The CD97-PPM1G axis dampens antiviral immunity by dephosphorylating IRF7 in type I interferon pathway

Huasong Chang; Wenjing Qi; Rukun Yang; Peili Hou; Ran Kang; Xiaoyu Liu; Yingying Li; Hongmei Wang; Hongbin He

doi:10.1371/journal.ppat.1014032

Abstract

The activation of type I interferon (IFN-I) signaling is crucial for defending host cells against viral infections. A comprehensive IFN-I response necessitates the activation of several cellular factors, among them Interferon Regulator Factor 7 (IRF7). Nonetheless, the mechanisms governing IRF7 inactivation in response to viral infection remain largely unknown. Here, we illustrate that Cluster of differentiation 97 (CD97), a G protein-coupled receptor, interacts with PPM1G via intracellular Arg-819 and Arg-822 residues. PPM1G then recruits and dephosphorylates IRF7, leading to its inhibition. CD97-mediated inactivation of IRF7 impedes its translocation into the nucleus and subsequent activation of IFN-I, ultimately promoting the viral replication. Moreover, mice lacking CD97 display heightened resistance to viral infection. The compound sanguinarine (SANG) hinders viral replication by dampening CD97 expression. This study provides a basis for CD97 as a potential antiviral target and SANG as a candidate antiviral small molecule drug.

Author summary

Upon viral infection, the body’s first line of defense is the IFN-I response, a critical immune signaling pathway that defends host cells against invading pathogens. However, this system is tightly regulated by innate immune-inhibiting molecules within the host. The significance of this study lies in its identification of the CD97-PPM1G axis as a key regulator that suppresses the IFN-I response. Specifically, the research elucidates the molecular mechanism by which CD97 recruits the phosphatase PPM1G, which subsequently dephosphorylates the transcription factor IRF7. This dephosphorylation inhibits IRF7, thereby suppressing the entire IFN-I signaling cascade. In addition, the study employed CD97 knockout mice, which demonstrated significantly enhanced resistance to viral infection, confirming its role in vivo. Furthermore, the compound sanguinarine effectively hinders viral replication by dampening CD97 expression. These findings not only advance our theoretical understanding of immune homeostasis but also provide a solid foundation for developing antiviral strategies for both humans and animals.

Citation: Chang H, Qi W, Yang R, Hou P, Kang R, Liu X, et al. (2026) The CD97-PPM1G axis dampens antiviral immunity by dephosphorylating IRF7 in type I interferon pathway. PLoS Pathog 22(3): e1014032. https://doi.org/10.1371/journal.ppat.1014032

Editor: Emily Hemann, The Ohio State University, UNITED STATES OF AMERICA

Received: June 5, 2025; Accepted: February 24, 2026; Published: March 3, 2026

Copyright: © 2026 Chang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The data that support the findings of this study are available within the article or the supporting information. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org/?pxid=PXD073631) via the iProX partner repository [DOI: https://doi.org/10.1093/nar/gky869 and https://doi.org/10.1093/nar/gkab1081] with the dataset identifier PXD073631. The RNA sequencing data have been deposited in NCBI under the accession code PRJNA1416578, http://www.ncbi.nlm.nih.gov/bioproject/1416578.

Funding: This work was supported by the National Natural Science Foundation of China (grant No. 32373000 to PLH), the Shandong Provincial Key Research and Development Program (grant No. 2022CXGC020711-2 to HMW), the Jinan Innovation Team (grant No. 202228060 to HBH), and the Jinan Research Pioneer Workshop (grant No. 202333059 to HMW). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

The innate immune response mediated by interferons plays a crucial role in controlling viral infections through the action of downstream interferon-stimulated genes (ISGs) [1]. When encountering viral infections, pattern-recognition receptors (PRRs) identify pathogen-associated molecular patterns (PAMPs), initiating the production of IFN-I via downstream signaling pathways. Viral RNA is identified by RIG-I-like receptors (RLRs), encompassing retinoic acid-inducible gene I (RIG-I), Melanoma Differentiation-Associated Protein 5 (MDA5), and Laboratory of Genetics and Physiology 2 (LGP2), culminating in MAVS-IRF3/IRF7-mediated transcription of specific genes and the generation of IFN-I [2,3].

IRF7 can be phosphorylated and activated through both the TBK1/IKKε and TRIF-dependent pathways, which are downstream of cytoplasmic RNA sensors [4]. Once activated, IRF7 translocates to the nucleus, forms dimers, and initiates transcriptional activation, promoting the expression of IFN-α and IFN-β [5]. As a crucial enhancer in the induction of IFN-I genes downstream of PRRs, the positive feedback loop involving IRF7-mediated IFN-I ensures the sustained production of substantial amounts of IFN-I. This robust response effectively combats infections caused by pathogenic microorganisms. Nevertheless, overactive immune responses can lead to severe pathological consequences, including the onset of autoimmune disorders [6]. Therefore, the signaling transduction of IFN-I requires precise modulation to prevent immune dysregulation and maintain physiological balance. The regulation of IRF7 is governed by a complex network of mechanisms, which can either amplify or attenuate the IFN-I signaling cascade. For instance, the neutral E3 ubiquitin-protein ligase 3 (NEURL3) facilitates K63-linked ubiquitination of IRF7 to enhance the innate antiviral response [7]. HSP70 exerts a negative regulation of IRF7 phosphorylation, leading to a reduction in IFN-I signaling [8]. The p200 family protein IFI204 is involved in the negative regulation of the IRF7-mediated IFN-I response following RNA virus infection by inhibiting the binding of IRF7 to its specific promoter [9]. However, the elaborate regulatory mechanisms of IRF7 still need to be investigated.

Protein phosphatases and kinases play a pivotal role in governing various cellular processes such as proliferation, differentiation, and stress responses [10]. They achieve this regulation by controlling reversible protein phosphorylation, which is a crucial post-translational modification [11]. PPM1G is a member of the PP2C family of serine/threonine (Ser/Thr) protein phosphatases [12]. Reported functions of PPM1G include regulating protein translation and cell growth by dephosphorylating 4E-binding protein 1 [13], participating in the exchange and dephosphorylation of H2A-H2B [14], and being involved in the dephosphorylation of MAVS/STING [15]. Nonetheless, it is yet to be determined whether PPM1G targets other innate immune proteins to modulate IFN-I signaling.

CD97 is a member of the Adhesion G protein-coupled receptors (AGPCR) family. We previously reported that CD97 negatively regulates RIG-I-mediated innate immune signaling in response to RNA viruses [16]. However, it remains unclear whether CD97 influences innate immunity through other mechanisms. Here, we demonstrated that CD97 signaling establishes a negative feedback loop, restraining IRF7-mediated IFN-I expression in a PPM1G-dependent manner, potentially aiding viruses in evading immune elimination. Our findings also offer mechanistic insight into the CD97-PPM1G axis-mediated posttranslational modification of IRF7, shedding light on its role in negatively regulating the IFN-I response.

Results

CD97 enhances the replication of BEFV, SeV, and H1N1

Our previous finding that CD97 promotes the replication of VSV and SARS-CoV-2 has led us to further investigate the broader impact and mechanistic role of CD97 in the replication of diverse RNA viruses [16]. To evaluate the impact of CD97 on Bovine Ephemeral Fever Virus (BEFV) replication, we generated Baby Hamster Syrian Kidney (BHK-21) cells overexpressing CD97, as confirmed by immunoblot analysis (Fig 1A). Upon BEFV infection in BHK-21 cells, the overexpression of CD97 led to a marked increase in the expression of BEFV nucleoprotein (N) compared to cells constructed with empty vector (EV) control (Fig 1B). Indeed, overexpression of CD97 significantly elevated the viral titer of BEFV at both 24 hours post-infection (hpi) and 48 hpi (Fig 1C). In contrast, knockdown of CD97 in BHK-21 cells results in a dramatic decrease in BEFV replication, as evidenced by reduced expression of BEFV N and viral titers (Fig 1D-1F). Furthermore, we generated A549 cells overexpressing CD97 to investigate the replication of Sendai virus (SeV) and Influenza A subtype H1N1 (Fig 1G). The results revealed that elevating CD97 expression led to an increase in SeV P and H1N1 M mRNA transcript (Fig 1H and 1I), suggesting that CD97 promotes SeV and H1N1 replication. As anticipated, silencing CD97 in A549 cells suppressed the replication of both viruses (Fig 1J-1L). Collectively, these findings suggest that CD97 facilitates the replication of BEFV, SeV, and H1N1.

Download:

Fig 1. CD97 promotes the replication of BEFV, SeV, and H1N1.

