Bat-specific adaptations in interferon signaling and GBP1 contribute to enhanced antiviral capacity

Gonzalez, Victoria; Lobb, Briallen; Côté, Jacob; Bhuinya, Arkadeb; Tubb, Adriana G.; Nuthalapati, Stephen S.; Asavajaru, Akarin; Zhou, Yan; Misra, Vikram; Falzarano, Darryl; Sweeney, Trevor R.; Gobeil, Sophie M. C.; Wang, Linfa; Doxey, Andrew C.; Banerjee, Arinjay

doi:10.1038/s41467-025-61254-7

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Published: 01 July 2025

Bat-specific adaptations in interferon signaling and GBP1 contribute to enhanced antiviral capacity

Nature Communications volume 16, Article number: 5735 (2025) Cite this article

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Abstract

Bats are reservoirs of emerging zoonotic viruses that may cause severe disease in humans and agricultural animals. However, it is poorly understood how bats can tolerate diverse viral infections. Here, we characterized type I interferon response pathways in kidney cell lines derived from two divergent bat species, Pteropus alecto and Eptesicus fuscus, identifying distinct mechanisms underlying their enhanced control of viral infection. We demonstrate the critical roles of STAT1/STAT2 in IFNβ signaling, along with species-specific adaptations that contribute towards a steady and ready antiviral state. Unlike in humans, bat IFNβ signaling processes resist the immune antagonistic properties of MERS-CoV which further explains the ability of bats to tolerate coronavirus infections. Transcriptomic analysis on interferon stimulated cell lines identified canonical and non-canonical interferon stimulated genes including two differentially expressed genes, IFIT1 and GBP1, that exhibit enhanced antiviral activity against a wide range of viruses, including the bat-derived Eptesipoxvirus. We have identified a functional (AV1) motif within E. fuscus GBP1 that restricts Eptesipoxvirus replication. Ultimately, our work provides important insights into the evolution of enhanced interferon-mediated antiviral responses in bats, contributing to their ability to resist viral diseases.

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Introduction

Bats are one of the most abundant and geographically diverse mammalian species in the world, with over 1480 species globally distributed on all continents with the exception of Antarctica^1,2. Accumulating data suggest that bats are hosts to multiple zoonotic viruses that have caused consequential disease outbreaks in humans, including severe-acute respiratory syndrome coronavirus (SARS-CoV), SARS-CoV-2, and Middle East respiratory syndrome coronavirus (MERS-CoV). Despite these viruses causing life-threatening disease in humans, naturally or experimentally infected bats do not demonstrate overt signs of disease^3,4,5. This suggests the evolution of enhanced antiviral capacity in reservoir bat species.

To successfully establish infection, a virus must surpass the host’s first line of defense, the innate immune system. Mammalian cells utilize conserved pattern recognition receptors (PRRs) to sense pathogen-associated molecular patterns (PAMPs), like viral nucleic acid. Following detection by PRRs, antiviral cytokines like interferons (IFNs) are secreted, which bind to their cognate receptors in paracrine and autocrine signaling loops. All type I IFNs, including IFNβ, bind to IFNAR1/2, leading to the activation of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway in humans⁶. This involves a series of phosphorylation events leading to the formation of a transcription factor complex composed of STAT1, STAT2, and IRF9, which migrates to the nucleus and induces the expression of antiviral interferon-stimulated genes (ISGs). These ISGs work alone or in concert to inhibit virus replication⁷. The IFN signaling cascade and the role of STAT proteins have not been mechanistically characterized in bat cells.

In this study, we generated species-specific bat IFNβ, which potently restricts VSV and MERS-CoV replication in kidney cells derived from two divergent bat species, the insectivorous big brown bat (Eptesicus fuscus) and the frugivorous black flying fox (Pteropus alecto). By applying a transcriptomic approach, we demonstrate a largely conserved type I IFN response across both bat species. However, lower expression levels of orthologous ISG transcripts, in addition to divergent transcripts, contributed to the overall antiviral IFNβ response in bat cells relative to human cells. GBP1 was one of the top upregulated transcripts in bat cells, and functional assays demonstrated that bat GBP1 is broadly protective against a range of RNA and DNA viruses compared to human GBP1. The antiviral function of bat GBP1 was dependent on a previously unknown and uncharacterized AV1 motif within the N-terminal domain, leading to restriction of the bat-derived Eptesipoxvirus.

Results

Generating functional species-specific bat IFNβ

To examine the type I IFN response in bat cells, we generated recombinant IFNβ from humans and two divergent bat species, Pteropus alecto and Eptesicus fuscus (Fig. 1A and Supplementary Fig. 1A). As a vehicle control, we generated secreted green fluorescent protein (GFP). We confirmed the expression and concentration of IFNβ using immunoblotting (Fig. 1B) and enzyme-linked immunosorbent assay (ELISA), respectively (Supplementary Fig. 1B). Next, we confirmed the function of IFNβ on species-matched cells from P. alecto (PaKiT03), E. fuscus (Efk3B), and humans (A549 and RPTEC). Each cell line was treated with serially diluted, species-matched IFNβ for 6 h, followed by infection with vesicular stomatitis virus that was engineered to express GFP (VSV-GFP; MOI 0.1). We used VSV-GFP inhibition data to determine functional units for IFNβ (Supplementary Fig. 1C). VSV-GFP replication was not inhibited in untreated or vehicle-treated cells (Supplementary Fig. 1C). However, we observed significant inhibition of VSV-GFP in cells pre-treated with IFNβ (Supplementary Fig. 1C). PaKiT03 and A549 cells required 10-fold more IFNβ than Efk3B and RPTEC cells (Supplementary Fig. 1C). We also validated IFNβ activity in multiple clonal populations of E. fuscus cell lines, such as Efk1F and Efk2A (Supplementary Fig. 1C)^8,9. Equivalent amounts of E. fuscus IFNβ were required for antiviral protection against VSV-GFP in all three clonal cell lines.

**Fig. 1: IFNβ potently restricts viral infection in *P. alecto* and *E. fuscus* cells.**

To determine if our recombinant IFNβ could induce the expression of canonical ISGs, we characterized the transcript levels of MX1, IFIT1, and RSAD2 by qRT-PCR. MX1, IFIT1, and RSAD2 transcript levels were upregulated in PaKiT03, A549, Efk3B, and RPTEC cells at 6 h post-IFNβ (1 U/mL) treatment (Fig. 1C). These data suggest that recombinant IFNβ can induce canonical ISGs and restrict VSV-GFP replication across the evaluated cell lines.

Antiviral efficacy of bat IFNβ

Next, we evaluated the IFNβ signaling kinetics in human and two divergent bat cell lines (Fig. 1D, E). We observed a significant reduction in GFP signal in bat cell lines that were treated with IFNβ for 1 h, while a similar effect was not observed until 2- and 4-h of IFNβ treatment in A549 and RPTEC human cells, respectively (Fig. 1E). We further investigated IFNβ-mediated protection using MERS-CoV, which is speculated to have evolved in insectivorous bats¹⁰. PaKiT03, Efk3B, Huh7, and RPTEC cells were either pretreated with IFNβ (10 U/mL) or treated following infection with MERS-CoV (MOI 0.1) (Fig. 1F). In PaKiT03, Efk3B, and RPTEC cells, viral load was substantially reduced upon IFNβ treatment in both pre- and post-treatment conditions compared to untreated cells. However, IFNβ was effective in Huh7 cells only when provided prophylactically (Fig. 1F). These data suggest that IFNβ can induce a robust antiviral state that is not antagonized by MERS-CoV in kidney cells derived from all three species.

