CREG1 restricts ALV-J replication via the mitochondrial dysfunction–driven activation of innate immunity and apoptosis

Background: Host antiviral defense relies on key regulatory genes that coordinate immune signaling and cellular homeostasis, yet their roles in J subgroup avian leukosis virus (ALV-J) infection remain poorly defined. Here, we identify cellular repressor of E1A-stimulated genes 1 (CREG1) as a key regulator of mitochondrial function and a critical immune-related gene involved in ALV-J infection. The objective of this study was to explore the effects and underlying mechanisms of CREG1 in the context of ALV-J infection.

Methods: In this study, transcriptomic analysis and RT-qPCR revealed that the expression of CREG1 is significantly upregulated in the spleen tissues of ALV-J infected chickens. By overexpressing and silencing CREG1 in cultured cells, and using Western blotting, transmission electron microscopy, immunofluorescence, and flow cytometry, we comprehensively validated its effects on viral replication, mitochondrial function, and apoptosis.

Results: Overexpression of CREG1 upregulates the expression of I-IFN and certain interferon-stimulated genes (ISGs), thereby suppressing viral replication. Mechanistically, overexpression of CREG1 induces mitochondrial dysfunction, characterized by a decrease in mitochondrial membrane potential (Δψm), reduced adenosine triphosphate (ATP) production and respiratory chain activity, enhanced mitophagy, and increased release of mitochondrial DNA (mtDNA), which in turn triggers the activation of innate immune responses. Mitochondrial dysfunction further leads to the cytosolic release of cytochrome c and an increase in reactive oxygen species (ROS) levels, thereby triggering a robust apoptotic response. Moreover, the regulation of mitochondrial function by CREG1 depends on its interaction with the mitochondrial chaperone protein heat shock protein 1 (HSPD1), and their co-expression synergistically amplifies the antiviral response.

Conclusions: Overall, we identify CREG1 as a potent antiviral gene and underscore the pivotal roles of mitochondria-mediated innate immunity and apoptosis during ALV-J infection.

1 Introduction

Avian leukosis virus (ALV), a retrovirus belonging to the family retroviridae, primarily infects avian species, especially chickens, and is closely associated with the onset of various immunosuppressive diseases and tumors (1). Certain ALV subtypes, such as J subgroup ALV (ALV-J), are capable of inducing malignant transformation of host cells, leading to the development of lymphomas or other forms of tumors, thereby directly impacting poultry health and productivity (2). Due to the variability of ALV and its immune evasion mechanisms, existing vaccines and antiviral drugs are often ineffective in preventing ALV-J infection (3, 4). Therefore, gaining a deeper understanding of the interactions between the virus and the host immune system, identifying genes that inhibit ALV-J replication or enhance the host immune response, and improving overall immunity are key to addressing ALV infection.

Mitochondria function as critical signaling platforms that integrate cellular metabolism with antiviral and innate immune responses (5). When mitochondria are damaged, they release various endogenous molecules, such as mitochondrial DNA (mtDNA), reactive oxygen species (ROS), and proteins, which have the capacity to activate the innate immune response (6). The release of mtDNA into the cytosol can be detected by cyclic GMP - AMP synthase (cGAS). Once recognized, cGAS catalyzes the production of cyclic GMP - AMP (cGAMP), a second - messenger molecule (7). Subsequently, cGAMP activates the stimulator of interferon genes (STING). This activation sets off a chain reaction that leads to the upregulation of both nuclear factor kappa - light - chain - enhancer of activated B cells (NF-κB) and proteins involved in the type I interferon (I-IFN) response (8). In addition to innate immunity, host cells utilize apoptosis as an antiviral defense mechanism to eliminate infected or damaged cells (9). When cells are subjected to stress or viral infection, mitochondria can initiate apoptosis via the mitochondrial pathway by modulating the B cell lymphoma 2 (BCL-2) family of proteins, leading to the release of pro-apoptotic factors such as cytochrome c (10). The increased permeability of the outer mitochondrial membrane facilitates the translocation of cytochrome c from the mitochondrial matrix into the cytosol, thereby activating the caspase family and triggering a cascade of signals that drive cell death (11).