(A) The establishment of a BHK-21 cell line stably expressing Flag-tagged CD97 was achieved. Stable overexpression of CD97 was confirmed by immunoblotting with Flag and CD97 antibodies. (B and C) BEFV-N levels and viral titers in BHK-21 cells overexpressing CD97 were determined by immunoblot and TCID₅₀. The cells were either mock-treated or infected with BEFV (MOI 0.1) for the indicated times. (D) CD97 knockdown was assessed by transfecting siRNA into BHK-21 cells. (E and F) BEFV-N levels and viral titers in BHK-21 cells with CD97 knockdown were measured by immunoblot and TCID₅₀. The cells were either mock-treated or infected with BEFV (MOI 0.1) for the indicated times. (G) A549 cells stably expressing Flag-tagged CD97 were established. (H and I) RT–qPCR analysis was performed to measure SeV P and H1N1 M transcripts in A549 cells overexpressing CD97, following infection with SeV (MOI 0.1) and H1N1(MOI 0.1) for 36 h. (J) CD97 knockdown was assessed by transfecting siRNA into A549 cells. (K and L) RT‒qPCR analysis was conducted to measure SeV P and H1N1 M transcripts in A549 cells with CD97 knockdown, following infection with SeV (MOI 0.1) and H1N1 (MOI 0.1) for 36 h. The results show the mean ± SEM from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data in (A, B, D, E, G, J) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.g001

CD97 attenuates the IFN-I response triggered by BEFV, SeV, and H1N1

As CD97 plays a role in virus replication, we investigated its involvement in the antiviral IFN-I response induced by BEFV, SeV, and H1N1. Following BEFV infection in CD97-overexpressing BHK-21 cells, IFN-β and ISG15 mRNA transcripts were diminished, whereas the knockdown of CD97 elevated their levels (Fig 2A and 2B). Similar outcomes were evident in H1N1-infected A549 cells (Fig 2C and 2D). To further confirm our findings, primary macrophages (PMs) were isolated from CD97-deficient (CD97^-/-) mice and wild-type (CD97^+/+) mice to investigate the IFN-I response. Flow cytometry was employed to confirm the CD97 deficiency (S1 Fig). As anticipated, CD97^-/- PMs exhibited increased IFN-β and ISG15 mRNA transcripts induced by BEFV (Fig 2E). Importantly, CD97^-/- PMs exhibited elevated IFN-β secretion, as detected by ELISA (Fig 2F). Likewise, CD97^-/- PMs presented a heightened IFN-I response induced by SeV (Fig 2G and 2H). These results were also demonstrated in bone marrow-derived macrophages (BMDM) (S2 Fig). Taken together, these findings suggest that CD97 negatively regulates the IFN-I response induced by BEFV, SeV, and H1N1.

Download:

Fig 2. CD97 dampens the IFN-I signaling response induced by BEFV, H1N1, and SeV.

(A and B) RT‒qPCR analysis of IFN-β and ISG15 transcripts was performed in (A) CD97-overexpressing BHK-21 cells and (B) BHK-21 cells with CD97 knockdown, both infected with either mock or BEFV (MOI 0.1) for 24 hours. (C) CD97-overexpressing A549 cells and (D) A549 cells with CD97 knockdown were analyzed after infection with H1N1 (MOI 0.1) for 36 hours. (E) RT‒qPCR analysis of IFN-β and ISG15 transcripts in CD97^-/- PMs that were either mock-treated or infected with BEFV (MOI 0.1) for 24 hours. (F) ELISA analysis of IFN-β in cellular supernatants under the same conditions as described in (E). (G and H) RT‒qPCR and ELISA analyses were conducted following infection with SeV (MOI 0.1) for 36 hours. The results show the mean ± SEM from three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

https://doi.org/10.1371/journal.ppat.1014032.g002

CD97 interacts with the protein phosphatase PPM1G

To delve deeper into the mechanisms underlying CD97 involvement in the antiviral IFN-I response, we employed immunoprecipitation coupled with mass spectrometry (IP-MS) to identify the proteins that interact with CD97. A schematic overview of the IP-MS approach is illustrated in Fig 3A. A total of 295 proteins were identified as interacting with CD97. Of these, 103 were exclusive to the CD97 interactome (S1 Data), while those in the EV interactome were considered nonspecific bindings (Fig 3B). Furthermore, the biological processes associated with CD97-interacting proteins were analyzed using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The GO analysis identified biological processes involved in protein-containing complex organization, protein import into the nucleus, and post-transcriptional regulation of gene expression (Fig 3C). Meanwhile, KEGG analysis identified processes including Human T-cell leukemia virus 1 infection, nucleocytoplasmic transport and Coronavirus disease- COVID-19 (Fig 3D). These data indicated that CD97 regulates viral replication through complex mechanisms.

Download:

Fig 3. CD97 interacts with PPM1G via arginine residues 819 and 822.

(A) Flow chart showing mass spectrometry. The schematic diagram was created in BioRender. Chang, H. (2026) https://BioRender.com/9yrgpl8. (B) The pie chart shows the protein differences between CD97 and EV groups. (C and D) The bubble chart shows of the GO and KEGG analysis analyses. (E and F) Co-IP analysis showing the interaction between CD97 and PPM1G after transfection for 36 hours with the indicated plasmids. (G) The interaction between truncated mutants of CD97 and PPM1G was performed using Co-IP. (H) Comparison of CD97 C-terminal sequences across different species. (I) Co-IP analysis of the interaction between the arginine residues mutant of CD97 and PPM1G. Data in (E-G, I) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.g003

We hypothesize that some of the 103 proteins interacting with CD97 may regulate IFN-I signaling, with PPM1G potentially mediating the dephosphorylation of key immune proteins in this pathway. To confirm the interaction between CD97 and PPM1G, HEK-293T cells were transfected with Flag-CD97 and HA-PPM1G plasmids, followed by a Co-immunoprecipitation (Co-IP) assay. The results showed that CD97 interacted with PPM1G, as demonstrated using either HA or Flag magnetic beads (Fig 3E and 3F). The endogenous interaction was further confirmed using specific antibody (S3A Fig). CD97 was mutated at the C-terminal and N-terminal regions as previously described [16]. Co-IP results showed that the C-terminal, but not N-terminal, of CD97 interacted with PPM1G (Fig 3G). The C-terminus of CD97 contains intracellular domains, the sequences of which were analyzed through cross-species comparisons (Fig 3H). We found that the sequence in the intracellular region is conserved. Previous studies indicated that the PRR motif of G protein-coupled receptors serves as an important binding site for intracellular signaling molecules [17]. To assess the interaction with PPM1G, arginine (R) residues at positions 819 and 822 of human CD97 were mutated to alanine (A), which resulted in a significant decrease in interaction with PPM1G, as observed in the Co-IP assay (Fig 3I). This suggests that Arg-819 and Arg-822 residues of CD97 are crucial for binding PPM1G. Taken together, these data suggest that PPM1G may be involved in the CD97-regulated antiviral innate immune response.

CD97 enhances virus replication through PPM1G-mediated downregulation of IFN-I

To investigate the mechanism of PPM1G involvement, we generated PPM1G-overexpressing BHK-21 cells to examine its role in the antiviral IFN-I response. Successful overexpression of PPM1G was confirmed by immunoblot analysis (Fig 4A). As anticipated, the overexpression of PPM1G suppressed IFN-β mRNA expression while increasing viral titers (Fig 4B and 4C). In contrast, PPM1G knockout promoted IFN-β mRNA levels and inhibited viral replication (Fig 4D-4F).

Download:

Fig 4. CD97 facilitates virus replication through PPM1G-mediated downregulation of IFN-I.

(A) BHK-21 cells stably expressing HA-tagged PPM1G was successfully established. Stable overexpression of PPM1G was confirmed through immunoblotting using Flag and PPM1G antibodies. (B) RT‒qPCR analysis of IFN-β transcripts in HeLa cells overexpressing PPM1G that were either mock-treated or infected with BEFV (MOI 0.1) for 24 hours. (C) TCID₅₀ analysis of BEFV was performed to determine the virus titer in BHK-21 cells overexpressing PPM1G. (D) Immunoblotting analysis was conducted on BHK-21 cells with established PPM1G knockout. (E) RT‒qPCR analysis of IFN-β transcripts following PPM1G knockout. (F) Viral titer detection in BHK-21 cells with PPM1G knockout. (G and H) BEFV N levels and viral titers were analyzed by immunoblotting and TCID₅₀ in CD97-overexpressing BHK-21 cells with PPM1G knockout. (I) HeLa cell line stably expressing HA-tagged PPM1G was confirmed through immunoblotting using Flag and PPM1G antibodies. (J) RT‒qPCR analysis of IFN-β transcripts in HeLa cells overexpressing PPM1G that were either mock-treated or infected with VSV (MOI 0.1) for 12 hours. (K) TCID₅₀ analysis was performed to determine the virus titer in HeLa cells overexpressing PPM1G. (L) Immunoblotting analysis was conducted on HeLa cells with established PPM1G knockout. (M) RT‒qPCR analysis of IFN-β transcripts following PPM1G knockout. (N) Viral titer detection in HeLa cells with PPM1G knockout. (O and P) VSV G levels and viral titers were analyzed by immunoblotting and TCID₅₀ in CD97-overexpressing HeLa cells with PPM1G knockout. The results show the mean ± SEM from three independent experiments. ns, not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data in (A, D, G, I, O, L) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.g004

Given the CD97-PPM1G interaction, we investigated whether CD97 suppresses IFN-I signaling via PPM1G to facilitate viral replication. Accordingly, we assessed BEFV replication in CD97-overexpressing PPM1G-knockout cells. The results showed that CD97 failed to significantly promote BEFV replication following PPM1G knockout, as evidenced by BEFV G expression levels and viral titers (Fig 4G and 4H). Similarly, we also generated PPM1G-overexpressing and PPM1G-knockout HeLa cells to assess VSV replication (Fig 4I and 4L). Consistent results demonstrated that CD97 promotes VSV replication by inhibiting IFN-I signaling through PPM1G (Fig 4J-4P). Collectively, these findings demonstrate that CD97 facilitates viral replication by suppressing IFN-I signaling through PPM1G.

CD97 promotes PPM1G-mediated dephosphorylation of IRF7

Since PPM1G suppresses IFN-I signaling, we speculated that it targets and negatively regulates a key adaptor in the innate immune pathway. To test our hypothesis, we performed Co-IP to investigate the interaction between PPM1G and key adaptor proteins in the IFN-I signaling pathway. The results demonstrated that PPM1G strongly interacts with IRF3 and IRF7, has a weak interaction with MAVS as previously reported [15], and shows no interaction with TBK1 (Fig 5A). To eliminate nonspecific interactions, we employed Co-IP with both HA magnetic beads and a PPM1G antibody to further validate the interactions between PPM1G and IRF7/IRF3. The results provided additional evidence supporting their interaction (Figs 5B, 5C, and S3A). Importantly, we discovered a direct interaction between PPM1G and IRF7/3 through GST-pull down assays (Fig 5D and 5E). Based on the characteristics of PPM1G, we hypothesized that it dephosphorylates IRF7 or IRF3. To validate our hypothesis, we evaluated the phosphorylation levels of IRF3 and IRF7 following the overexpression of PPM1G. The results demonstrated that PPM1G decreases the phosphorylation of IRF7 in a dose-dependent manner, but does not affect IRF3 phosphorylation during BEFV infection (Fig 5F). Moreover, PPM1G significantly suppressed IRF7 phosphorylation at 12 and 24 h post-infection with BEFV but had no significant effect on IRF3 or NF-κB p65 phosphorylation (S3B Fig). These findings indicate that PPM1G binds to IRF7 and suppresses its phosphorylation triggered by viral infection.