Characterizing the bat IFNβ signaling pathway

The role and importance of the JAK-STAT signaling pathway during virus infection in bats has not been studied. In humans, a critical step in the JAK-STAT pathway involves the phosphorylation of STAT1 and STAT2 proteins by tyrosine kinases at positions Y701 and Y690, respectively^11,12. To evaluate the role of STAT1/2 phosphorylation in bat cell signaling, we pre-treated PaKiT03, EfK3B, A549, and RPTEC cells with Staurosporine, a broad-spectrum kinase inhibitor, followed by stimulation with IFNβ (10 U/mL) and VSV-GFP infection (MOI 0.1). Staurosporine treatment reduced the phosphorylation of STAT1/2 at positions Y701 and Y690, respectively (Supplementary Fig. 2A). Phosphorylation or activation of STAT1 and STAT2 in human cells leads to nuclear localization of both proteins prior to driving the expression of ISGs^11,12,13. Treating human and bat cells with Staurosporine reduced nuclear STAT1/2 in IFNβ-treated cells (Supplementary Fig. 2B–D). Staurosporine treatment also reduced the expression of IFIT1 and MX1 ISG transcripts in IFNβ-treated cells (Supplementary Fig. 3A, B). In addition, VSV-GFP was able to replicate to higher levels in all cell lines treated with Staurosporine (Supplementary Fig. 3C, D). Taken together, our data demonstrate that phosphorylation is critical for IFNβ signaling in bat cells.

Next, we investigated the rate of phosphorylation and nuclear translocation of STAT1/2 upon IFNβ treatment for 5 to 20 min. Phosphorylation of Y701-STAT1 occurred within 20 min of treatment in PaKiT03, A549, and RPTEC cells (Fig. 2A and Supplementary Fig. 4). Interestingly, Efk3B cells had higher basal levels of pY701-STAT1, suggesting differences between bat cells (Fig. 2A). Phosphorylation of Y690-STAT2 occurred within 5 min of IFNβ treatment in PaKiT03 and A549 cells, and by 20 min in RPTEC cells. Phosphorylation of Y690-STAT2 could not be evaluated in Efk3B cells due to the lack of cross-reactivity of the antibody, which may be due to sequence divergence around the phosphorylation site of STAT2 in E. fuscus bats (Supplementary Fig. 5). We also corroborated our STAT1/2 phosphorylation data using confocal microscopy to demonstrate the kinetics of IFNβ treatment-mediated nuclear translocation of STAT1/2 across different times in all cell lines. Across all cell lines, STAT1/2 localized to the nucleus within 20 min of IFNβ treatment, but P. alecto cells overall demonstrated a faster trend for STAT1/2 nuclear translocation (5 min) compared to E. fuscus and human cells (10–20 min) (Fig. 2B and Supplementary Fig. 6). However, basal levels of nuclear STAT1 and STAT2 could be detected in unstimulated E. fuscus cells, suggesting that these cells may be primed to respond to IFNβ.

**Fig. 2: STAT1 and STAT2 play an antiviral role in bats.**

To determine the importance of STAT1/2 in IFNβ signaling, we attempted to knock down these proteins in human and bat cells, followed by IFNβ treatment. PaKiT03, EfK3B, and A549 cells were treated with siRNA directed against STAT1/2 individually or in combination for 48 h. After 48 h, human and bat cell lines were treated with their species-matched IFNβ for 6 h, followed by infection with VSV-GFP (MOI 0.1) (Fig. 2C, D and Supplementary Fig. 7). While knocking down STAT1 levels reduced IFNβ-mediated protection in PaKiT03 cells, Efk3B and A549 cells were fully protected from VSV-GFP infection (Fig. 2C, D and Supplementary Fig. 7A, E), suggesting STAT1-independent signaling mechanisms¹⁴. However, knockdown of STAT2 expression levels led to loss of protection across all human and bat cell lines, suggesting a more critical and conserved role of STAT2 in IFNβ-mediated antiviral protection in these three species (Fig. 2C, D and Supplementary Fig. 7B, E). Knocking down expression levels of both STAT1/2 in PaKiT03 and A549 cells led to increased virus replication in both cell lines (Fig. 2C, D and Supplementary Fig. 7C, E). However, knocking down both STAT1/2 in Efk3B cells was lethal (Supplementary Fig. 7D), suggesting an important combined role of STAT1 and STAT2 in maintaining cell viability in these cells.

MERS-CoV is a human pathogen of bat origin and can inhibit IFN signaling in human cells^15,16. We next assessed whether MERS-CoV could modulate IFNβ signaling in P. alecto and E. fuscus cells (Fig. 2E and Supplementary Fig. 8). MERS-CoV infection alone suppressed the phosphorylation of pY701-STAT1 in both human (Huh7 and RPTEC) cell lines (Fig. 2E and Supplementary Fig. 8). IFNβ treatment alone induced the phosphorylation of pY701-STAT1 across all cell lines pretreated with IFNβ followed by MERS-CoV infection (Fig. 2E and Supplementary Fig. 8). However, treating MERS-CoV infected human cells with IFNβ (post-treatment condition) led to reduced levels of pY701-STAT1 phosphorylation compared to IFNβ treated cells. In bat cells however, treating MERS-CoV infected cells with IFNβ (post-treatment condition) did not lead to a loss of pY701-STAT1 levels, suggesting that bat cells are able to resist MERS-CoV infection mediated inhibition of STAT1 phosphorylation (Fig. 2E and Supplementary Fig. 8). Pretreating human or P. alecto bat cells with IFNβ maintains pY690-STAT2 levels upon MERS-CoV infection; however, treating MERS-CoV infected cells with IFNβ leads to partial loss of pY690-STAT2 levels in both Huh7 and P. alecto cells (Fig. 2E and Supplementary Fig. 8). Taken together, these data demonstrate that bat cells are differentially sensitive to MERS-CoV infection mediated modulation of IFNβ signaling, compared to virally permissive human cells. Of note, consistent with other studies¹⁷, MERS-CoV is likely able to shut down host translation in Huh7 cells as evidenced by the reduced actin and GAPDH levels (Fig. 2E). We did not observe this phenomenon in either bat cell line or the human RPTEC cells.

Discovering IFNβ-driven ISGs in two divergent bat species

Next, we identified the top expressed ISGs in cells from two divergent bat species upon stimulation with species-matched IFNβ (Fig. 3A). We also stimulated two human cell lines, A549 and RPTEC, with human IFNβ for comparison. IFNβ treatment induced the expression of several genes representing multiple signaling pathways across all cell lines (Fig. 3B and Supplementary Fig. 9A). There were limited perturbations between the kidney cell lines along principal component one (PC1), which accounts for more than 67% of sample variation. Gene enrichment analysis identified conserved and unique processes shared between all cell types, where 55 processes like type I IFN signaling pathway, regulation of viral entry into host cells, and defense response to virus were shared among all four cell lines (Supplementary Data 1). Of note, 136 processes were uniquely upregulated in bat cell lines upon IFNβ stimulation (Supplementary Data 1).

**Fig. 3: Global transcriptional response in human and bat cells upon IFNβ stimulation.**

Next, we analyzed the top differentially expressed processes in bat cells and determined the fold enrichment (Fig. 3C). For antiviral GO processes like defense response to virus, innate immune response, negative regulation of viral genome replication, response to virus, and type I IFN signaling pathway, fold enrichment was the highest in both bat cell lines compared to human cells (Fig. 3C and Supplementary Data 2). Both human and bat cell lines had comparable enrichment scores for GO process associated with positive regulation of IκB kinase/NFκB signaling (Fig. 3C and Supplementary Data 2). We also noticed differences in enrichment scores between the two bat cell lines. E. fuscus- derived cells had a higher enrichment score compared to P. alecto cells for GO processes associated with defense response to virus, response to virus, type I IFN signaling, IFNγ-mediated signaling pathway, positive regulation of IFNβ, and IL27-mediated signaling pathway (Fig. 3C and Supplementary Data 2). In contrast, P. alecto-derived cells had a higher enrichment score compared to E. fuscus cells for GO processes associated with response to IFNβ, response to IFNα, and antigen processing and presentation of endogenous peptide antigen via MHC class I (Fig. 3C and Supplementary Data 2). Indeed, our data demonstrate that while both bat cells mount a robust IFNβ-mediated response compared to human cells, differences exist between bat cells and likely between bat species.