In this study, we explored the role of cellular repressor of E1a-stimulating gene 1 (CREG1) during viral infection, based on its elevated expression in tissues and cells infected with ALV-J. CREG1 was initially discovered as a transcriptional repressor that antagonizes E1A oncoprotein 3-induced transcription and cell transformation (12). Studies have demonstrated that CREG1 not only activates lysosomal autophagy to shield the heart from ischemia/reperfusion (MI/R) injury but also promotes the phenotypic transformation of myocardial fibroblasts after myocardial infarction by regulating the expression of cell division control protein 42 (CDC42) (13, 14). Recent studies suggest that CREG1 enhances lysosome-associated membrane protein 2 (LAMP2) expression through an F-box protein 27 (FBXO27)-dependent pathway, thereby improving autophagy and mitigating the progression of diabetic cardiomyopathy (15). In addition to its role in autophagy regulation, CREG1 has been found to localize to mitochondria, where it interacts with heat shock protein 1 (HSPD1) to regulate mitophagy, thereby maintaining skeletal muscle homeostasis (16). Thus far, the biological role and underlying molecular mechanisms of CREG1 during ALV-J infection remain enigmatic.

Here, we demonstrate that CREG1 overexpression markedly restricts ALV-J replication by enhancing host innate immune responses. Mechanistically, CREG1 disrupts mitochondrial homeostasis, promotes mtDNA release, and activates the cGAS-STING signaling pathway, thereby amplifying I-IFN responses and interferon-stimulated genes (ISGs) expression. In parallel, CREG1 increases mitochondrial permeability, triggering cytochrome c release, caspase activation, and apoptosis, which further limits viral replication. Moreover, the interaction between CREG1 and HSPD1 cooperatively strengthens antiviral responses. Collectively, these findings provide new insights into the unique antiviral mechanism of CREG1 during ALV-J infection, advancing our understanding of potential strategies to enhance antiviral responses in poultry.

2 Materials and methods

2.1 Pathological chicken tissues

The chicken tissues used for transcriptome sequencing or quantitative reverse transcription polymerase chain reaction (RT-qPCR) in this study were previously collected (17), initially preserved in liquid nitrogen, and subsequently stored at -80 °C. The ALV-J-infected and control chickens had been validated in our previous study (17).

2.2 Cell culture

DF-1 and HD11 cells are preserved in our laboratory. All the cells were cultured at 37 °C and 5% CO₂. HD11 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, # 11875093). DF-1 and MSB-1 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, # 11965092). 10% fetal bovine serum (FBS, ABW, # AB-FBS-1050S), 5% chicken serum (only for MSB-1 cells), 100U/mL penicillin and 100µg/mL streptomycin were supplied to the base medium.

2.3 Virus infection

The ALV-J strain, SCAU-HN06, was kindly provided by Professor Cao Weisheng from South China Agricultural University. A 10⁴ TCID₅₀/0.1 mL of ALV-J SCAU-HN06 were used in this study. Cells were infected with ALV-J SCAU-HN06 in without DMEM. Following 2h of incubation, washed by 1×PBS, and the media was replaced with DMEM, supplemented with 1% FBS.

2.4 Plasmids and RNA interference

The coding sequence (CDS) of CREG1, ITGAV or HSPD1 was cloned into the pcDNA3.1 expression vector, which contains an N-terminal ATG start codon and either a 3×Flag tag (Flag-CREG1/ITGAV) or a 3×HA tag (HA-HSPD1). Small interfering RNAs (siRNAs) targeting ZEB2, BCL2L10, ITGAV, CTSB, CREG1, HSPD1, as well as negative control RNAs (siNC), were synthesized by Gene Create Company (Wuhan, China). Cells were transfected with plasmids or siRNAs by lipofectamine 3000 (Invitrogen, # L3000015) according to the manufacturers’ instructions. The overexpression or knockdown efficiency was checked 36–48 h after transfection using RT-qPCR. Targeting sequences were listed in Supplementary Table 1.

2.5 RNA isolation and RT-qPCR

Total RNA was isolated using TRIzol reagent (Invitrogen, # 15596026), followed by cDNA synthesis with reverse transcription kit (EZBioscience, A0010CGQ) and diluted 5-fold. cDNA amplification was performed using SYBR Green (Vazyme Biotech, # Q713-02) with the QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific, USA). The results were calculated by 2^ΔΔCt method with the normalization to GAPDH. All primers were synthesized from Tsingke Biological Technology (Beijing, China) and primers sequences were shown in Supplementary Table 2.

