression levels in transfected Lenti-X 293T cells.The expected HA-tagged X protein sizes ranged between 10.64 and 16.89 kDa.(B,D) Myc-tagged human TRIF expression levels in transfected Lenti-X 293T cells.The expected Myctagged human TRIF size was 120 kDa.(E) Relative values of TRIF expression.Relative value was calculated from the corrected area of the TRIF band with Compass for Simple Western software version 6.3.0.

Figure 6 .
Figure 6.Orthohepadnavirus X protein degrades TIR-domain-containing adaptor protein inducing IFN-β (TRIF).(A,C) HA-tagged Orthohepadnavirus X protein expression levels in transfected Lenti-X 293T cells.The expected HA-tagged X protein sizes ranged between 10.64 and 16.89 kDa.(B,D) Myctagged human TRIF expression levels in transfected Lenti-X 293T cells.The expected Myc-tagged human TRIF size was 120 kDa.(E) Relative values of TRIF expression.Relative value was calculated from the corrected area of the TRIF band with Compass for Simple Western software version 6.3.0.