Several upregulated genes identified in both bat cell lines are known to play a canonical antiviral role in human and bat cells, including genes like IFI6, ISG15, STAT1, OAS1, IRF7, RSAD2, IRF9, and TRIM5 (Fig. 3D and Supplementary Data 3, 4)^7,18. We also observed a significant upregulation of ISG15 and RPT4 transcripts in our cells upon IFNβ stimulation, and both ISG15 and RTP4 were recently identified as antiviral genes in select bat species (Fig. 3D and Supplementary Fig. 9B)^19,20. Function enrichment analysis of upregulated genes in all four cell lines also demonstrated that genes that negatively regulate the type I IFN response, like ADAR, OAS1, OAS3, and USP18 were also enriched for in both bat cells, suggesting a likely difference in the regulation of IFNβ signaling (Fig. 3D and Supplementary Data 3, 4)^21,22,23. Consistent with the literature, we observed the upregulation of well-studied IFN-stimulated genes in human cells, including OAS2, OAS3, TRIM5, GBP1, HERC5, and HERC6, among others (Fig. 3D and Supplementary Data 3, 4)^24,25,26,27.

Next, we identified the top upregulated DEGs in bat cells for which we had detected orthologous genes in human cells (Fig. 3E). Due to the divergent transcriptional profile, human A549 cells were removed from the analysis, and we focused our analyses on the three kidney-derived cell lines (Fig. 3E and Supplementary Fig. 9C). For the top upregulated DEGs, gene expression levels in A549 cells were generally consistently higher than human RPTEC cells and the two bat cell lines, except for MX2, HERC6, XAF1, and BST2, where gene expression levels were lower than or similar to levels in the other cells (Supplementary Fig. 9C). Human RPTEC cells upregulated over 60% (17/27 genes) of the top upregulated orthologous DEGs observed in bat cells to higher levels than either bat cell line (Fig. 3E). Unlike the other cell lines, Efk3B cells had increased basal expression of HERC6, XAF1, and DTX3L transcripts which were further upregulated upon IFNβ treatment (Fig. 3E). Expansion of both BST2 and PARP14 genes within the genomic locus of E. fuscus have been reported, where both genes have been triplicated in the E. fuscus genome^28,29. In our study, we observed an upregulation of transcript levels for all three variants of BST2 and PARP14 upon IFNβ stimulation of Ek3B cells (Fig. 3E and Supplementary Data 3, 4). We did not detect multiple transcript variants of BST2 or PARP14 in RPTEC or PakiT03 cells, so we plotted transcript levels for all three variants of BST2 and PARP14 found in Efk3B cells against the transcript levels of a single variant that we detected in RPTEC and PakiT03 cells (Fig. 3E). We also analyzed the expression levels of bat specific DEGs that we could not directly map back to the human genome annotation, such as IFI27-like, TRIM5a-like, OAS1, SP140-like, LOC112483779 (unnamed), DDX58, and DDX60L, (Supplementary Fig. 9D, E and Supplementary Data 4). Additionally, IFI44 and IFI44L have been lost in P. alecto (Supplementary Fig. 9D). Bat specific DEGs also include several unannotated and uncharacterized genes such as LOC102882974, LOC112476441, LOC129151230, and LOC129150681 (Supplementary Fig. 9E). Our data demonstrate that duplicated genes like BST2 and PARP14 in E. fuscus encode for mRNA transcripts upon stimulation with IFNβ, which suggests that duplicated genes in bats may have functional implications and warrant further investigation.

Antiviral potency of bat IFNβ-stimulated genes

Next, we characterized two ISGs that were upregulated in bat cells upon IFNβ stimulation, interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) and guanylate binding protein 1 (GBP1) (Supplementary Fig. 9D, E and Supplementary Data 4). Across mammalian species, IFIT proteins are known to inhibit a broad range of viruses; however, the number and identity of these genes vary substantially^30,31. For instance, the IFIT locus in humans encodes five genes (IFIT1, 1B, 2, 3, and 5), while four and six genes can be found within rats and mice, respectively³⁰. Mice and other rodents have lost the IFIT1 ortholog and have duplicated IFIT1B^32,33. We identified two IFIT1-like genes (LOC103296399 and LOC103300075) in the E. fuscus genome that map back to human IFIT1B (Supplementary Figs. 10, 11). In P. alecto bats, we identified one IFIT1 gene (LOC102878285) that mapped back to the human genome (Supplementary Figs. 10, 12).

The antiviral function of IFIT1 and IFIT1B relies on the discrimination of non-self RNA from host mRNA due to the lack of 2’O-methylation of the first or second ribose of the 5’ cap (cap1 and cap2, respectively)^34,35. IFIT1-mediated antiviral activity is also enhanced through protein–protein interactions like the binding of IFIT1 with IFIT3^36,37. Amino acid sequence comparison of human and P. alecto IFIT1 and IFIT3 showed 68.6 and 75.5% sequence identity, respectively (Supplementary Fig. 13). Key residues involved in human IFIT1-cap0 mRNA binding, including R38, L46, W147, K151, E176, and Y218³⁸, are conserved across at least 15 bat species (Supplementary Fig. 14, arrows). In our study, we observed that like human IFIT1, P. alecto IFIT1 inhibits translation of reporter mRNAs bearing the luciferase gene flanked by SARS-CoV-2 5ʹ and 3ʹ untranslated regions when the RNA had a cap0 at the 5’ end but not a cap1 (Fig. 3F). However, a luciferase reporter mRNA with Zika virus 5’ and 3’ untranslated regions was more resistant to P. alecto IFIT1 inhibition, consistent with a role for stable RNA secondary structure at the 5’ end of the viral genome to evade IFIT1 restriction^36,38,39.

We and others have previously identified a motif in the C-terminal end of human IFIT1 that is critical for interaction with IFIT3^36,37, and we demonstrated that hetero-oligomerization with IFIT3 is necessary for the full antiviral activity of IFIT1. Human IFIT1 and 3 interact through a highly conserved Y(E)XXL motif in the C-terminal region of each protein³⁶. While the Y(E)XXL motif is conserved across 15 bat IFIT1s, the corresponding motif in IFIT3 contains a Y (human)→F (bat) substitution (F₄₄₀EKEL), which is also conserved across diverse bat species (Supplementary Figs. 14, 15). To test if this substitution affected IFIT1 and IFIT3 interaction, we used P. alecto as a model to analyse the association of IFIT1 and IFIT3 by size exclusion chromatography (SEC) as we previously described for humans³⁶. P. alecto IFIT1 and IFIT3 co-elute earlier during SEC than either protein individually, confirming that they form a stable complex despite the Y → F substitution in the IFIT3 binding motif (Fig. 3G). Mutation of the Y₄₆₀(E)XXL motif in P. alecto IFIT1 to E₄₆₀(E)XXE prevented its association with IFIT3 during SEC (Fig. 3H), confirming that the interaction is through this conserved motif. Thus, IFIT1 antiviral function against SARS-CoV-2, along with its interaction with IFIT3 are conserved in bats despite the evolution of a F₄₄₀EKEL motif in bat IFIT3, compared to Y₄₄₀EKEL in humans.