Download:

Fig 5. CD97 enhances the ability of PPM1G to dephosphorylate IRF7.

(A) Co-IP analysis showing the interaction between PPM1G and adaptors (MAVS, TBK1, IRF3, and IRF7) following transfection with the indicated plasmids and using Flag magnetic beads in HEK-293T cells. (B and C) Reverse validation of the interaction between PPM1G and IRF7/IRF3 was performed using Co-IP with HA magnetic beads. (D and E) GST pull-down analysis of the direct interaction between PPM1G and IRF7/IRF3. (F) BHK-21 cells were transfected with different doses of PPM1G (0.6, 1.2, 1.8 µg per 2.5mL in a 6-well plate) after BEFV infection 24 h to analyze the phosphorylation of IRF7/IRF3 by immunoblotting. (G) HEK-293T cells were transfected with PPM1G and its enzymatically inactive mutant (D496A) to assess the phosphorylation of IRF7/IRF3 following IKKε transfection. (H-K) Dual-luciferase reporter assays were used to evaluate the impact of (H and I) IRF7-induced or (J and K) IRF3-induced promoter activity of IFN-β and ISRE in the presence of PPM1G and its D496A mutant. This was achieved through the transfection of pRL-TK, pIFNβ-Luc, pISRE-Luc, and the indicated plasmids. The results show the mean ± SEM from three independent experiments. ns, not significant, *, p < 0.05; **, p < 0.01; ***, p < 0.001. (L) The synergistic effect of CD97 and PPM1G on IRF7 phosphorylation during VSV infection was detected by immunoblot. (M) The binding of endogenous PPM1G to IRF7 in HeLa cells infected with VSV was determined by immunoprecipitation using anti-PPM1G (or an IgG isotype control). (N) The effect of CD97 overexpression on the phosphorylation and translocation of IRF7 was evaluated through nuclear and cytoplasmic separation. Data in (A-G, L-N) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.g005

Previous studies have shown that the aspartic acid residue at position 496 is essential for its dephosphorylation activity [15]. We generated the D496A mutant of PPM1G to examine its impact on IRF7 phosphorylation and found that, unlike the wild-type PPM1G, the PPM1G-D496A mutant failed to suppress IKKε-induced IRF7 phosphorylation (Fig 5G). This result suggests that PPM1G decreases IRF7 phosphorylation through its enzymatic activity. Moreover, we evaluated the effect of PPM1G and PPM1G-D496A on IRF7- and IRF3-induced IFN-β and Interferon-Stimulated Response Element (ISRE) promoter activities. The results indicated that PPM1G inhibits the activity of the IFN-β and ISRE promoters induced by IRF7, whereas PPM1G-D496A does not affect these promoters (Fig 5H and 5I). However, neither PPM1G nor PPM1G (D496A) had any effect on the IFN-β and ISRE promoter activities induced by IRF3 (Fig 5J and 5K). To further prove the relevance of PPM1G activity, we analyzed its effect on constitutively active phosphomimetic IRF7/3. The results showed that PPM1G did not inhibit the activation of the IFN-β and ISRE promoters by IRF7/3(5D) (S3C-S3F Fig). These data demonstrate that PPM1G specifically inhibits IRF7 phosphorylation.

To further verify the role of CD97 in PPM1G-mediated IRF7 dephosphorylation, we performed co-transfection experiments with CD97 and PPM1G. The results demonstrated that co-expression of CD97 and PPM1G synergistically suppressed IRF7 phosphorylation during VSV infection (Fig 5L). Overexpression of CD97 failed to inhibit IRF7 phosphorylation upon PPM1G knockout (S4A and S4B Fig), confirming that CD97 suppresses IRF7 phosphorylation via PPM1G. Using co-immunoprecipitation with differentially tagged IRF7/3, we further confirmed that the inhibition of IRF7, but not IRF3, dimerization by CD97 is dependent on PPM1G (S4C and S4D Fig). Furthermore, in line with the observation that CD97 knockout promotes IFN-I production in BMDMs, we confirmed that CD97 knockout also enhances IRF7 phosphorylation (S4E Fig). We also assessed whether CD97 influences the interaction between PPM1G and IRF7. Overexpression of CD97 was found to enhance the binding between PPM1G and IRF7 (Fig 5M). Since phosphorylated IRF7 translocates to the nucleus to initiate IFN-β transcription, we next examined the impact of CD97 on this process using a nucleocytoplasmic separation assay. The results revealed that CD97 overexpression markedly reduced the nuclear translocation of phosphorylated IRF7 following VSV infection, without affecting the distribution of total IRF7 (Fig 5N). Collectively, these findings suggest that CD97 promotes PPM1G-mediated dephosphorylation of IRF7, thereby suppressing the IFN-I response.

CD97 promotes the transcription of PPM1G through IKZF1

To investigate whether CD97 regulates PPM1G through distinct mechanisms, we first examined PPM1G transcription following CD97 overexpression. The results showed that CD97 overexpression significantly increased the mRNA levels of PPM1G (Fig 6A). Similarly, PPM1G protein expression was elevated under both mock and BEFV infection conditions upon CD97 overexpression (Fig 6B). Conversely, CD97 knockout suppressed PPM1G expression in both HeLa cells and PMs (Fig 6C and 6D), suggesting that CD97 promotes PPM1G transcription. To identify the transcription factors involved in PPM1G regulation, we constructed a luciferase reporter driven 2000 bp fragment of the human PPM1G promoter. Five truncated promoter variants (designated M1 to M5) were generated and their luciferase activities evaluated in HEK-293T cells (Fig 6E). The results indicated that promoters M1, M2, and M5 exhibited high luciferase activity, suggesting that the key regulatory region is located between positions -1223 bp and -639 bp (Fig 6F).

Download:

Fig 6. CD97 enhances the transcription of PPM1G via IKZF1.

(A) RT‒qPCR analysis of PPM1G transcripts in BHK-21 cells overexpressing CD97 following BEFV infection. (B) Immunoblotting analysis of PPM1G expression in BHK-21 cells either mock-treated or infected with BEFV. (C and D) The expression of PPM1G was analyzed in CD97 knockout HeLa cells and (D) PMs, either mock-treated or infected with VSV. (E) PPM1G promoter truncation mutant (M1 to M5) model. (F) Various truncated PPM1G promoter constructs and a Renilla luciferase reporter vector were transfected into HEK-293T cells, and then analyzed for dual luciferase activity. (G) A heatmap of transcriptome analysis for samples with CD97 overexpression and BEFV infection in BHK-21 cells. (H) Model showing the interaction site between the indicated transcription factors and the PPM1G promotor region. (I and J) The indicated protein expression was analyzed in BHK-21 cells overexpressing CD97, either mock-treated or infected with BEFV or VSV. (K) PPM1G-M5 promoter constructs and IKZF1, along with a Renilla luciferase reporter vector, were transfected into HEK-293T cells, followed by analysis of dual luciferase activity. (L and M) RT‒qPCR and immunoblotting analysis of PPM1G expression were performed in HeLa cells overexpressing IKZF1 following VSV infection. (N) RT‒qPCR analysis of PPM1G mRNA transcripts was conducted in CD97-overexpressing HeLa cells with IKZF1 silencing. Data in (B-D, I, J, M) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.g006

We next performed transcriptomic analysis in CD97-overexpressing cells (Fig 6G) and predicted potential transcription factor binding sites within the PPM1G promoter using the JASPAR vertebrate database (Fig 6H). By integrating these datasets, five candidate transcription factors were identified. Among them, only IKZF1 expression was significantly upregulated by CD97 overexpression under both BEFV and VSV infection conditions, while the expression of SOX10, NEUROD1, CTF1, and Cdx2 remained unchanged (Fig 6I and 6J). Further analysis showed that IKZF1 overexpression enhanced the luciferase activity of the PPM1G-M5 promoter construct (Fig 6K), and increased both the mRNA and protein levels of PPM1G during VSV infection (Fig 6L and 6M). To evaluate whether CD97 regulates PPM1G transcription through IKZF1, we silenced IKZF1 expression using siRNA [18]. The results demonstrated that CD97 could no longer significantly upregulate PPM1G mRNA levels upon IKZF1 knockdown (Fig 6N). Collectively, these findings suggest that CD97 promotes PPM1G transcription via the transcription factor IKZF1.

CD97 deficiency in mice are more resistant to viral infections in vivo

To further investigate the role of CD97 in host defense against viral infection in vivo, we administered nasal infections with H1N1 to both CD97^-/- and CD97^+/+ mice. RT‒qPCR assays showed that CD97 deficiency significantly reduced H1N1 M mRNA levels while increasing IFN-β mRNA levels in lung and nasal turbinate tissues (Fig 7A-7D). H&E staining of the lungs following H1N1 infection showed that CD97^-/- mice exhibited less thickening of alveolar walls compared to CD97^+/+ mice (Fig 7E). Additionally, we observed that CD97^+/+ mice began to lose weight on day 2, reaching a minimum by day 9, whereas CD97^-/- mice experienced almost no weight loss following low-dose H1N1 infection (Fig 7F). When the infection dose was increased, the results revealed that CD97^-/- mice exhibited significantly improved survival compared to CD97^+/+ mice, as shown by the survival assay (Fig 7G). Collectively, these findings demonstrate that CD97^-/- mice exhibit elevated IFN-I levels, which contribute to the attenuation of viral replication in vivo.