2.6 Western blotting

Total protein was extracted using IP lysis buffer containing 1% PMSF solution (Beyotime Biotech, # ST506) and phosphatase inhibitors (MCE, # HY-K0021). Quantification of protein was conducted with bicinchoninic acid (BCA) assay kit (Beyotime Biotech, P0009). Equal amounts of protein were separated by 10% SDS-PAGE electrophoresis (Beyotime Biotech, # P0804S) and then transferred to 0.45 μm PVDF membranes (Beyotime Biotech, # FFP22). After being blocked by 5% non-fat milk in TBST for 1h, membranes were incubated with corresponding primary antibodies at 4°C overnight. Subsequently, the membranes were washed with TBST three times, followed by incubation with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were detected with enhanced chemiluminescence detection kit (Beyotime Biotech, # P0018M) and visualized using ODYSSEY^®FC Imager System (LI-COR, USA) and quantified by gel analysis using ImageJ software (National Institutes of Health, USA). The antibodies used in this study were listed in Supplementary Table 3.

2.7 RNA-seq library construction and sequencing

Total RNA was isolated using TRIzol reagent (Invitrogen). Subsequently, library construction and RNA sequencing (RNA-seq) were performed using the Illumina NovaSeq 6000 platform (Illumina, USA). Raw sequencing reads were processed to remove adaptor sequences, poly-N sequences, and low-quality reads. The cleaned reads were then mapped to GenBank to identify known chicken mRNA. RNA-seq and analysis were conducted by BIOMARKER technologies (Beijing, China).

2.8 Immunoprecipitation and mass spectrometry

Total protein was collected from Flag-CREG1 or HA-HSPD1 overexpression cells using IP lysis buffer (Beyotime Biotech, # P0013). After centrifugation, total cellular protein was incubated with anti-Flag (Proteintech, # 66008-4-Ig) or anti-HA (Proteintech, # 51064-2-AP) M2 magnetic beads (Sigma-Aldrich, # M8823) overnight at 4 °C. The precipitate was washed three times with lysis buffer and then boiled in SDS-PAGE loading buffer (Beyotime Biotech, # P0286). The supernatant was then extracted and subjected to immunoblotting with the appropriate antibodies.

Total protein was collected from Flag-CREG1 overexpression DF-1 cells using IP lysis buffer (Beyotime Biotech). The cell lysate (1000 μg) was incubated overnight at 4 °C with IgG (Abcam) or Flag (Proteintech) antibodies and protein A/G beads (Thermo Fisher Scientific, # 88802). After incubation, the beads were washed and resuspended in 2× loading buffer. Each sample was separated using a 10% SDS–PAGE gel and visualized using mass spectrometry-compatible silver staining (Invitrogen). Similar conditions utilizing chicken IgG antibody (Bioss) were applied as the control lane for each gel. Mass spectrometry analyses were conducted using an LC–MS/MS system (Ekspert™ nanoLC, ABSciex Triple TOF™ 5600-plus) to identify proteins potentially interacting with CREG1.

2.9 Data independent acquisition quantitative proteomic techniques

Overexpression of CREG1 or the control group EGFP in DF-1 cells, followed by ALV-J infection at 24 h, with cells harvested at 48 h post-infection. DIA quantitative proteomic techniques were performed as previously described (18). DIA analysis was provided by Guangzhou Banianmedical Biotechnology Co., Ltd. (Guangzhou, China).

2.10 Confocal microscope

DF-1 cells were transfected with the Flag-CREG1 expression plasmid for 24h, then infected with ALV-J for an additional 24h. Cells were harvested and fixed in 4% paraformaldehyde, permeabilized with 0.1% saponin, blocked for 30 min with 10% goat serum, and incubated anti-Flag at 4 °C overnight. After each step, it is necessary to wash the cells three times with a washing solution. Subsequently, samples were incubated FITC-labeled Goat Anti-Mouse IgG (Beyotime Biotech, # A0568) for 1h, and DAPI was used to stain the nuclei. Images were visualized by confocal laser microscopy (Leica).

2.11 Detection of mitophagy by confocal microscopy

To evaluate mitophagy, cells were co-infected with Ad-GFP-LC3 (Hanbio, # HBLV-1010) for 24 h, followed by staining with MitoTracker Red (200 nM, Beyotime, # C1032) at 37 °C for 30 min in the dark. Images were acquired using a laser scanning confocal microscope (Leica). Colocalization indicates the occurrence of mitophagy.

2.12 Transmission electron microscopy

Cells were transfected with Flag-CREG1, si-CREG1, or the corresponding controls for 24 h, followed by ALV-J infection for 48 h. Cells were harvested and fixed using 2% glutaraldehyde for 24 h at 4°C. The samples underwent various processing steps, including fixation, gradient alcohol dehydration, and displacement, before being imaged with a transmission electron microscope (JEM-2000EX TEM, Japan).