In human cells, GBPs like GBP1, GBP2, and GBP5 are known to confer protection against a range of pathogens, including bacteria, protozoa, and viruses like VSV and HSV-1^40,41. In contrast to data from human cells, a recent study in mice demonstrated that the entire chromosome 3 cluster, which includes GBP1, GBP2, GBP3, GBP5, and GBP7 did not encode for proteins with antiviral properties against influenza A virus, HSV-1, or lymphotropic choriomeningitis virus, suggesting that the antiviral capacity of GBPs may be species dependent or virus specific⁴². In our study, we assessed the expression level and antiviral capacity of GBP1 from E. fuscus and P. alecto bats, along with humans. We observed higher basal levels of expression of GBP1 transcripts in unstimulated E. fuscus cells, with low levels of induction upon IFNβ treatment (Fig. 3I). IFNβ treatment in P. alecto cells did not induce GBP1 transcript expression (Fig. 3I). In human cells, transcript levels for GBP1 were upregulated upon IFNβ stimulation, which is consistent with other studies (Fig. 3I)⁴³. ClustalW alignment of bat and human GBP1 sequences identified conserved motifs within the GTPase domain that have been identified in human GBP1 (Supplementary Fig. 16)^44,45. In addition, the CaaX motif was also present in GBP1 of both bat species. The CaaX motif allows human GBP1, GBP2 and GBP5 to undergo isoprenylation⁴⁶, a post-translational modification that is thought to promote the translocation of human GBPs to intracellular membranes and enable interaction with other proteins^47,48. The presence of a CaaX motif in bat GBP1 sequences suggests that the GTPase activity and isoprenylation are likely conserved across bat species.

Next, we compared the electrostatic surface potential of GBP1 from human (Hu), P. alecto (Pa), and E. fuscus (Ef) (Fig. 4A). The left side of HuGBP1 and EfGBP1 has a similar charge distribution, while PaGBP1 possesses a more positive patch at the head of the protein (Fig. 4A, arrows). Modeling of the right side demonstrated further differences between the GBP1 proteins, where the head region in HuGBP1 is more positively charged than EfGBP1 and PaGBP1 (Fig. 4A, arrows). One of the roles of HuGBP1 is to hydrolyze GTP to GDP and GMP, which causes a conformational change that promotes pathogen-targeting function of the protein^47,48,49. These functions include recruitment and deposition on pathogen-containing vacuoles, disruption of actin filament formation, and/or interaction with alternative proteins⁴¹. Modeling the dimerization interface of GBP1 using APBS revealed a more positive interface of PaGBP1 compared to HuGBP1 and EfGBP1 (Fig. 4A, arrows).

**Fig. 4: The antiviral capacity of human and bat GBP1.**

We investigated the antiviral activity of GBP1 across the three species. Human, P. alecto, and E. fuscus GBP1 were expressed in human HEK293T cells (Fig. 4B). GBP1 from all three mammalian species localized within the cytoplasm of cells (Fig. 4C). To investigate the antiviral activity of GBP1, we expressed varying levels of human and bat GBP1 in human A549 cells to avoid interference from other bat cellular antiviral proteins and to assess the direct acting antiviral capacity of human and bat GBP1 against a range of different RNA and DNA viruses like VSV, HSV-1, influenza A virus (H1N1/PR8), SARS-CoV-2, MERS-CoV, Vaccina virus (VACV), and Eptesipoxvirus (EfPV) (Fig. 4D and Supplementary Fig. 17). Ectopic expression of HuGBP1 and EfGBP1 significantly inhibited the replication of VSV-GFP, while a modest effect was observed for PaGBP1 (Fig. 4D and Supplementary Fig. 17A–C). Upon infection with HSV-1, GBP1 from all species minimally suppressed virus replication, but HuGBP1 was effective at the lowest concentration (Fig. 4D and Supplementary Fig. 17D–F). A downward trend was observed upon infection with H1N1 in cells expressing GBP1 from all species, where EfGBP1 was effective at low concentrations (Fig. 4D and Supplementary Fig. 17G, H). For infection with SARS-CoV-2, HuGBP1 and EfGBP1 worked at similar concentrations of 250 ng to inhibit virus infection, while higher concentrations were required for PaGBP1 (Fig. 4D and Supplementary Fig. 17I, J). For MERS-CoV infection, HuGBP1 was more protective compared to either bat GBP1 (Fig. 4D and Supplementary Fig. 17K, L). Interestingly, when GBP1-expressing cells were infected with a bona fide E. fuscus bat-derived virus, Eptesipoxvirus, EfGBP1 completely abolished virus replication at the lowest concentration, while HuGBP1 and PaGBP1 were effective at higher concentrations compared to EfGBP1 (Fig. 4D and Supplementary Fig. 17M, N). Finally, to determine whether EfGBP1 was equally potent in suppressing the replication of a distant poxvirus that is not speculated to have evolved in bats, we tested all GBP1 proteins against VACV. All three GBPs were only partially effective in suppressing VACV replication at high levels of GBP1 expression (Fig. 4D and Supplementary Fig. 17O, P).

Given the unique and potent antiviral activity of EfGBP1 against E. fuscus-derived EfPV, we next determined the functional motifs within the GTPase domain of EfGBP1 since this domain is important for the antiviral activity of HuGBP1^44,45. The four critical domains (G1–G4) identified in human HuGBP1 are highly conserved across the 14 bat species we compared (Supplementary Fig. 16). However, we discovered an additional putative motif of interest in the N-terminus of GBP1 that we named AV1. The AV1 motif differs between the four bat families compared to humans (Fig. 4E and Supplementary Fig. 16). To investigate whether this AV1 motif had any effect on the antiviral activity of bat GBP1, we generated P. alecto and E. fuscus AV1 deletion (Δ_18–20) and substitution variants (A₁₈AA) (Fig. 4E). For E. fuscus AV1 deletion variant we saw a complete loss of GBP1-mediated protection (Supplementary Fig. 18A); however, due to the reduced expression of the PaGBP1 deletion variant (Supplementary Fig. 18B, C), only the substitution variants were further characterized. APBS predictions identified significant changes in electrostatic potential when the AV1 motif is mutated to A₁₈AA, where the site becomes more neutral in PaGBP1 and EfGBP1 (Fig. 4E). In cells infected with EfPV, no significant difference in antiviral activity was observed between PaGBP1(A₁₈AA) and wild-type PaGBP1 (Fig. 4F, G and Supplementary Fig. 18A, B). In cells expressing EfGBP1 and infected with EfPV, EfGBP1(A₁₈AA) displayed no antiviral activity compared to wildtype EfGBP1 (Fig. 4F, G and Supplementary Fig. 18B). Thus, in this study we identified a functional motif within the GBP1 protein that is critical for antiviral function and our results demonstrate species-specific activity of this AV1 motif in bats.

Discussion

In this study, we utilized species-specific IFNβ to evaluate ISG induction in bat cells. While the human type I IFN pathway is extensively studied⁷, its applicability to bats remains uncertain. Recent work has demonstrated the activation of the JAK-STAT pathway in Rousettus aegyptiacus nasal epithelial cells following RoIFNλ1 stimulation⁵⁰, in addition to the requirement of IFNAR and phosphorylation of STAT1 upon stimulation of pulmonary cells from the Greater horseshoe bat (Rhinolophus ferrumequinum) with IFNω⁵¹. Similar results have been obtained for P. alecto, where signaling with bat IFNα requires IFNAR1/2⁵². However, the precise role of STATs during virus infection in bats remains unclear. We have demonstrated that phosphorylation is required for IFIT1 and MX1 expression and for antiviral protection against VSV-GFP infection in P. alecto and E. fuscus cells (Supplementary Fig. 3). We noticed that E. fuscus kidney-derived cells express basal levels of nuclear STAT1/2, suggesting that these cells may be primed to respond faster to IFNβ (Fig. 2B). In addition, P. alecto kidney-derived cells mounted a rapid response upon IFNβ stimulation (Fig. 2B), in line with previous work that has shown a faster ISG response to virus infection in P. alecto cells due to elevated levels of IRF1, 3, and 7^53,54. We further demonstrated that the JAK-STAT pathway is not directly antagonized by MERS-CoV during infection in P. alecto or E. fuscus cells, highlighting the ability of bat cells to resist MERS-CoV infection mediated shutdown of innate IFN dependent antiviral responses. Our analyses were limited to two bat species and two cell types. Future studies in diverse bat species and primary cells will fully characterize the breadth of IFNβ-mediated antiviral responses in bats⁵⁵.