Download:

Fig 7. CD97-deficient mice are more resistant to viral infections in vivo.

(A-D) RT‒qPCR analysis of IFN-β and H1N1 M in lungs and nasal turbinates of CD97^+/+ and CD97^-/- mice that were administered 10 MLD₅₀ of H1N1 via nasal inhalation and analyzed after 3 days. The results show the mean ± SEM from three independent experiments. ns, not significant; N.D., Not Detected; ***, p < 0.001. (E) H&E staining of lung sections of CD97^+/+ and CD97^-/- mice treated with 10 MLD₅₀ of H1N1 via nasal inhalation conducted after 6 days. Scale bars: 50 µm. (F) Body weight measurements of CD97^+/+ and CD97^-/- mice (n = 5) were recorded over 12 days following administration 10 MLD₅₀ of H1N1 via nasal inhalation. (G) Survival curve analysis of CD97^+/+ and CD97^-/- mice (n = 5) was performed over 14 days following administered 50 MLD₅₀ of H1N1 via nasal inhalation.

https://doi.org/10.1371/journal.ppat.1014032.g007

The compound SANG attenuates viral replication by suppressing CD97 expression

To further validate the role of CD97 on viral replication, we employed the compound SANG to investigate its impact on CD97-mediated viral replication, as previously described [16]. The results showed that SANG significantly reduced the expression of CD97 at concentrations of 1 µM and 2 µM in A549 cells (Fig 8A). A549 cells were then infected with H1N1 and SeV, followed by treatment with 1 µM SANG. The results indicated that SANG inhibited the mRNA levels of H1N1 M and SeV P. (Fig 8B and 8C). Moreover, we examined the IFN-I response and IRF7 phosphorylation following SANG treatment. As expected, SANG decreased viral protein and CD97/PPM1G expression, and enhanced IRF7 phosphorylation upon SeV and BEFV infection (S5A and S5B Fig). Consistently, SANG also promoted the IFN-I response upon viral infection (S5C and S5D Fig). Most importantly, SANG improved by approximately 20% survival in CD97^+/+ mice but did not further increase survival in CD97^-/- mice (Fig 8D and 8E). Taken together, these data suggest that SANG inhibits viral replication by suppressing the expression of CD97.

Download:

Fig 8. SANG inhibits viral replication by suppressing the expression of CD97 (A) Immunoblot analysis of CD97 treated with SANG (0-2 μM) (B and C) RT–qPCR analysis of H1N1 M and SeV P mRNA in A549 cells treated with DMSO (NC) and SANG (1μM) after 36 hours of H1N1 or SeV infection.

The results show the mean ± SEM from three independent experiments. *, p < 0.05; **, p < 0.01. (D and E) Survival curve analysis of CD97^+/+ and CD97^-/- mice (n = 5) administered 50 MLD₅₀ of H1N1 via nasal inhalation, and treated with SANG (30mg/kg) intragastrically for 14 days. Data in (A) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.g008

Discussion

To maintain effective antiviral immunity while preventing autoimmune pathology, organisms require precise regulation of innate immune responses to balance immune activation and suppression [19]. IRF7, a master transcriptional regulator of IFN-I signaling, undergoes phosphorylation via upstream innate immune pathways [20]. In this study, we identified CD97 as a critical negative regulator of IRF7-mediated IFN-I signaling (Fig 9). Our findings demonstrate that the protein phosphatase PPM1G specifically dephosphorylates IRF7, consequently suppressing IFN-I production and facilitating viral replication. CD97 overexpression promoted PPM1G recruitment to enhance IRF7 dephosphorylation. Furthermore, CD97 upregulated PPM1G expression through activation of the transcription factor IKZF1, resulting in sustained IRF7 inactivation.

Download:

Fig 9. The model depicting the mechanism by which CD97 attenuates the antiviral IFN-I response via PPM1G.

Upon viral infection, sensors such as RIG-I recognize viral RNA and activate the MAVS signaling pathway, leading to IRF7 phosphorylation. PPM1G interacts with and dephosphorylates IRF7 to inhibit the antiviral IFN-I signaling response. This function is modest in the absence of CD97. However, CD97 overexpression enhances the interaction between PPM1G and IRF7, leading to significant dephosphorylation of IRF7, which suppresses IFN-I signaling and promotes viral replication. Additionally, CD97 increases the transcription of the PPM1G through transcription factor IKZF1, resulting in higher levels of PPM1G expression. Furthermore, the compound SANG inhibits virus replication by suppressing CD97. The schematic diagram was created in BioRender. Chang, H. (2026) https://BioRender.com/i8aztqm.

https://doi.org/10.1371/journal.ppat.1014032.g009

Protein dephosphorylation is essential for the termination of signaling, with protein phosphatases serving as the key enzymes responsible for removing phosphate groups from target proteins [21]. Innate immune signaling can be regulated by protein phosphatases through the dephosphorylation of immune adaptor proteins. For example, PPM1B serves as a negative regulator of antiviral innate immunity by directly dephosphorylating TBK1, thereby suppressing its activity and downstream signaling [22]; PPP6C-mediated dephosphorylation of STING serves as a critical regulatory mechanism to limit excessive STING-dependent cytokine production, thereby preventing potential autoimmune pathogenesis [23]; PP1α, PP1β, and PP1γ subunits inhibit IRF7 phosphorylation triggered by Newcastle disease virus infection [24]. In this study, we demonstrated that PPM1G directly interacts with IRF7 and suppresses its phosphorylation triggered by various viruses, which facilitate viral replication by dampening the IFN-I signaling response. Our findings provide novel mechanistic insights into the regulatory role of protein phosphatases in IRF7-mediated innate immune responses.

Phosphorylation constitutes the primary activation mechanism for IRF7, governing its nuclear translocation, dimerization, and transcriptional activity in IFN-I responses. Studies have reported that protein kinases such as TBK1 [25], IKKε [26], IRAK1 [27], and IKKα [28] are the key kinases for catalyzing the phosphorylation of IRF7. However, the mechanisms underlying the negative regulation of IRF7 have remained largely unclear. Our results demonstrate that CD97 facilitates the recruitment of PPM1G to suppress IRF7 phosphorylation triggered by various viruses, thereby downregulating IFN-I signaling. In addition, the TLR3 agonist poly(I:C), added to the cell culture medium, was used to assess IRF7 activation via alternative pathways. We found that the CD97-PPM1G axis consistently promoted IRF7 dephosphorylation (S6 Fig). This suggests that the influence of CD97-PPM1G on IRF7 may not be limited to the IFN-I pathway, warranting further investigation. Although our previous findings showed that CD97 suppresses RIG-I-mediated innate immune response [16], the promotion of viral replication by CD97 significantly decreased following PPM1G knockout, suggesting that CD97 modulates IFN-I signaling through multiple mechanisms.

CD97 interacts with various cellular molecules via its cellular domain to regulate tumorigenic processes [29] and cell communication [30]. However, the interaction between CD97 and protein phosphatase in regulating targeted molecules remains obscure. GPR54, another GPCR, recruits calcineurin to dephosphorylate TBK1, thereby negatively regulating virus-induced IFN-I signaling [31]. Additionally, GRP54 interacts with PP2A through the PRR motif in its C-terminal domain [17]. We identified R819 and R822 as critical conserved residues mediating the specific interaction between CD97 and protein phosphatase PPM1G. These findings suggest that CD97, analogous to GPR54’s recruitment of calcineurin, recruits PPM1G to dephosphorylate IRF7. This function could be mediated by CD97 on the plasma membrane or in its internalized form. As previously reported, PPM1G, while primarily nuclear, exhibits some cytoplasmic signal [13,32]. This observation suggests that its recruitment likely involves translocation to the cytoplasm induced by viral infection or IFN-I signaling. This nucleo-cytoplasmic shuttling behavior is shared by other phosphatases, such as PP2A [33] and Pez [34]. These proposed mechanisms warrant further investigation.

Notably, CD97 was also found to promote the mRNA transcription of PPM1G. We used a CD97(R819/822A) mutant to assess IFN-I signaling. Compared to wild-type CD97, the mutant significantly abrogated the inhibition of IFN-I signaling (S7A and S7B Fig). However, it still promoted PPM1G expression to a similar extent (S7C Fig). These results suggest that the recruitment of PPM1G by CD97 is essential for IFN-I inhibition. Through RNA sequencing and prediction analyses, we identified IKZF1 as the transcription factor regulating PPM1G, with this process being mediated by CD97. Our study provides the first evidence that CD97 modulates the IKZF1-PPM1G-IRF7 regulatory axis to control antiviral IFN-I signaling. These findings establish a novel mechanism by which CD97 coordinates with protein phosphatases to fine-tune innate immune responses. Furthermore, we observed that viral infection downregulates CD97 expression at a later stage, suggesting a host-driven feedback strategy. We propose that viral infection or ligand [30] may activate CD97 at both the mRNA and protein levels. A feedback mechanism may then function to balance the immune response by suppressing low-level IFN-I signaling, the precise regulation of which warrants further investigation.

In summary, our study establishes PPM1G as a critical checkpoint in IFN-I signaling that negatively regulates IRF7 activation. We elucidated a novel immunoregulatory axis between the CD97 and PPM1G, demonstrating their coordinated control of antiviral responses. Using CD97-deficient mice and the compound SANG, we validated CD97’s immunomodulatory function, offering a promising avenue for the treatment of viral diseases and drug design.