2.13 Mitochondrial membrane potential, ROS, and superoxide detection

2.13.1 JC-1 staining

Mitochondrial membrane potential (Δψm) was assessed using the JC-1 dye (Beyotime Biotech, # C2003S) according to the manufacturer’s instructions. Briefly, cells were harvested and incubated with JC-1 working solution at 37 °C for 20 min in the dark. After washing twice with JC-1 buffer, fluorescence was measured by flow cytometry. Mitochondrial depolarization was indicated by a decrease in the red/green fluorescence intensity ratio.

2.13.2 MitoROS detection

MitoROS was assessed using the MitoSOX™ Red mitochondrial superoxide indicator (Beyotime Biotech, #S0061S). Cells adhering to coverslips in a 35 mm dish were treated with 2 mL of the working solution of MitoSOX reagent for 30 min at 37 °C and 5% CO2. After incubation, cells were washed three times with PBS. The fluorescence intensity was measured using flow cytometry with excitation/emission at 510/580 nm.

2.13.3 TMRE staining

TMRE (tetramethylrhodamine ethyl ester) staining was used to evaluate mitochondrial membrane potential. Cells were incubated with 200 nM TMRE (Beyotime Biotech, #C2001S) at 37 °C for 20 min in the dark, then washed with PBS. The fluorescence intensity was detected by flow cytometry using the PE channel (excitation/emission: 550/575 nm).

2.14 Flow Cytometry for detecting apoptosis

The prepared cells were added to 500 mL of 1× Annexin V buffer and gently resuspended. The cell suspensions were then incubated with 5 mL of Annexin V-FITC/APC (Beyotime Biotech, # C1383S)/(Abcam, # ab236215) and 5 mL of propidium iodide staining solution at 25 °C for 15 min in the dark before being analyzed by flow cytometry.

2.15 Generation of mitochondria-deficient ρ⁰ cells

Cells were treated with 50 ng/mL ethidium bromide (Sigma, # E1510), supplemented with 50 μg/mL uridine (MCE, HY-B1449) and 1 mM sodium pyruvate (MCE, HY-W015913) for 5 d, as previously described (19). Control cells were cultured under identical conditions without exposure to ethidium bromide. The deletion of mitochondrial DNA was verified by staining cells with MitoTracker Red (Beyotime, # C1032) or by RT-qPCR to detect the total cellular mtDNA content.

2.16 Mitochondrial cytosolic separation

The cell mitochondria isolation kit (Beyotime Biotech, # C3601) is used for isolating mitochondria and cytoplasm from cultured cells. Briefly, collect the processed cells, wash once with cold PBS, then add 1-2.5 mL of mitochondrial isolation reagent to 20–50 million cells, gently resuspend the cells, and incubate on ice for 10–15 min. Transfer the cell suspension to a suitably sized glass homogenizer and homogenize for 10–30 strokes. Carefully transfer the homogenate to a new centrifuge tube and centrifuge at 11,000 g for 10 min at 4 °C. Carefully remove the supernatant. The pellet contains the isolated mitochondria.

2.17 Cytosolic DNA extraction

Cytosolic extracts were isolated primarily as previously described (20). Briefly, cells were resuspended in 500 µL of buffer containing 150 mM NaCl, 50 mM HEPES, and 40 µg/mL Digitonin, followed by agitation at 4 °C for 15 min. The homogenates were centrifuged at 2000×g for 5 min, and the supernatant, containing the cytosolic fraction, was transferred to a fresh tube. The pellet from this centrifugation was saved for western blot analysis. To ensure the cytosolic fraction was free of nuclear, membrane, and mitochondrial contaminants, it was further purified by three additional rounds of centrifugation at 2000×g. The purity of both the cytosolic and pellet fractions was verified by western blotting. Total DNA was extracted from our cytosolic fraction using DNA extraction kit (Omega). Nuclear DNA primers were used to detect very small amounts of nuclear DNA (β-actin) in the cytosolic fractions and were used for normalization.

2.18 ROS assay

To determine ROS production, cells were treated with 10 mM 2’,7’-Dichlorodihydrofluorescein (DCFH-DA) following the manufacturer’s protocols (Beyotime Biotech, # S0035S). Set the microplate reader to an excitation wavelength of 488 nm and an emission wavelength of 525 nm to measure the fluorescence intensity of DCF.

2.19 Quantification of intracellular nitric oxide (NO) levels

Cellular nitric oxide (NO) levels were determined using a commercial detection kit based on nitrate reductase activity (Beyotime, Cat# S0024) following the supplier’s guidelines. After treatment, cells were collected, lysed, and centrifuged to obtain the supernatants. Nitrate in the samples was enzymatically converted to nitrite, which then reacted with Griess reagent to generate a colored product. The absorbance at 540 nm was recorded using a microplate spectrophotometer, and NO concentrations were quantified using a sodium nitrite standard curve.