Metagenomic analyses have demonstrated that immune genes, like ISGs, are more likely to undergo selection, with bats experiencing a high rate of selection within immune genes compared to other mammals⁵⁶. As there are over 1480 species of bats, the potential differential function of conventional and atypical ISGs is worth investigating, as it may shed light on the ability of bats to tolerate viral infections. As seen in Fig. 3, both bat species induced a conserved type I IFN response upon stimulation with IFNβ. However, both bat species upregulated orthologous transcripts to a lower degree than either human cell line (Fig. 3C), suggesting that lower levels of ISGs may contribute to an antiviral state. Many of the upregulated genes in bats contributed to different biological processes (Supplementary Data 1), suggesting that the bat antiviral response likely differs in quality and intensity when compared to humans. Lineage-specific differences in MX2 antiviral activity have been identified, where, unlike the human counterpart, P. vampyrus MX2 is unable to restrict HIV-1 infection, whereas MX2 from closely related P. alecto can^57,58. Additionally, three BST2 and PARP14 paralogs were upregulated in E. fuscus cells (Fig. 3E). BST2 gene duplications have been identified within the Vespertilionidae bat family^28,29, with previous studies on Marburg virus and Nipah virus suggesting a distinct role for each BST2 paralog in orchestrating an antiviral response^28,59,60. While duplications are common in the Vespertilionidae bat family, deletion of genes like IFI44 and IFI44L (Supplementary Fig. 9D) have been reported for the Pteropodidae bat family⁶¹. Notably, IFI44L was readily upregulated in E. fuscus cells upon stimulation, demonstrating additional variation in the antiviral response across bats.

In R. aegyptiacus cells, the overexpression of IFIT1 inhibits Ebolavirus and Marburg virus replication, though it is unclear how this antiviral activity compares to human IFIT1⁶². A study of IFIT1 across 39 mammals revealed diverse antiviral phenotypes—while human and P. alecto IFIT1 could bind cap0 RNAs and strongly inhibit Venezuelan equine encephalitis virus, chimpanzee IFIT1 lacked this ability³¹. We observed similar activity between P. alecto and human IFIT1 in inhibiting the translation of mRNA bearing the untranslated regions of SARS-CoV-2 and Zika virus (Fig. 3F)³⁹. We did not observe differential activity of IFIT1 between P. alecto and humans (Fig. 3G and Supplementary Fig. 9B), unlike recent work on ISG15 and RPT4^19,20. These findings further highlight the evolutionary arms race between bat immune genes and viruses.

In humans, induction of GBP1 upon type I and II IFN signaling has been reported⁶³; with in vivo studies demonstrating induction through type I IFN signaling pathways⁴³. Recent work in mice has demonstrated that the antiviral activity of GBP1 may not be shared across mammals⁴² leading us to investigate whether this gene was functional within both bat species. Both Hu- and EfGBP1 downregulated VSV-GFP replication, while PaGBP1 was unable to do so (Fig. 4D). Previous work using GBP1 from the Chinese tree shew (Tupaia belangeri chinensis) demonstrated that the restriction activity of GBP1 was due to its competition with the viral nucleocapsid (N) protein in binding to phosphoprotein (P) of VSV⁶⁴. Whether the interaction of PaGBP1 with VSV-P is less favorable compared to human and E. fuscus GBP1 remains unknown. In contrast, both bat GBP1s were less effective against HSV-1 than HuGBP1. HSV-1 is a human-derived isolate and has co-evolved alongside humans for millions of years⁶⁵. In the future, the antiviral potency of bat GBP1 against bat-derived herpesviruses, such as the Gammaherpesvirus from E. fuscus⁶⁶ will shed further light on the co-evolution of bat GBP1 and their viruses.

To our knowledge, no study has evaluated the efficacy of human and bat GBP1 against two coronaviruses of bat origin that have caused outbreaks in humans. We observed differential activity against SARS-CoV-2 and MERS-CoV, where restriction against the former was more apparent at lower expression levels of Hu- and EfGBP1 (Fig. 4D). Previous reports have demonstrated the interaction of chicken GBP1 with the nucleocapsid protein of infectious bronchitis virus, leading to its degradation via the autophagy pathway⁶⁷. The conserved or divergent mechanisms by which bat GBP1 elicits its antiviral effect against a range of different RNA and DNA viruses is the focus of ongoing research within our laboratory. Our work with GBP1 has also led to the identification of a functional (AV1) motif within EfGBP1 that enables it to potentially restrict an E. fuscus-derived poxvirus, EfPV (Fig. 4F, G). Previous work on GBP2 and Ectromelia virus (ECTV), a poxvirus that closely resembles Mpox, demonstrated GBP2 as a mild inhibitor of ECTV in mice⁶⁸. This was attributed to the ability of the poly(A) polymerase catalytic subunit of ECTV to bind to GBP2, antagonizing its restriction activity⁶⁹. Although ECTV and EfPV are distantly related viruses, future work may evaluate whether the evolution of the AV1 site in EfGBP1 prevents EfPV from antagonizing its antiviral activity. Importantly, the functional role of the AV1 site within human GBP1 can now also be explored based on our discoveries. Overall, our study highlights the evolutionary differences in the IFNβ response between humans and bats, and between bat species. By investigating the arms race between bat GBP1 and multiple RNA and DNA viruses, we have identified a motif (AV1) within GBP1, and this knowledge can one day be harnessed to better understand and control viral infections in humans.

Methods

Cell culture

Pteropus alecto kidney (PakiT03), Eptesicus fuscus kidney (Efk3B), Huh7, HEK293T, and Vero76 cells were grown in Dulbecco’s Minimal Essential Medium with high glucose (DMEM; Gibco, #11965118) supplemented with 10% fetal bovine serum (FBS; Sigma-Aldrich, #12107 C), 1% Penicillin/Streptomycin (Pen-Strep; Gibco, #15140122), and 1% GlutaMax (Gibco, #35050061). Human alveolar basal epithelial (A549) cells were grown in Ham’s F-12 (Kaighn’s) medium (Gibco, #21127022) supplemented with 10% FBS and 1% Pen-Strep. PakiT03, Efk3B, A549, Huh7, and HEK293T cells were used up to passage 50. A549-ACE2 cells (A549 cells overexpressing human angiotensin-converting enzyme 2; ACE2) were provided by Dr. Colpitts’ laboratory, with clonal population B9 used⁷⁰. A549-ACE2 cells were maintained in Ham’s F-12 (Kaighn’s) medium supplemented with 10% FBS, 1% Pen-Strep, and 10 ug/mL blasticidin (info). Human, life-extended renal epithelial cell line isolated from the proximal tubule of a male patient (RPTEC-hTERT; ATCC, #CRL-4031) were grown in DMEM:F-12 media (ATCC; #30-2006) supplemented with the hTERT growth kit (ATCC; #ACS-4007). RTPEC cells were used up to passage 10. Drosophila Schneider 2 (S2) cells (Thermo Scientific, #R69007) were grown in Schneider’s Drosophila medium (Life Technologies, #21720024) supplemented with 10% FBS and 1% Pen-Strep. Madin-Darby canine kidney cells (MDCK II; Sigma-Aldrich, #00062107) were grown in Minimal Essential Medium (MEM; Sigma-Aldrich, #M4655) supplemented with 5% FBS, 1% Pen-Strep, and 1% GlutaMax.

Chemicals

Staurosporine (Cell Signaling, #9953S) was reconstituted in DMSO and used at 500 nM/mL at the times indicated.