Materials and methods

Ethics statement

All animal experimentation was conducted in strict accordance with the State Council-approved People’s Republic of China Regulations on the Administration of Experimental Animal Affairs (1 November 1988). Mice were handled in compliance with the animal use policies set by the Institutional Animal Care and Use Committee (IACUC) at Shandong Normal University (permission number: AEECSDNU2024074).

Cell lines, mice and viruses

The cell lines used in this study included human cervical carcinoma cell (HeLa), Baby Hamster Kidney (BHK-21), Human alveolar epithelial cells (A549), and Human embryonic kidney cells (HEK-293T). These cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Shandong Sparkjade Biotechnology, CA0004–500ML) within a humidified incubator at 37°C with 5% CO₂. The medium was enriched with 10% heat-inactivated fetal bovine serum (FBS; ExCell Bio, FSP500) and a penicillin-streptomycin solution (100 μg/mL; Beijing Labgic Technology, Bl505A).

B6;129P2-Adgre5tm1Dgen/JC57BL/6J mice, sourced from the Jackson Laboratory (JAX stock code 005788), were used in this study. Genotyping was performed according to a JAX-optimized protocol (28954). The BEFV and VSV strain utilized in this research was propagated in BHK-21 cells, while H1N1 and SeV strains were propagated in chicken embryos.

Antibodies and chemicals

Rabbit anti-Flag-Tag antibody (AB0028), Rabbit anti-HA-Tag antibody (AB0025), Rabbit anti-β-actin antibody (AB0061), Rabbit anti-p-IRF3 antibody (CY6575), Rabbit anti-IRF3 antibody (CY5779), Rabbit anti-IRF7 antibody (CY5645), Rabbit anti-VSV-G antibody (AB0053), Rabbit anti-PPM1G antibody (CY7286), Rabbit anti-GAPDH antibody (AB0038), Rabbit anti-LaminA/C antibody (CY9727), Rabbit anti-GST antibody (AB0055), Rabbit anti-IKZF1 antibody (CY8190), Rabbit anti-SOX10 antibody (CY5826), Rabbit anti-NEUROD1 antibody (CY6781), and Rabbit anti-Cdx2 antibody (CY5117) were purchased from Abways Biotechnology Co.,Ltd. Rabbit anti-p-IRF7 antibody (24129S) was purchased from Cell Signaling Technology. Rabbit anti-CD97 antibody (clone ERP4427; ab108368) was purchased from Abcam. Rabbit anti-CD97 antibody (66972–1-Ig) was purchased from Proteintech Group, Inc. Sanguinarine (HY-N0052) were purchased from MedChemExpress.

Generation of stable overexpressing and knockout cells

To generate CD97-overexpressing BHK-21 and A549 cells, as well as PPM1G-overexpressing HeLa cells, genes were synthesized from dsDNA templates of various species via PCR. Lentivirus packaging was performed to transfect the corresponding plasmids, following previously described protocols [16]. After three days, cells were selected using puromycin. The overexpression and knockout of the genes were confirmed through immunoblotting.

Viral infection

Six- to eight-week-old C57BL/6J mice were infected with H1N1 via intranasal injection, as previously described [35]. Mouse weight was monitored for 12 days following the intranasal injection of H1N1 (10 MLD₅₀), and survival was tracked for 14 days after intranasal injection of H1N1 (50 MLD₅₀). RT‒qPCR was conducted to assess the expression of IFN-β and H1N1 M in various organs. Additionally, samples from virus-infected mice were subjected to H&E staining at the specified time points.

siRNA silencing and RT–qPCR

siRNA was transfected into BHK-21 or A549 cells at a final concentration of 30 pM using the Attractene transfection reagent (301007; QIAGEN). The siRNA sequences used were provided by Beijing Tsingke Biotechnology Co., Ltd., and specific sequences are listed in S1 Table.

Following the method described by Chang et al. [36], RT–qPCR was employed to measure mRNA levels. After virus challenge, total RNA was extracted with the Total RNA Extraction Kit (AC0205-B, Shandong Sparkjade Biotechnology Co., Ltd). Complementary DNA (cDNA) was synthesized using the RT Mix Kit with gDNA Clean for qPCR Ver.2 (AG11728, Accurate Biology). mRNA expression levels were quantified with the ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme Biotech) and analyzed using the 2^-ΔΔCT method. Primer sequences for qPCR were designed with Primer Premier 6.0 software and are listed in S1 Table.

Immunoblotting and co-immunoprecipitation (Co-IP)

The specified plasmids transfected HEK-293T cells for 36 hours. Washing the cells with PBS, the cells were lysed in RIPA lysis buffer (New Cell & Molecular Biotech, WB3100) and then kept on ice for 20 minutes. Co-IP was performed using anti-HA-tag mAb-magnetic beads (M132-11, MBL Life Science) or anti-DDDDK-tag mAb-magnetic beads (M185-11R, MBL Life Science) at 4°C for 2 hours. For immunoprecipitation of endogenous PPM1G, magnetic beads coupled with specific commercially PPM1G antibody. Following immunoprecipitation, the beads were washed three times with lysis buffer. The immunoprecipitates were eluted using SDS loading buffer (Beyotime, P0015) and denatured at 95°C for 10 minutes. To reduce nonspecific binding, the blots were blocked with Tris-buffered saline-Tween (TBST) containing 5% nonfat dry milk. Primary antibodies specific to the proteins of interest were applied, and the membranes were washed. Enhanced chemiluminescence reagents (ED0015-C; Shandong Sparkjade Biotechnology) were used as per the manufacturer’s instructions to visualize the membranes, as previously described [37]. For a pull-down assay, purified GST-PPM1G or GST from Escherichia coli and attached to glutathione (GSH) agarose beads was incubated with HA-tagged IRF7/IRF3, which had been expressed in HEK-293T cells.

Dual-luciferase reporter assay

HEK-293T cells (2 × 10⁵) were seeded in 24-well plates and transfected with pRL-TK (10 ng; a Renilla luciferase plasmid), firefly luciferase (100 ng), and various concentrations of either control or protein-expressing plasmids. After 24 hours, the cells were harvested, and firefly and Renilla luciferase activities were measured using a dual-luciferase reporter assay system kit (DD1205-01; Vazyme Biotech) and a SpectraMax M5 microplate reader, following the manufacturer’s instructions. The relative fluorescence intensity of firefly luciferase was normalized to that of Renilla luciferase.

Nuclear and cytoplasmic extraction (NE-PER)

NE-PER analysis was conducted using NE-PERTM kits (YB370765) purchased from Thermo Fisher Scientific. Briefly, cells were lysed with cold CER reagents, and the cytoplasmic supernatant was obtained through centrifugation. The precipitate was then resuspended in cold NEB reagents for further lysis and centrifuged to isolate the nucleus. Both the cytoplasmic and nuclear fractions were subsequently boiled for immunoblot analysis.

Quantification and statistical analysis

Statistical analysis was performed using GraphPad Prism software (version 8.0; GraphPad, San Diego, CA, USA). Two-way analysis of variance (ANOVA) was used to assess statistical significance, while one-way ANOVA was employed for comparisons among multiple samples. Results are presented as means with standard error of the mean (SEM) and are representative of at least three independent experiments. A p value of 0.05 or less was considered statistically significant.

Supporting information

S1 Fig. Flow cytometric verification of CD97 knockout in PMs.

PMs from two wild-type and two CD97 knockout mice were analyzed. After gating on live (FSC/SSC) and single cells (FSC-A/FSC-H), the absence of CD97 expression in KO cells was confirmed compared to WT controls, using an isotype-matched antibody for gating.

https://doi.org/10.1371/journal.ppat.1014032.s001

(TIF)

S2 Fig. CD97 inhibits IFN-I signaling response induced by SeV and BEFV in BMDMs.

(A and B) RT–qPCR analysis of IFN-β and ISG15 transcripts in CD97^+/+ and CD97^-/- BMDMs. Cells were mock-treated or infected with BEFV (MOI 0.1) for 24 hours or SeV (MOI 0.1) for 36 hours. The results show the mean ± SEM from three independent experiments. ***, p < 0.001.

https://doi.org/10.1371/journal.ppat.1014032.s002

(TIF)

S3 Fig. PPM1G dephosphorylates IRF7.

(A) Endogenous co-immunoprecipitation and immunoblotting analysis with an PPM1G antibody. (B) immunoblotting analysis of the indicated protein in BHK-21 cells infected with BEFV for the specified durations. (C-F) Dual-luciferase reporter assays measuring the promoter activity of IFN-β (C, E) and ISRE (D, F) induced by the constitutively active mutants IRF7(5D) (C, D) or IRF3(5D) (E, F), in the presence or absence of PPM1G. The results show the mean ± SEM from three independent experiments. ns, not significant. Data in (A, B) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.s003

(TIF)

S4 Fig. CD97 facilitates the PPM1G-mediated dephosphorylation of IRF7.

(A, B) Immunoblotting analysis of indicated proteins in PPM1G-deficient (A) HeLa and (B) BHK-21 cells after infection with VSV or BEFV, respectively. (C, D) PPM1G-knockout HeLa cells were co-transfected with plasmids encoding Flag- and HA-tagged IRF7 (C) or IRF3 (D), together with a CD97 overexpression plasmid or control. Dimerization was assessed by co-IP following VSV infection. (E) Immunoblotting analysis of CD97^+/+ and CD97^-/- BMDMs that were mock-treated or infected with VSV. Data in (A-E) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.s004

(TIF)

S5 Fig. SANG potentiates the IFN-I signaling response.

(A, B) Immunoblotting analysis of indicated proteins in (A) HeLa and (B) BHK-21 cells following SANG treatment and infection with SeV or BEFV, respectively. (C, D) RT-qPCR analysis of IFN-β and ISG15 mRNA levels in cells treated under the same conditions as in (A) and (B), respectively. The results show the mean ± SEM from three independent experiments. ***, p < 0.001. Data in (A-B) are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.s005

(TIF)

S6 Fig. CD97 and PPM1G inhibits IRF7 phosphorylation induced by poly(I:C).