2.20 Measurement of intracellular ATP levels

ATP levels were measured using an ATP Assay Kit (Beyotime, Cat# S0027) according to the manufacturer’s instructions. Briefly, cells were lysed with the lysis buffer provided in the kit, and the lysates were centrifuged at 12,000 × g for 5 min at 4 °C. The supernatants were collected, and equal volumes of the samples and the luciferase reagent were mixed in a white 96-well plate. Luminescence was immediately measured using a microplate reader. ATP concentrations were calculated based on a standard curve and normalized to the total protein content of each sample.

2.21 Measurement of NADH-CoQ reductase and cytochrome C oxidase activities

The activities of NADH-CoQ reductase (Complex I) and Cytochrome C Oxidase (Complex IV) were determined using commercial assay kits from Solarbio (Cat# BC0510 and Cat# BC0940) following the manufacturer’s protocols. Briefly, cells were collected and lysed using the extraction buffer provided in the kit under ultrasonic homogenization. After centrifugation, the enzyme activity was measured using the resulting supernatant. The absorbance changes were recorded at the specified wavelength using a microplate reader. Enzyme activities were calculated based on the extinction coefficient and normalized to the total protein content.

2.22 Enzyme-linked immunosorbent assay

The supernatants were collected from CREG1-overexpressing or CREG1-knockdown cells 48 h post ALV-J infection. The release of chicken IL-1β (Mlbio, # ml042758), TNF-α (Mlbio, # ml002790), IL-10 (Mlbio, # ml059830), IL-4 (Mlbio, # ml059838), IFN-γ (Mlbio, # ml023435), IFN-β (Mlbio, # ml059828), and cGAMP (Cayman chemical, # 501700) was measured using quantitative ELISA kits according to the manufacturer’s instructions.

2.23 Statistical analysis

Statistical analysis was performed using GraphPad Prism 8 software (GraphPad Software, Inc.). All data in this study were generated from at least three independent replicated experiments. Data are presented as the mean ± standard deviation (SD). Statistical significance was tested using the two-tailed Student’s t test and is indicated by p values; *p < 0.05, **p < 0.01 and ***p < 0.001 were considered to indicate significance.

3 Results

3.1 CREG1 is a potential antiviral molecule against ALV-J infection

To investigate the key regulatory factors involved in the ALV-J infection process, we first performed bulk transcriptome sequencing on ALV-J-infected spleen tissues (Supplementary Figures S1A–D). ALV-J infection affects multiple signaling pathways, including immune regulation (e.g., Toll-like receptor signaling pathway and intestinal immune network for IgA production), cell signaling transduction (Calcium signaling pathway), and metabolic regulation (Porphyrin metabolism, Tyrosine metabolism, and Glycine, serine and threonine metabolism) (Figures 1A, B). These findings suggest that ALV-J infection may promote viral replication by affecting multiple key pathways, while also being associated with inflammation, immune suppression, or alterations in host physiological functions.

Figure 1

Figure 1. Overexpression of CREG1 inhibits the replication of ALV-J. (A). Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of differentially upregulated genes in RNA-seq data from ALV-J infected spleen and the control group. n = 4. (B). Volcano plot showing differentially expressed genes (DEGs) in the RNA-seq data from ALV-J-infected spleen tissues compared with the control group. DEGs were defined as those with |log₂ fold change| ≥ 1 and adjusted p-value ≤ 0.01. Red dots represent upregulated DEGs, while green dots represent downregulated DEGs. n = 4. (C, D). Relative mRNA expression levels of ZEB2, CSTB, BCL2L10, CREG1, and ITGAV were sequentially detected by RT-qPCR. Expression levels of specific genes in spleen tissues from ALV-J-infected chickens and control groups (C). Expression levels of specific genes in DF-1 cells after 48 h of siRNA treatment (D). n = 4/6. (E). After siRNA treatment of specific genes in DF-1 cells, the cells were infected with ALV-J. The expression level of the viral protein gp85 was measured by RT-qPCR 48 hours post-infection (hpi). Statistical significance was determined by comparing all other groups with the si-NC control. n = 6. (F). After siRNA treatment of specific genes in DF-1 cells, the cells were infected with ALV-J. At 48 hpi, the expression levels of the viral envelope protein env and β-actin were detected by western blotting. The left panel shows the immunoblot, and the right panel shows the relative expression levels of env (env/actin). Statistical significance was determined by comparing all other groups with the si-NC control. n = 3. (G). After transfection of Flag-CREG1 or control (Flag-pcDNA3.1 without insert was used as a negative control) into DF-1 cells, the expression level of CREG1 was measured by RT-qPCR at 48 hpi. n = 6. (H). DF-1 cells were transfected with Flag-CREG1 or control, followed by ALV-J infection. The expression level of the viral protein gp85 was then assessed by RT-qPCR at 48 hpi. n = 6. (I). Flag-CREG1 or control was transfected into DF-1 cells, followed by ALV-J infection. At 48 hpi, the expression levels of the viral envelope protein env and β-actin were analyzed by western blotting. The left panel shows the immunoblot, and the right panel shows the statistical analysis of relative env expression (env/actin). n = 3. For (C-I), the data are presented as mean ± SD. Statistical significance was determined using unpaired Student’s t-test, with ns indicating no significant difference, *p < 0.05, **p < 0.01, and ***p < 0.001.