Recombinant IFNβ

Recombinant V5 and 6x-His-tagged IFNβ proteins for P. alecto, E. fuscus, and human were cloned into pMT-BiP/V5-His and generated by transfecting Drosophila S2 cells following the manufacturer’s instructions (Thermo Scientific, K513001). Briefly, Drosophila S2 cells were transfected with 19 μg of the pMT-BiP/V5-His plasmid containing Human, P. alecto or E. fuscus IFNβ and 1 μg of pCoBlast. Following 24 h of transfection, cells were washed and incubated for 48 h before blasticidin selection (25 μg/mL) was added. Resistant clones were then stimulated with 500 μM of copper sulfate for 72 h. A vehicle control, supernatant containing secreted GFP, was also generated following this procedure. Proteins were characterized by immunoblotting for the V5-epitope tag (Supplementary Data 5) and quantified by ELISA as per the manufacturer’s instructions (Abcam, #ab285248). To determine the efficacy and protective unit for each recombinant IFNβ, cells were treated with serially diluted, species-matched IFNβ or vehicle for 6 h and then infected with VSV-GFP at a multiplicity of infection (MOI) of 0.1. Infection was visualized at 10X magnification on a Leica DMI6000B fluorescent microscope 16 h post-infection (hpi). The last dilution of IFNβ in which 100% protection was observed was classified as 1 unit (U) of protection.

Viruses

Genetically engineered vesicular stomatitis virus encoding a green fluorescent protein (VSV-GFP) cassette was propagated in Vero76 cells and stored at −80 °C. Genetically engineered herpes simplex virus 1 encoding GFP (HSV-1-K26-GFP) was kindly provided by Dr. Karen Mossman (McMaster University) and was propagated in HeLa cells and stored at −80 °C. Influenza A virus stain A/Puerto Rico/8/34 (H1N1; PR8) was propagated in 11-day-old embryonated eggs and stored at −80 °C. Vaccinia virus VR-2153 (VACV; ATCC, vr-2153) was propagated in HeLa cells and stored at 80 °C. Middle East respiratory syndrome coronavirus (MERS-CoV; isolate EMC/2012), severe-acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and Eptesipoxvirus (EfPV) were propagated in Vero76 cells and stored at −80 °C. All work with infectious MERS-CoV and SARS-CoV-2 was completed at VIDO-InteVac in a containment level 3 laboratory and was approved by the institutional biosafety committee. Standard operating procedures approved by the institutional biosafety committee were followed for sample inactivation.

Tissue culture infectious dose 50 assay (TCID₅₀)

The supernatants from HSV-1, MERS-CoV, and SARS-CoV-2- infected cells were titrated in triplicate on Vero76 cells using a tissue culture infectious dose 50 assay. Meanwhile, MDCK II cells were used to titrate H1N1. Briefly, 3 × 10⁴ cells were seeded per well of a 96-well plate for 24 h. Media was then removed from the wells in exchange for 50 μL of 1:10 serially diluted supernatant containing virus. Following a 1 h incubation at 37 °C, the inoculum was replaced with DMEM supplemented with 2% FBS. The plates were incubated at 37 °C for 3 (HSV-1, H1N1) or 5 days (MERS-CoV, SARS-CoV-2) and cytopathic effect was monitored under a light microscope. The TCID₅₀/mL was calculated using the Spearman and Karber algorithm.

Plaque assay

Confluent monolayers of Vero76 or MDCK II cells in six-well plates were infected with 800 μL of tenfold serially diluted virus for 1 h at 37 °C. Following inoculation, 2% CMC or Avicel overlay medium was added. Plates were incubated for 2 days for H1N1, 3 days for VACV, and 4 days for EfPV. Cells were fixed for 30 min with 10% neutral buffer formalin and stained with 0.1% Toluidine blue (Fluka, #89640).

Quantitative real-time PCR

Cells were untreated or treated with species-matched IFNβ for the appropriate period of time. Following IFNβ treatment, RNA extraction was performed as per the manufacturer’s instructions for the RNeasy Plus Mini Kit (Qiagen, #74136). Four hundred nanograms of purified RNA was reverse transcribed into cDNA following the manufacturer’s instructions for the iScript gDNA Clear cDNA Synthesis Kit (Bio-Rad, #1725035). Samples were incubated in a T100 thermal cycler (Bio-Rad, #1861096) for 5 min at 25 °C, 20 min at 46 °C, and 1 min at 95 °C. qRT-PCR reactions were then carried out using 10 μL of the SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, #1725274), and 4 μM of the forward and reverse primers per reaction tube. Primers utilized are described in Supplementary Data 6. qRT-PCR reactions were completed using a StepOne Real-Time PCR System (Applied Biosystems). Samples were run in duplicate, with Ct values normalized to cellular Gapdh.

Immunoblots

Protein lysates were either collected in 1X sample buffer or in RIPA buffer (Sigma-Aldrich, #R0278-50ML) containing 3X Halt protease and phosphatase inhibitor cocktail (Thermo Scientific, #78442) for bat-derived cells and 2X Halt protease and phosphatase inhibitor cocktail for human-derived cell lines for phospo-protein analysis. Following boiling for 10 min at 96 °C, proteins were separated by SDS-PAGE using homemade 10% polyacrylamide gels and semi-dry transferred to a 0.2 µm nitrocellulose membrane using the Trans-Blot Turbo Transfer System (Bio-Rad, #1704270). Membranes were blocked using intercept blocking solution (Licor Biosciences, #927-60001) and probed with primary antibodies (Supplementary Data 5) diluted in 50% intercept blocking solution and 50% TBS overnight on a rocker. The appropriate secondary antibody (Supplementary Data 5) diluted in 50% intercept blocking solution and 50% TBS was then added for 60 min. Membranes were imaged and analyzed using the Odyssey imager (Licor Biosciences) and Image Studio Software (Licor Biosciences).

Immunofluorescence

Cells were fixed for 20 min at −20 °C with 100% MeOH and blocked using a homemade solution (PBS, 10% newborn calf serum, and 0.1% Tween-20). Cells were then stained using the appropriate primary and secondary antibodies (Supplementary Data 5) and mounted onto a glass slide. Visualization of stained cells occurred by confocal microscopy (Leica).

Knockdown of the JAK-STAT Pathway

Dicer-ready silencing RNA (siRNA) targeting human, P. alecto, and E. fuscus STAT1 or STAT2 were designed and obtained through Integrated DNA Technologies (IDT; Supplementary Data 6). A final concentration of 25 nM was transfected to knock down STAT1, while 75 nM was transfected to knock down STAT2 using Lipofectamine 3000 (Thermo Scientific). Double knockdown of STAT1 and STAT2 occurred by transfecting both siRNA at a concentration of 50 nM and 75 nM, respectively, in PaKiT03 and A549 cells. Scrambled non-specific siRNA (IDT) was used as a negative control. Immunoblotting was performed to confirm knockdown efficiency.

RNA sequencing

PaKiT03, Efk3B, A549, and RPTEC cells were untreated or treated with 1 U/mL of species-matched IFNβ for 6 h (n = 3/condition). RNA extraction was performed as per the manufacturer’s instructions for the RNeasy Plus Mini Kit (Qiagen, #74136) and shipped to McMaster Genomics Facility, Farncombe Institute at McMaster University for sequencing. Sample quality was first assessed using a Bioanalyzer (Agilent), then enriched (NEBNext Poly(A) mRNA Magnetic Isolation Module; NEB). Library preparations were conducted (NEBNext Ultra II Directional RNA Library Prep Kit; NEB), and library fragment size distribution was verified (Agilent TapeSection D1000; Agilent). Libraries were quantified by qPCR, pooled in equimolar amounts, and qPCR and fragment size distribution verification were conducted again. Libraries were then sequenced on an Illumina NextSeq 2000 using a paired-end, 2 × 50 bp configuration.