Immunoblotting analysis of IRF7 phosphorylation in control and CD97/PPM1G-overexpressing HeLa cells after stimulation with poly(I:C) for 12 h. Data are representative of three independent experiments.

https://doi.org/10.1371/journal.ppat.1014032.s006

(TIF)

S7 Fig. The impact of a CD97 mutant on IFN-I and PPM1G.

(A, B) RT-qPCR analysis of IFN-β and ISG15 mRNA level in A549 cells overexpressing wild-type CD97 or the CD97(R819/822A) mutant after infection with (A) H1N1 or (B) SeV. (C) RT-qPCR analysis of PPM1G mRNA in A549 cells overexpressing the indicated CD97 constructs. The results show the mean ± SEM from three independent experiments. **, p < 0.01; ***, p < 0.001; not significant.

https://doi.org/10.1371/journal.ppat.1014032.s007

(TIF)

S1 Table. Sequences used for qPCR and siRNA in this study.

https://doi.org/10.1371/journal.ppat.1014032.s008

(DOCX)

S1 Data. List of Candidate interactors of CD97 detected by mass spectrometry.

(XLSX)

Acknowledgments

https://doi.org/10.1371/journal.ppat.1014032.s009

We thank iProX [38,39] for depositing our proteomic data.

References

1. Chen K, Liu J, Cao X. Regulation of type I interferon signaling in immunity and inflammation: A comprehensive review. J Autoimmun. 2017;83:1–11. pmid:28330758
- View Article
- PubMed/NCBI
- Google Scholar
2. Barral PM, Sarkar D, Su Z, Barber GN, DeSalle R, Racaniello VR, et al. Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: key regulators of innate immunity. Pharmacol Ther. 2009;124(2):219–34. pmid:19615405
- View Article
- PubMed/NCBI
- Google Scholar
3. Chow KT, Gale M Jr, Loo Y-M. RIG-I and Other RNA Sensors in Antiviral Immunity. Annu Rev Immunol. 2018;36:667–94. pmid:29677479
- View Article
- PubMed/NCBI
- Google Scholar
4. Guo Y, Li R, Tan Z, Shi J, Fu Y, Song Y, et al. E3 ubiquitin ligase ASB8 negatively regulates interferon via regulating TBK1/IKKi homeostasis. Mol Immunol. 2020;121:195–203. pmid:32298923
- View Article
- PubMed/NCBI
- Google Scholar
5. Dalskov L, Narita R, Andersen LL, Jensen N, Assil S, Kristensen KH, et al. Characterization of distinct molecular interactions responsible for IRF3 and IRF7 phosphorylation and subsequent dimerization. Nucleic Acids Res. 2020;48(20):11421–33. pmid:33205822
- View Article
- PubMed/NCBI
- Google Scholar
6. Young C, Singh M, Jackson KJL, Field MA, Peters TJ, Angioletti-Uberti S, et al. A triad of somatic mutagenesis converges in self-reactive B cells to cause a virus-induced autoimmune disease. Immunity. 2025;58(2):412-430.e10. pmid:39818208
- View Article
- PubMed/NCBI
- Google Scholar
7. Qi F, Zhang X, Wang L, Ren C, Zhao X, Luo J, et al. E3 ubiquitin ligase NEURL3 promotes innate antiviral response through catalyzing K63-linked ubiquitination of IRF7. FASEB J. 2022;36(8):e22409. pmid:35792897
- View Article
- PubMed/NCBI
- Google Scholar
8. Lee KJ, Lee H, Joo CH. Negative Regulation of IKKε-Mediated IRF7 Phosphorylation by HSP70. J Immunol. 2020;204(9):2562–74. pmid:32169844
- View Article
- PubMed/NCBI
- Google Scholar
9. Cao L, Ji Y, Zeng L, Liu Q, Zhang Z, Guo S, et al. P200 family protein IFI204 negatively regulates type I interferon responses by targeting IRF7 in nucleus. PLoS Pathog. 2019;15(10):e1008079. pmid:31603949
- View Article
- PubMed/NCBI
- Google Scholar
10. Pang K, Wang W, Qin J-X, Shi Z-D, Hao L, Ma Y-Y, et al. Role of protein phosphorylation in cell signaling, disease, and the intervention therapy. MedComm (2020). 2022;3(4):e175. pmid:36349142
- View Article
- PubMed/NCBI
- Google Scholar
11. Kamada R, Kudoh F, Ito S, Tani I, Janairo JIB, Omichinski JG, et al. Metal-dependent Ser/Thr protein phosphatase PPM family: Evolution, structures, diseases and inhibitors. Pharmacol Ther. 2020;215:107622. pmid:32650009
- View Article
- PubMed/NCBI
- Google Scholar
12. Dai J, Zhang J, Sun Y, Wu Q, Sun L, Ji C, et al. Characterization of a novel human protein phosphatase 2C family member, PP2Ckappa. Int J Mol Med. 2006;17(6):1117–23. pmid:16685424
- View Article
- PubMed/NCBI
- Google Scholar
13. Liu J, Stevens PD, Eshleman NE, Gao T. Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1). J Biol Chem. 2013;288(32):23225–33. pmid:23814053
- View Article
- PubMed/NCBI
- Google Scholar
14. Kimura H, Takizawa N, Allemand E, Hori T, Iborra FJ, Nozaki N, et al. A novel histone exchange factor, protein phosphatase 2Cgamma, mediates the exchange and dephosphorylation of H2A-H2B. J Cell Biol. 2006;175(3):389–400. pmid:17074886
- View Article
- PubMed/NCBI
- Google Scholar
15. Yu K, Tian H, Deng H. PPM1G restricts innate immune signaling mediated by STING and MAVS and is hijacked by KSHV for immune evasion. Sci Adv. 2020;6(47):eabd0276. pmid:33219031
- View Article
- PubMed/NCBI
- Google Scholar
16. Chang H, Hou P, Wang X, Xiang A, Wu H, Qi W, et al. CD97 negatively regulates the innate immune response against RNA viruses by promoting RNF125-mediated RIG-I degradation. Cell Mol Immunol. 2023;20(12):1457–71. pmid:37978243
- View Article
- PubMed/NCBI
- Google Scholar
17. Evans BJ, Wang Z, Mobley L, Khosravi D, Fujii N, Navenot J-M, et al. Physical association of GPR54 C-terminal with protein phosphatase 2A. Biochem Biophys Res Commun. 2008;377(4):1067–71. pmid:18977201
- View Article
- PubMed/NCBI
- Google Scholar
18. Krönke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014;343(6168):301–5. pmid:24292625
- View Article
- PubMed/NCBI
- Google Scholar
19. Deng C, Chen D, Yang L, Zhang Y, Jin C, Li Y, et al. The role of cGAS-STING pathway ubiquitination in innate immunity and multiple diseases. Front Immunol. 2025;16:1522200. pmid:40028324
- View Article
- PubMed/NCBI
- Google Scholar
20. Kazzaz SA, Shaikh KA, White J, Zhou Q, Powell WH, Harhaj EW. Phosphorylation of aryl hydrocarbon receptor interacting protein by TBK1 negatively regulates IRF7 and the type I interferon response. J Biol Chem. 2024;300(1):105525. pmid:38043800
- View Article
- PubMed/NCBI
- Google Scholar
21. Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell. 2007;28(5):730–8. pmid:18082598
- View Article
- PubMed/NCBI
- Google Scholar
22. Zhao Y, Liang L, Fan Y, Sun S, An L, Shi Z, et al. PPM1B negatively regulates antiviral response via dephosphorylating TBK1. Cell Signal. 2012;24(11):2197–204. pmid:22750291
- View Article
- PubMed/NCBI
- Google Scholar
23. Ni G, Ma Z, Wong JP, Zhang Z, Cousins E, Major MB, et al. PPP6C Negatively Regulates STING-Dependent Innate Immune Responses. mBio. 2020;11(4):e01728-20. pmid:32753499
- View Article
- PubMed/NCBI
- Google Scholar
24. Wang L, Zhao J, Ren J, Hall KH, Moorman JP, Yao ZQ, et al. Protein phosphatase 1 abrogates IRF7-mediated type I IFN response in antiviral immunity. Eur J Immunol. 2016;46(10):2409–19. pmid:27469204
- View Article
- PubMed/NCBI
- Google Scholar
25. Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature. 2005;434(7034):772–7. pmid:15800576
- View Article
- PubMed/NCBI
- Google Scholar
26. Chau T-L, Gioia R, Gatot J-S, Patrascu F, Carpentier I, Chapelle J-P, et al. Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated?. Trends Biochem Sci. 2008;33(4):171–80. pmid:18353649
- View Article
- PubMed/NCBI
- Google Scholar
27. Uematsu S, Sato S, Yamamoto M, Hirotani T, Kato H, Takeshita F, et al. Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction. J Exp Med. 2005;201(6):915–23. pmid:15767370
- View Article
- PubMed/NCBI
- Google Scholar
28. Hoshino K, Sugiyama T, Matsumoto M, Tanaka T, Saito M, Hemmi H, et al. IkappaB kinase-alpha is critical for interferon-alpha production induced by Toll-like receptors 7 and 9. Nature. 2006;440(7086):949–53. pmid:16612387
- View Article
- PubMed/NCBI
- Google Scholar
29. Yin Y, Xu X, Tang J, Zhang W, Zhangyuan G, Ji J, et al. CD97 Promotes Tumor Aggressiveness Through the Traditional G Protein-Coupled Receptor-Mediated Signaling in Hepatocellular Carcinoma. Hepatology. 2018;68(5):1865–78. pmid:29704239
- View Article
- PubMed/NCBI
- Google Scholar
30. Hilbig D, Sittig D, Hoffmann F, Rothemund S, Warmt E, Quaas M, et al. Mechano-Dependent Phosphorylation of the PDZ-Binding Motif of CD97/ADGRE5 Modulates Cellular Detachment. Cell Rep. 2018;24(8):1986–95. pmid:30134161
- View Article
- PubMed/NCBI
- Google Scholar
31. Huang H, Xiong Q, Wang N, Chen R, Ren H, Siwko S, et al. Kisspeptin/GPR54 signaling restricts antiviral innate immune response through regulating calcineurin phosphatase activity. Sci Adv. 2018;4(8):eaas9784. pmid:30101190
- View Article
- PubMed/NCBI
- Google Scholar
32. Foster WH, Langenbacher A, Gao C, Chen J, Wang Y. Nuclear phosphatase PPM1G in cellular survival and neural development. Dev Dyn. 2013;242(9):1101–9. pmid:23723158
- View Article
- PubMed/NCBI
- Google Scholar
33. Arif M, Wei J, Zhang Q, Liu F, Basurto-Islas G, Grundke-Iqbal I, et al. Cytoplasmic retention of protein phosphatase 2A inhibitor 2 (I2PP2A) induces Alzheimer-like abnormal hyperphosphorylation of Tau. J Biol Chem. 2014;289(40):27677–91. pmid:25128526
- View Article
- PubMed/NCBI
- Google Scholar
34. Wadham C, Gamble JR, Vadas MA, Khew-Goodall Y. Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation. J Cell Sci. 2000;113 (Pt 17):3117–23. pmid:10934049
- View Article
- PubMed/NCBI
- Google Scholar
35. Sima M, Lv C, Qi J, Guo J, Luo R, Deng X, et al. Anti-inflammatory effects of theaflavin-3’-gallate during influenza virus infection through regulating the TLR4/MAPK/p38 pathway. Eur J Pharmacol. 2023;938:175332. pmid:36265612
- View Article
- PubMed/NCBI
- Google Scholar
36. Chang H, Wu H, Hou P, Aizaz M, Yang R, Xiang A, et al. DLG1 promotes the antiviral innate immune response by inhibiting p62-mediated autophagic degradation of IKKε. J Virol. 2023;97(12):e0150123. pmid:37982618
- View Article
- PubMed/NCBI
- Google Scholar
37. Wang S, Wei R, Ma X, Guo J, Aizaz M, Li F, et al. The host protein CALCOCO2 interacts with bovine viral diarrhoea virus Npro, inhibiting type I interferon production and thereby promoting viral replication. Virulence. 2024;15(1):2289764. pmid:38047736
- View Article
- PubMed/NCBI
- Google Scholar
38. Ma J, Chen T, Wu S, Yang C, Bai M, Shu K, et al. iProX: an integrated proteome resource. Nucleic Acids Res. 2019;47(D1):D1211–7. pmid:30252093
- View Article
- PubMed/NCBI
- Google Scholar
39. Chen T, Ma J, Liu Y, Chen Z, Xiao N, Lu Y, et al. iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res. 2022;50(D1):D1522–7. pmid:34871441
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Chen K, Liu J, Cao X. Regulation of type I interferon signaling in immunity and inflammation: A comprehensive review. J Autoimmun. 2017;83:1–11. pmid:28330758
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Barral PM, Sarkar D, Su Z, Barber GN, DeSalle R, Racaniello VR, et al. Functions of the cytoplasmic RNA sensors RIG-I and MDA-5: key regulators of innate immunity. Pharmacol Ther. 2009;124(2):219–34. pmid:19615405
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Chow KT, Gale M Jr, Loo Y-M. RIG-I and Other RNA Sensors in Antiviral Immunity. Annu Rev Immunol. 2018;36:667–94. pmid:29677479
View Article
PubMed/NCBI
Google Scholar