Next, we investigated the impact of several upregulated genes, including ZEB2, BCL2L10, ITGAV, CTSB, and CREG1, on viral replication. Notably, these genes were also highly expressed in certain immune cell subsets in single-cell sequencing data of ALV-infected peripheral blood mononuclear cells (21). The expression of these genes was induced in the ALV-J-infected spleen, as confirmed by RT-qPCR (Figure 1C). To verify the effect of these genes on viral replication efficiency, we performed gene knockdown using small interfering RNA (siRNA) in the chicken fibroblast cell line (DF-1), followed by infection with ALV-J for 48 h. The knockdown efficiency was detected by RT-qPCR (Figure 1D). We found that depletion of CREG1 significantly enhanced viral replication, while depletion of ITGAV notably suppressed viral replication. No significant effect on viral replication was observed for the other genes (Figures 1E, F). To further confirm the roles of CREG1 and ITGAV in viral replication, we constructed expression plasmids for Flag-CREG1 and Flag-ITGAV (Figure 1G and Supplementary Figure S2A). The results demonstrated that CREG1 overexpression (OE-CREG1) markedly inhibited viral replication, while ITGAV overexpression markedly promoted viral replication (Figures 1H, I and Supplementary Figures S2B–D). Thus, these results suggest that CREG1 may be a potential antiviral molecule in the course of ALV-J infection.

3.2 CREG1 initiates innate immune responses

To further explore the role of CREG1 in antiviral immune responses, we performed quantitative proteomics analyses on CREG1-overexpressing ALV-J-infected DF-1 cells. Surprisingly, CREG1 induced the expression of a significant number of ISGs, including IFIT5, MX1, ZNFX1, CCL4, ISG12 (2), OASL and others (Figure 2A and Supplementary Figures S3A, B). Strikingly, in this proteomics data, we found that the gene encoding the ALV-J envelope glycoprotein, env, appeared in the downregulated group, further confirming the inhibitory effect of CREG1 on the virus (Figure 2A). We confirmed the induction of certain ISGs in CREG1-overexpressing ALV-J-infected cells using RT-qPCR (Figures 2B, C). Upregulation of ISG expression suggests that CREG1 may have activated the interferon pathway. In fact, in the results of the quantitative protein analysis, we also found that interferon induced with helicase C domain 1 (IFIH1, also known as MDA5) and STING1 were upregulated (Figure 2A). Activation of MDA5 or STING promotes the production of I-IFN, further activating the antiviral immune response (22). To verify this hypothesis, we examined the activation of the interferon pathway after transfecting Flag-CREG1 and subsequently infecting with ALV-J. The results indicated that in DF-1 and HD11 cells (chicken macrophage cell line), OE-CREG1 induced the activation of IFN-β through the cGAS-STING pathway (Figures 2D–F), accompanied by key downstream signaling events, including phosphorylation of Janus kinase (JAK) and signal transducer and activator of transcription 1 (STAT1) (Figures 2G, H and Supplementary Figure S3C). Although the overexpression of CREG1 did not cause differential expression of MDA5 (Supplementary Figure S3D). Interestingly, when we stimulated the cells with I-IFN, the expression of CREG1 did not significantly increase, suggesting that CREG1 is not a classic ISG (Supplementary Figure S3E). Moreover, in the single-cell omics data, CREG1 is highly expressed in the Th1-like cell subpopulation (21). We speculate that CREG1 might also activate the antiviral immune response of T cells. To verify this, we transfected Flag-CREG1 into the chicken T lymphocyte cell line MSB-1, followed by ALV-J infection, and subsequently evaluated the secretion of IFN-γ, interleukin-10 (IL-10), and IL-4. Unexpectedly, the results showed that CREG1 did not effectively activate T cells (Supplementary Figure S3F). Meanwhile, the deletion of CREG1 significantly weakened the production of IFN-β and the expression of ISGs (Supplementary Figures S3G, H). Overall, these data suggest that CREG1 initiates the innate immune responses.