Raw reads were processed with fastp v0.21.0, with the first 14 nucleotides trimmed based on an initial fastp run. Samples run in duplicate on two lanes during sequencing were then merged together. Transcriptome indices based on the RefSeq annotations for Pteropus alecto (GCF_000325575.1), Eptesicus fuscus (GCF_027574615.1), and Homo sapiens (GCF_000001405.40) were created with salmon v1.4.0 using their genomes as a decoy. Salmon transcript quantification mapped against the appropriate index was performed for all samples. In R v4.1.1, tximport v1.22.0 pulled the gene and transcript IDs from the appropriate gff files, and mapped the salmon abundance counts to the gene level. Genes with less than a length-scaled TPM of 10 across all samples were removed. Three samples were removed due to large deviations in expression profile detected via a PCA plot (DESeq2 v1.34.0). Differential gene expression analysis on the remaining samples, separated by mock versus interferon-beta treatment, was done with DESeq2. P-values were filtered with results using an alpha of 0.05. Significantly differentially expressed genes (DEGs) had an adjusted p value less than 0.05. OrthoFinder v2.5.2 was applied pair-wise across the three species, with each bat gene set being mapped back to human genes, and any remaining unmapped genes in an orthogroup between the two bat species also being mapped together. All genes in an orthogroup were defined as each other’s orthologs. A PCA for the expression profiles was created with plotPCA based on scaled transcripts per million (TPM). pheatmap v1.0.12 was used to create heatmaps between the three species, also based on scaled TPM, with sample values further scaled across each gene using scale.

Function/pathway annotation of the H. sapiens DEGs was done with the online DAVID webserver in Aug. 2023. These annotations were mapped to bat genes based on the OrthoFinder-defined orthogroups. Phyper was used in R to calculate the hypergeometric pvalue, subsequently adjusted with p.adjust using the Benjamini & Hochberg correction method.

Antiviral efficacy of GBP1

Human, P. alecto, and E. fuscus GBP1 cDNA was synthesized (GenScript) and cloned into the p3X-FLAG7.1 backbone using NotI, BamHI, KpnI, and/or Sall restriction sites. A549-ACE2 cells (1.5 × 10⁵ cells/well) seeded in a 12- well plate were transfected with 250–1000 ng of each construct using Lipofectamine 3000 (Thermo Scientific)⁷⁰. After 24 h, cells were infected with VSV-GFP (MOI 0.01), HSV-1 (MOI 0.01), H1N1 (MOI 0.01), SARS-CoV-2 (MOI 0.01), MERS-CoV (MOI 0.1), EfPV (MOI 0.01), H1N1 (MOI 0.01), or VACV (MOI 0.01). A low MOI was chosen to assess the antiviral role of GBP1 during multiple rounds of virus replication in culture and to not overwhelm the culture system with too much virus that could potentially bypass GBP1-mediated restriction. As A549 cells are less permissive to MERS-CoV, a higher MOI was utilized. To assess infection, microscopy was performed for VSV-GFP, while the TCID₅₀ assay or plaque assay was performed for the remaining viruses.

Site-directed mutagenesis

E. fuscus and P. alecto GBP1 mutants were generated using the QuikChange II site-directed mutagenesis kit (Agilent Technologies), following the protocol modified by Wang and Malcom⁷¹. Briefly, specific primers were designed to introduce the desired mutations into the GBP1 gene (Supplementary Data 7). Target regions were amplified using these primers. The PCR products were then treated with DpnI to digest the parental DNA template, and the resulting mutant plasmids were transformed into E. coli Stbl3 cells. Positive clones were selected on LB agar plates with ampicillin, and mutations were confirmed by Sanger sequencing (National Research Council, Canada). For the alanine insertions, a similar protocol was followed using the GBP1 plasmids with deletions as template.

Phylogenetic analyses

IFIT protein sequences were aligned with MUSCLE v3.8.31. Gblocks site selection was done in SeaView v5.0.5 using the least stringent options. We used RAxML v8.2.12 with the PROTGAMMAAUTO setting and 100 bootstrap runs to create the phylogenetic tree. The best-scoring tree used JTT likelihood with empirical base frequencies. The tree was subsequently visualized in iTOL v7.1 and midpoint rooted.

IFIT protein purification and analysis

Human IFIT1 and IFIT3 expression plasmids were previously described³⁶. Wildtype and mutant sequences for Pteropus alecto IFIT1 (XM_006925964) and IFIT3 (XM_006925965) were synthesized by Twist Biosciences and cloned into pET28b (NdeI and XhoI) plasmid to encode full-length proteins with an N-terminal 6X-His tag for purification. Template plasmids for T7 transcription of a firefly luciferase reporter gene flanked by Zika virus (ZIKV-Fluc) or β-globin (Globin-Fluc) untranslated regions were previously described³⁶. A plasmid containing a short fragment of SARS-CoV-2 non-structural protein 1 (NSP1) separated from the firefly luciferase reporter gene by a 2 A StopGo sequence, flanked by the SARS-CoV-2 5ʹ- and 3’UTRs and 5ʹ and 3ʹ hammer head and hepatitis D virus ribozymes respectively (SARS-CoV-2-Fluc), under the control of a T7 promoter was a gift from T. Peacock, The Pirbright Institute.

Recombinant His-tagged human and bat IFIT1 and IFIT3 proteins were expressed in Rosetta (DE3) E. coli (Novagen) for 16 h at 22 °C after induction of protein synthesis with 1 mM isopropyl β-D-1- thiogalactopyranoside. Cells were centrifuged and resuspended on ice with lysis buffer (400 mM KCl, 10% glycerol, 1 mM DTT) supplemented with 5 mg/ml lysozyme (Invitrogen) and EDTA-free cOmplete Protease Inhibitor Cocktail tablets (Roche). Lysis buffers included 30 mM HEPES (IFIT1) or 20 mM Tris-HCl pH7.5 (IFIT3). Lysates were clarified by centrifugation following lysis by sonication before purification on Ni-NTA Agarose beads (Cube Biotech). IFIT1 and IFIT3 were further purified using an ÄKTA FPLC system (Cytiva) on MonoS or MonoQ 5/50 GL columns (Cytiva), respectively, in HEPES-based lysis buffer (MonoQ- IFIT1) or Tris-based lysis buffer (MonoS- IFIT3) before being stored at –70 °C. Size exclusion chromatography (SEC) was performed on a Superdex 200 Increase 10/300 GL column in SEC buffer (20 mM Tris-HCl pH7.5, 200 mM KCl, 1 mM DDT. 200 µL of 1 mg/ml wildtype or mutant IFIT1 or IFIT3 were injected into the SEC column to determine peak elution volumes. IFIT1:IFIT3 assembly: 1 mg/ml each of wildtype or mutant IFIT1 and IFIT3 were incubated in a 200 µL of SEC buffer for 1 h at 4 °C before injecting onto the column. Peak fractions were subsequently analysed by SDS-PAGE and Coomassie staining.