[10] View Article

[11] PubMed/NCBI

[12] Google Scholar

[ref4] 4. Guo Y, Li R, Tan Z, Shi J, Fu Y, Song Y, et al. E3 ubiquitin ligase ASB8 negatively regulates interferon via regulating TBK1/IKKi homeostasis. Mol Immunol. 2020;121:195–203. pmid:32298923
View Article
PubMed/NCBI
Google Scholar

[14] View Article

[15] PubMed/NCBI

[16] Google Scholar

[ref5] 5. Dalskov L, Narita R, Andersen LL, Jensen N, Assil S, Kristensen KH, et al. Characterization of distinct molecular interactions responsible for IRF3 and IRF7 phosphorylation and subsequent dimerization. Nucleic Acids Res. 2020;48(20):11421–33. pmid:33205822
View Article
PubMed/NCBI
Google Scholar

[18] View Article

[19] PubMed/NCBI

[20] Google Scholar

[ref6] 6. Young C, Singh M, Jackson KJL, Field MA, Peters TJ, Angioletti-Uberti S, et al. A triad of somatic mutagenesis converges in self-reactive B cells to cause a virus-induced autoimmune disease. Immunity. 2025;58(2):412-430.e10. pmid:39818208
View Article
PubMed/NCBI
Google Scholar

[22] View Article

[23] PubMed/NCBI

[24] Google Scholar

[ref7] 7. Qi F, Zhang X, Wang L, Ren C, Zhao X, Luo J, et al. E3 ubiquitin ligase NEURL3 promotes innate antiviral response through catalyzing K63-linked ubiquitination of IRF7. FASEB J. 2022;36(8):e22409. pmid:35792897
View Article
PubMed/NCBI
Google Scholar

[26] View Article

[27] PubMed/NCBI

[28] Google Scholar

[ref8] 8. Lee KJ, Lee H, Joo CH. Negative Regulation of IKKε-Mediated IRF7 Phosphorylation by HSP70. J Immunol. 2020;204(9):2562–74. pmid:32169844
View Article
PubMed/NCBI
Google Scholar

[30] View Article

[31] PubMed/NCBI

[32] Google Scholar

[ref9] 9. Cao L, Ji Y, Zeng L, Liu Q, Zhang Z, Guo S, et al. P200 family protein IFI204 negatively regulates type I interferon responses by targeting IRF7 in nucleus. PLoS Pathog. 2019;15(10):e1008079. pmid:31603949
View Article
PubMed/NCBI
Google Scholar

[34] View Article

[35] PubMed/NCBI

[36] Google Scholar

[ref10] 10. Pang K, Wang W, Qin J-X, Shi Z-D, Hao L, Ma Y-Y, et al. Role of protein phosphorylation in cell signaling, disease, and the intervention therapy. MedComm (2020). 2022;3(4):e175. pmid:36349142
View Article
PubMed/NCBI
Google Scholar

[38] View Article

[39] PubMed/NCBI

[40] Google Scholar

[ref11] 11. Kamada R, Kudoh F, Ito S, Tani I, Janairo JIB, Omichinski JG, et al. Metal-dependent Ser/Thr protein phosphatase PPM family: Evolution, structures, diseases and inhibitors. Pharmacol Ther. 2020;215:107622. pmid:32650009
View Article
PubMed/NCBI
Google Scholar

[42] View Article

[43] PubMed/NCBI

[44] Google Scholar

[ref12] 12. Dai J, Zhang J, Sun Y, Wu Q, Sun L, Ji C, et al. Characterization of a novel human protein phosphatase 2C family member, PP2Ckappa. Int J Mol Med. 2006;17(6):1117–23. pmid:16685424
View Article
PubMed/NCBI
Google Scholar

[46] View Article

[47] PubMed/NCBI

[48] Google Scholar

[ref13] 13. Liu J, Stevens PD, Eshleman NE, Gao T. Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1). J Biol Chem. 2013;288(32):23225–33. pmid:23814053
View Article
PubMed/NCBI
Google Scholar

[50] View Article

[51] PubMed/NCBI

[52] Google Scholar

[ref14] 14. Kimura H, Takizawa N, Allemand E, Hori T, Iborra FJ, Nozaki N, et al. A novel histone exchange factor, protein phosphatase 2Cgamma, mediates the exchange and dephosphorylation of H2A-H2B. J Cell Biol. 2006;175(3):389–400. pmid:17074886
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref15] 15. Yu K, Tian H, Deng H. PPM1G restricts innate immune signaling mediated by STING and MAVS and is hijacked by KSHV for immune evasion. Sci Adv. 2020;6(47):eabd0276. pmid:33219031
View Article
PubMed/NCBI
Google Scholar

[58] View Article

[59] PubMed/NCBI

[60] Google Scholar

[ref16] 16. Chang H, Hou P, Wang X, Xiang A, Wu H, Qi W, et al. CD97 negatively regulates the innate immune response against RNA viruses by promoting RNF125-mediated RIG-I degradation. Cell Mol Immunol. 2023;20(12):1457–71. pmid:37978243
View Article
PubMed/NCBI
Google Scholar

[62] View Article

[63] PubMed/NCBI

[64] Google Scholar

[ref17] 17. Evans BJ, Wang Z, Mobley L, Khosravi D, Fujii N, Navenot J-M, et al. Physical association of GPR54 C-terminal with protein phosphatase 2A. Biochem Biophys Res Commun. 2008;377(4):1067–71. pmid:18977201
View Article
PubMed/NCBI
Google Scholar

[66] View Article

[67] PubMed/NCBI

[68] Google Scholar

[ref18] 18. Krönke J, Udeshi ND, Narla A, Grauman P, Hurst SN, McConkey M, et al. Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science. 2014;343(6168):301–5. pmid:24292625
View Article
PubMed/NCBI
Google Scholar

[70] View Article

[71] PubMed/NCBI

[72] Google Scholar

[ref19] 19. Deng C, Chen D, Yang L, Zhang Y, Jin C, Li Y, et al. The role of cGAS-STING pathway ubiquitination in innate immunity and multiple diseases. Front Immunol. 2025;16:1522200. pmid:40028324
View Article
PubMed/NCBI
Google Scholar