Figure 2

Figure 2. CREG1 triggers innate immune response activation. (A). Overexpression of CREG1 or control in DF-1 cells infected with ALV-J, with proteomic results at 48 hpi. The heatmap shows the results of several key differential proteins, including ISGs and the viral protein env. Z-score normalized log2 (FPKM) values, n = 3. (B, C). Overexpression of CREG1 or control in DF-1 (B) or HD11 (C) cells infected with ALV-J, with RT-qPCR analysis of ISGs mRNA expression at 48 hpi, including IFIT5, ISG12-2, ZNFX1, MX1, OASL, and CCL4. n = 6. (D). Overexpression of CREG1 or control in DF-1 (left) or HD11 (right) cells followed by ALV-J infection, with STING1 mRNA expression levels measured by RT-qPCR at 48 hpi, n = 6. (E, F). CREG1 or control overexpression in DF-1 or HD11 cells, followed by ALV-J infection, with cell supernatants harvested at 48 hpi for cGAMP (E) and IFN-β (F) quantification by enzyme-linked immunosorbent assay (ELISA), n = 6. (G, H). CREG1 or control overexpression in DF-1 followed by ALV-J infection, with western blot analysis at 48 hpi to assess the activation of the I-IFN signaling pathway, including TBK1, P-TBK1 (Ser172), STAT1, P-STAT1 (Ser727), JAK, P-JAK (Tyr1022), and actin. The left panel shows the immunoblotting results (G), while the right panel presents the densitometric analysis of relative protein expression (H). n = 3. (I). DF-1 cells were treated with blank, CREG1 overexpression alone, CREG1 + DMSO, or CREG1 + RU.521 (5 μM RU.521 for 2 h), followed by ALV-J infection. At 48 hpi, western blot was used to measure the viral protein env and actin. The upper panel shows the immunoblotting results, while the lower panel presents the densitometric analysis of relative protein expression. n = 3. (J). DF-1 cells were subjected to blank treatment, CREG1 overexpression alone, CREG1 + FLAG, or CREG1 + SOCS3 co-transfection, followed by ALV-J infection. At 48 hpi, viral envelope protein (env) and actin were assessed using western blot. The upper panel shows the immunoblotting results, while the lower panel presents the densitometric analysis of relative protein expression. n = 3. For (B, F, H, J), the values are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student’s t test.

To further investigate whether the antiviral effect of CREG1 depends on the innate immune responses, we pretreated OE-CREG1 cells with the cGAS inhibitor, RU.521, followed by virus infection. Interestingly, when the type I-IFN response is inhibited, the suppressive effect of CREG1 on viral replication was partially diminished (Figure 2I). Meanwhile, since suppressor of cytokine signaling 3 (SOCS3) negatively regulates the JAK-STAT pathway (23), we found that co-transfection of SOCS3 and CREG1 resulted in a partial attenuation of the antiviral activity of CREG1 (Figure 2J). Therefore, we conclude that the antiviral effect of CREG1 is partially dependent on the innate immune response induced by the cGAS-STING-TBK1 pathway.

3.3 CREG1 induces mitochondrial dysfunction

Next, we begin to investigate the reasons for CREG1 activation of the innate immune response. Mitochondria are not only the energy metabolism factories of the cell, but also, when mitochondrial dysfunction occurs, they release mtDNA, which activate the STING pathway, promote the production of I-IFN, and subsequently initiate innate immune responses and regulate the activity of immune cells (24, 25). Meanwhile, previous reports have identified that CREG1 localizes to mitochondria and regulates the motility of skeletal muscle through mitochondrial autophagy (16). Therefore, we hypothesize that CREG1 may trigger innate immune pathways by affecting mitochondrial function. To this end, we transfected Flag-CREG1 into DF-1 cells and examined its subcellular localization by immunofluorescence, with or without ALV-J infection. However, we found that CREG1 was not localized to mitochondria in chicken cells. Instead, the exogenously introduced CREG1 was mainly distributed in the cytoplasm and nucleus (Supplementary Figure S4A). We then investigated whether the overexpression of CREG1 affects mitochondrial biological function under viral infection conditions. Transmission electron microscopy (TEM) analysis showed that the number of abnormal mitochondria was significantly increased in OE-CREG1 cells (Figures 3A–C). Compared to the control group, OE-CREG1 cells exhibited mitochondrial damage, such as cristae shortening and disintegration, reduced matrix electron density, and mitophagy (Figures 3A–C). Mitochondrial ROS (MitoSOX), mitochondrial membrane potential (TMRE and JC-1 staining), were also assessed in OE-CREG1 DF-1 cells by flow cytometry (Figures 3D–F). The results showed that mitochondrial superoxide levels were significantly reduced in OE-CREG1 cells (Figure 3G). In addition, TMRE and JC-1 assays revealed that CREG1 induction led to a significant decrease in mitochondrial membrane potential (Figures 3H, I). To determine whether mitochondrial dysfunction had occurred, we evaluated key mitochondrial functions such as ATP synthesis and respiratory chain activity. As expected, a reduction in ATP synthesis capacity and respiratory chain activity was observed in OE-CREG1 DF-1 cells compared with the control group (Figures 3J–L). In contrast, the knockdown of CREG1 resulted in effects opposite to those induced by CREG1 overexpression (Figures 3D–L).