In vitro transcription and luciferase assay

Globin-Fluc and ZIKV-Fluc plasmids were linearized with HindIII, while SARS2-Fluc was linearized with XhoI. RNA was transcribed with T7 polymerase (New England Biolabs), using 1 µg of linearized template DNA in transcription buffer (40 mM HEPES pH 7.5, 32 mM MgOAc, 40 mM DTT, 2 mM Spermidine, 10 mM NTPs and 0.8 U/µl Ribolock (Thermo Scientific), and incubated overnight at 37 °C. Following template DNA digestion using DNase I (Thermo Scientific) unincorporated nucleotides were removed using Amersham MicroSpin G-50 Columns (Cytiva). RNA was purified by acidic-phenol extraction and ethanol precipitation. RNA was capped using the ScriptCap and ScriptCap 2′-O-methyltransferase system (CellScript). IFIT1 and IFIT3, where indicated, was combined with 100 ng of RNA in buffer containing 20 mM Tris-HCl pH7.5, 150 mM KCl, 5% glycerol, 1 mM DTT, 10 U/µL RNase inhibitor and 0.5 mg/ml BSA and incubated for 15 min at 30 °C. The RNA protein mixture was added to in vitro translation reactions using the Flexi® Rabbit Reticulocyte Lysate (RRL) System (Promega), with a final concentration of 100 nM of IFIT1 or IFIT1:IFIT3 complex and incubated for 90 min at 30 °C and then placed on ice to halt the reaction. Firefly luciferase signal was measured on the GloMax® Discover microplate reader (Promega), by adding an equal volume of ONE-Glo™ luciferase assay reagent (Promega). Luciferase values were normalized to a no-IFIT control.

Electrostatic potential of GBP1 at the solvent-accessible molecular surface

Using the sequences of P. alecto and E. fuscus GBP1 proteins, residues 18–20 were modified to AAA (alanine mutant). For each of the wildtype and mutant sequences, AlphaFold3 was used to predict three-dimensional structures⁷². Protonation states of the titratable residues were assigned at physiological pH using PROPKA^73,74. The structures were prepared for electrostatic calculations by assigning atomic charges and radii using PDB2PQR⁷⁵. The electrostatic potential for each protein structure was calculated by solving the continuum electrostatics equations with the adaptive Poisson–Boltzmann solver (APBS)⁷⁵. Calculated electrostatics were mapped onto the solvent-accessible molecular surface of each protein using ChimeraX⁷⁶.

Statistics and software

Graphpad Prism v10.2.3 was used to perform significance tests, with the statistical test used specified where appropriate. MacVector software was used to perform multiple sequence alignments. ImageJ software was used to analyze microscopy images to quantify nuclear translocation and GFP expression.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The transcriptomic dataset has been deposited to NCBI under the accession code PRJNA1255723 (or https://www.ncbi.nlm.nih.gov/sra/PRJNA1255723). The following genomes were used as the reference to analyze the transcriptomics data: GCF_000325575.1 (Pteropus alecto), GCF_027574615.1 (Eptesicus fuscus), and GCF_000001405.40 (Homo sapiens). The DESeq2 expression data generated from the transcriptomic analysis performed this study are provided in the Source Data file. Source data are provided with this paper.

Code availability

All codes and scripts that were used for data analysis is available at https://github.com/doxeylab/gonzalez-et-al⁷⁷.

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Acknowledgements

V.G. is supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) scholarship (#569587-2022). Research within A.B.’s lab is supported by an NSERC Discovery Grant (RGPIN-2022-03010), Canadian Institutes of Health Research (CIHR)—Institute for Infection and Immunity Early Career Research grant (PTT-192089), CIHR - Pandemic Preparedness and Health Emergencies Early Career Investigator grant (PEE-183995), and CIHR-Institute for Infection and Immunity, Project grant (PJT-195787). This research was undertaken, in part, thanks to funding from the Canada Research Chairs Program awarded to A.B. (CRC-2024-00291). J.C. is supported by an NSERC undergraduate student research award (BRPC-539885). Research within S.G.’s lab is supported by an NSERC Discovery Grant (RGPIN-2023-03546), an NSERC Discovery Launch Supplement (DGECR-2023-00091) and a Canada Biomedical Research Fund and Biosciences Research Infrastructure Fund (CBRF2-2023-00176). S.G. holds a Tier 2 Canada Research Chair in Structural Systems Biology (CRC-2022-00099). Y.Z. is supported by NSERC (RGPIN-2019-04578) and CIHR (PJT-166138). T.R.S. is supported by a Wellcome Trust/Royal Society Sir Henry Dale Fellowship (202471/A/16/Z) and BBSRC grants (BB/X011038/1 and BBS/E/PI/230001 A). We would like to thank B. Haagmans and R. Fouchier, Erasmus Medical Center, for providing MERS-CoV (isolate hCoV-EMC/2012). VIDO receives operational funding from the Government of Saskatchewan through Innovation Saskatchewan and the Ministry of Agriculture, and from the Canada Foundation for Innovation through the Major Science Initiatives Fund. We would like to thank members of the Laboratory of Zoonotic Viruses and Comparative Immunology at VIDO for their feedback during weekly laboratory meetings.

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Laboratory of Zoonotic Viruses and Comparative Immunology, Vaccine and Infectious Disease Organization (VIDO), University of Saskatchewan, Saskatoon, SK, Canada
Victoria Gonzalez, Arkadeb Bhuinya, Akarin Asavajaru, Yan Zhou, Darryl Falzarano & Arinjay Banerjee
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, SK, Canada
Victoria Gonzalez, Arkadeb Bhuinya, Yan Zhou, Vikram Misra, Darryl Falzarano & Arinjay Banerjee
Department of Biology, University of Waterloo, Waterloo, ON, Canada
Briallen Lobb, Andrew C. Doxey & Arinjay Banerjee
Département de Biochimie, de Microbiologie et de Bio-informatique, Faculté des Sciences et de Génie Université Laval, Québec, QC, Canada
Jacob Côté & Sophie M. C. Gobeil
Institut de Biologie Intégrative et des Systèmes, Université Laval, Québec, QC, Canada
Jacob Côté & Sophie M. C. Gobeil
PROTEO, Le Regroupement Québécois de Recherche sur la Fonction, L’Ingénierie et les Applications des Protéines, Université Laval, Québec, QC, Canada
Jacob Côté & Sophie M. C. Gobeil
Centre de Recherche en Infectiologie de l’Université Laval, Université Laval, Québec, QC, Canada
Jacob Côté & Sophie M. C. Gobeil
The Pirbright Institute, Guildford, Surrey, UK
Adriana G. Tubb, Stephen S. Nuthalapati & Trevor R. Sweeney
Department of Virology, University of Cambridge, Cambridge, UK
Trevor R. Sweeney
Programme in Emerging Infectious Disease, Duke-NUS Medical School, Singapore, Singapore
Linfa Wang
Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, ON, Canada
Arinjay Banerjee
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada
Arinjay Banerjee

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Victoria Gonzalez
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Briallen Lobb
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Contributions

V.G. and A.B. designed the study. V.G. assembled the figures. VG performed all experiments except for the following: Arkadeb Bhuinya (ABh) performed site-directed mutagenesis, A.G.T., S.S.N., and T.R.S. performed IFIT1-related experiments, and A.B. cloned and made stable S2 cells that secrete human and bat IFNβ. A.A. expanded cell lines for laboratory stocks, optimized cell culture protocols, and trained students. B.L. and A.D. analysed raw RNA-sequencing data. J.C. and S.M.C.G. performed predication and structural analyses for GBP1. Y.Z. provided H1N1-PR8 virus strain, V.M. provided EfPV strain, and D.F. provided MERS-CoV strain. L.W. provided the PakiT03 cell line. V.G. and A.B. wrote the manuscript. All authors contributed to the text.

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Correspondence to Arinjay Banerjee.

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Competing interests

A.B. is a co-inventor of the Efk3B cell line that is sold through Kerafast, USA. S.G. is named in a patent regarding coronavirus monoclonal antibodies (WO2022060906A1). The invention provides methods for using the inventive antibodies in prophylactic and/or therapeutic methods to prevent or treat coronavirus infection. The remaining authors declare no competing interests.

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Gonzalez, V., Lobb, B., Côté, J. et al. Bat-specific adaptations in interferon signaling and GBP1 contribute to enhanced antiviral capacity. Nat Commun 16, 5735 (2025). https://doi.org/10.1038/s41467-025-61254-7

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Received: 12 December 2024
Accepted: 16 June 2025
Published: 01 July 2025
DOI: https://doi.org/10.1038/s41467-025-61254-7

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