[74] View Article

[75] PubMed/NCBI

[76] Google Scholar

[ref20] 20. Kazzaz SA, Shaikh KA, White J, Zhou Q, Powell WH, Harhaj EW. Phosphorylation of aryl hydrocarbon receptor interacting protein by TBK1 negatively regulates IRF7 and the type I interferon response. J Biol Chem. 2024;300(1):105525. pmid:38043800
View Article
PubMed/NCBI
Google Scholar

[78] View Article

[79] PubMed/NCBI

[80] Google Scholar

[ref21] 21. Hunter T. The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell. 2007;28(5):730–8. pmid:18082598
View Article
PubMed/NCBI
Google Scholar

[82] View Article

[83] PubMed/NCBI

[84] Google Scholar

[ref22] 22. Zhao Y, Liang L, Fan Y, Sun S, An L, Shi Z, et al. PPM1B negatively regulates antiviral response via dephosphorylating TBK1. Cell Signal. 2012;24(11):2197–204. pmid:22750291
View Article
PubMed/NCBI
Google Scholar

[86] View Article

[87] PubMed/NCBI

[88] Google Scholar

[ref23] 23. Ni G, Ma Z, Wong JP, Zhang Z, Cousins E, Major MB, et al. PPP6C Negatively Regulates STING-Dependent Innate Immune Responses. mBio. 2020;11(4):e01728-20. pmid:32753499
View Article
PubMed/NCBI
Google Scholar

[90] View Article

[91] PubMed/NCBI

[92] Google Scholar

[ref24] 24. Wang L, Zhao J, Ren J, Hall KH, Moorman JP, Yao ZQ, et al. Protein phosphatase 1 abrogates IRF7-mediated type I IFN response in antiviral immunity. Eur J Immunol. 2016;46(10):2409–19. pmid:27469204
View Article
PubMed/NCBI
Google Scholar

[94] View Article

[95] PubMed/NCBI

[96] Google Scholar

[ref25] 25. Honda K, Yanai H, Negishi H, Asagiri M, Sato M, Mizutani T, et al. IRF-7 is the master regulator of type-I interferon-dependent immune responses. Nature. 2005;434(7034):772–7. pmid:15800576
View Article
PubMed/NCBI
Google Scholar

[98] View Article

[99] PubMed/NCBI

[100] Google Scholar

[ref26] 26. Chau T-L, Gioia R, Gatot J-S, Patrascu F, Carpentier I, Chapelle J-P, et al. Are the IKKs and IKK-related kinases TBK1 and IKK-epsilon similarly activated?. Trends Biochem Sci. 2008;33(4):171–80. pmid:18353649
View Article
PubMed/NCBI
Google Scholar

[102] View Article

[103] PubMed/NCBI

[104] Google Scholar

[ref27] 27. Uematsu S, Sato S, Yamamoto M, Hirotani T, Kato H, Takeshita F, et al. Interleukin-1 receptor-associated kinase-1 plays an essential role for Toll-like receptor (TLR)7- and TLR9-mediated interferon-{alpha} induction. J Exp Med. 2005;201(6):915–23. pmid:15767370
View Article
PubMed/NCBI
Google Scholar

[106] View Article

[107] PubMed/NCBI

[108] Google Scholar

[ref28] 28. Hoshino K, Sugiyama T, Matsumoto M, Tanaka T, Saito M, Hemmi H, et al. IkappaB kinase-alpha is critical for interferon-alpha production induced by Toll-like receptors 7 and 9. Nature. 2006;440(7086):949–53. pmid:16612387
View Article
PubMed/NCBI
Google Scholar

[110] View Article

[111] PubMed/NCBI

[112] Google Scholar

[ref29] 29. Yin Y, Xu X, Tang J, Zhang W, Zhangyuan G, Ji J, et al. CD97 Promotes Tumor Aggressiveness Through the Traditional G Protein-Coupled Receptor-Mediated Signaling in Hepatocellular Carcinoma. Hepatology. 2018;68(5):1865–78. pmid:29704239
View Article
PubMed/NCBI
Google Scholar

[114] View Article

[115] PubMed/NCBI

[116] Google Scholar

[ref30] 30. Hilbig D, Sittig D, Hoffmann F, Rothemund S, Warmt E, Quaas M, et al. Mechano-Dependent Phosphorylation of the PDZ-Binding Motif of CD97/ADGRE5 Modulates Cellular Detachment. Cell Rep. 2018;24(8):1986–95. pmid:30134161
View Article
PubMed/NCBI
Google Scholar

[118] View Article

[119] PubMed/NCBI

[120] Google Scholar

[ref31] 31. Huang H, Xiong Q, Wang N, Chen R, Ren H, Siwko S, et al. Kisspeptin/GPR54 signaling restricts antiviral innate immune response through regulating calcineurin phosphatase activity. Sci Adv. 2018;4(8):eaas9784. pmid:30101190
View Article
PubMed/NCBI
Google Scholar

[122] View Article

[123] PubMed/NCBI

[124] Google Scholar

[ref32] 32. Foster WH, Langenbacher A, Gao C, Chen J, Wang Y. Nuclear phosphatase PPM1G in cellular survival and neural development. Dev Dyn. 2013;242(9):1101–9. pmid:23723158
View Article
PubMed/NCBI
Google Scholar

[126] View Article

[127] PubMed/NCBI

[128] Google Scholar

[ref33] 33. Arif M, Wei J, Zhang Q, Liu F, Basurto-Islas G, Grundke-Iqbal I, et al. Cytoplasmic retention of protein phosphatase 2A inhibitor 2 (I2PP2A) induces Alzheimer-like abnormal hyperphosphorylation of Tau. J Biol Chem. 2014;289(40):27677–91. pmid:25128526
View Article
PubMed/NCBI
Google Scholar

[130] View Article

[131] PubMed/NCBI

[132] Google Scholar

[ref34] 34. Wadham C, Gamble JR, Vadas MA, Khew-Goodall Y. Translocation of protein tyrosine phosphatase Pez/PTPD2/PTP36 to the nucleus is associated with induction of cell proliferation. J Cell Sci. 2000;113 (Pt 17):3117–23. pmid:10934049
View Article
PubMed/NCBI
Google Scholar

[134] View Article

[135] PubMed/NCBI

[136] Google Scholar

[ref35] 35. Sima M, Lv C, Qi J, Guo J, Luo R, Deng X, et al. Anti-inflammatory effects of theaflavin-3’-gallate during influenza virus infection through regulating the TLR4/MAPK/p38 pathway. Eur J Pharmacol. 2023;938:175332. pmid:36265612
View Article
PubMed/NCBI
Google Scholar

[138] View Article

[139] PubMed/NCBI

[140] Google Scholar

[ref36] 36. Chang H, Wu H, Hou P, Aizaz M, Yang R, Xiang A, et al. DLG1 promotes the antiviral innate immune response by inhibiting p62-mediated autophagic degradation of IKKε. J Virol. 2023;97(12):e0150123. pmid:37982618
View Article
PubMed/NCBI
Google Scholar

[142] View Article

[143] PubMed/NCBI

[144] Google Scholar

[ref37] 37. Wang S, Wei R, Ma X, Guo J, Aizaz M, Li F, et al. The host protein CALCOCO2 interacts with bovine viral diarrhoea virus Npro, inhibiting type I interferon production and thereby promoting viral replication. Virulence. 2024;15(1):2289764. pmid:38047736
View Article
PubMed/NCBI
Google Scholar

[146] View Article

[147] PubMed/NCBI

[148] Google Scholar

[ref38] 38. Ma J, Chen T, Wu S, Yang C, Bai M, Shu K, et al. iProX: an integrated proteome resource. Nucleic Acids Res. 2019;47(D1):D1211–7. pmid:30252093
View Article
PubMed/NCBI
Google Scholar

[150] View Article

[151] PubMed/NCBI

[152] Google Scholar

[ref39] 39. Chen T, Ma J, Liu Y, Chen Z, Xiao N, Lu Y, et al. iProX in 2021: connecting proteomics data sharing with big data. Nucleic Acids Res. 2022;50(D1):D1522–7. pmid:34871441
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

Figures

Abstract

Author summary

Introduction

Results

CD97 enhances the replication of BEFV, SeV, and H1N1

CD97 attenuates the IFN-I response triggered by BEFV, SeV, and H1N1

CD97 interacts with the protein phosphatase PPM1G

CD97 enhances virus replication through PPM1G-mediated downregulation of IFN-I

CD97 promotes PPM1G-mediated dephosphorylation of IRF7

CD97 promotes the transcription of PPM1G through IKZF1

CD97 deficiency in mice are more resistant to viral infections in vivo

The compound SANG attenuates viral replication by suppressing CD97 expression

Discussion

Materials and methods

Ethics statement

Cell lines, mice and viruses

Antibodies and chemicals

Generation of stable overexpressing and knockout cells

Viral infection

siRNA silencing and RT–qPCR

Immunoblotting and co-immunoprecipitation (Co-IP)

Dual-luciferase reporter assay

Nuclear and cytoplasmic extraction (NE-PER)

Quantification and statistical analysis

Supporting information

S1 Fig. Flow cytometric verification of CD97 knockout in PMs.

S2 Fig. CD97 inhibits IFN-I signaling response induced by SeV and BEFV in BMDMs.

S3 Fig. PPM1G dephosphorylates IRF7.

S4 Fig. CD97 facilitates the PPM1G-mediated dephosphorylation of IRF7.

S5 Fig. SANG potentiates the IFN-I signaling response.

S6 Fig. CD97 and PPM1G inhibits IRF7 phosphorylation induced by poly(I:C).

S7 Fig. The impact of a CD97 mutant on IFN-I and PPM1G.

S1 Table. Sequences used for qPCR and siRNA in this study.

S1 Data. List of Candidate interactors of CD97 detected by mass spectrometry.

References