Figure 3

Figure 3. CREG1 disrupts mitochondrial integrity and function. (A–C). Transmission electron microscopy (TEM) images of HD11 cells transfected with Flag-CREG1 or control and infected with ALV-J for 48 hpi (A). The lower panels show magnified views of the corresponding upper panels. Green arrows indicate morphologically normal mitochondria; red arrows indicate swollen mitochondria or those with cristae loss; yellow arrows indicate mitophagy. Scale bars: 1.0 μm or 5.0 μm (upper panels), 500 nm or 1.0 μm (lower panels). Quantification of the number of mitochondria (B) and the percentage of damaged mitochondria (C). (D–F). Flow cytometry histograms or dot plots of MitoSOX red (D), TMRE (E), and JC-1 (F) staining in CREG1-overexpressing or knockdown cells and corresponding control groups at 48 h post ALV-J infection. n = 6. (G, H). Quantification of relative fluorescence intensity of MitoSOX red (G) and TMRE staining (H). (I). The bar graph represents the ratio of red fluorescence to green fluorescence. (J–L). After 48 h of ALV-J infection, ATP (J), NADH-CoQ Reductase (K), and Cytochrome C Oxidase (L) levels were measured in CREG1-overexpressing or knockdown cells and the control group, respectively. n = 6. For (B, C, G–L), the values are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student’s t test.

Mitochondrial dysfunction may trigger mitophagy to remove damaged mitochondria. We analyzed the status of mitophagy under in OE-CREG1 or si-CREG1 DF-1 cells. We performed subcellular fractionation of mitochondria and cytoplasm and found that ectopic expression of CREG1 led to an upregulation of Parkin (RBR E3 ubiquitin protein ligase) in the mitochondrial fraction, accompanied by a decrease in the outer membrane protein TOMM20 (Figure 4A). In contrast, Parkin expression in the cytoplasmic fraction was reduced (Figure 4A). Furthermore, analysis of the autophagy-related proteins LC3 and p62 in total cellular lysates revealed that ectopic expression of CREG1 increased LC3-II levels and decreased p62 levels (Figure 4B), whereas siRNA-mediated knockdown of CREG1 produced the opposite effects (Figure 4C). These results suggest that CREG1 may induce mitophagy. To confirm the findings, we further assessed the mitophagy flux in OE-CREG1 cells. Colocalization analysis of GFP-LC3 with MitoTracker Deep Red (MTDR) showed that CREG1 promotes the delivery of LC3B to mitochondria (Supplementary Figure S4B). Taken together, these results indicate that the presence of CREG1 promotes mitochondrial dysfunction and subsequently induces mitophagy.

Figure 4

Figure 4. CREG1 induces mtDNA release into the cytosol and promotes mitophagy. (A). CREG1-overexpressing DF-1 cells were infected with ALV-J and collected at 48 hpi for mitochondrial and cytosolic fractionation. The expression of Parkin, TFAM, TOMM20, and VDAC1 in the mitochondrial fraction, as well as Parkin and TFAM in the cytosolic fraction, was analyzed by western blotting. Immunoblot image (left), statistical grayscale plot (right). n = 3. (B). The expression levels of P62 and LC3-I/II in total cellular proteins from CREG1-overexpressing or CREG1-knockdown cells were analyzed by western blotting. Immunoblot image (left), statistical grayscale plot (right). n = 3. (C). CREG1-knockdown cells were infected with ALV-J and harvested at 48 hpi for mitochondrial and cytosolic fractionation and subsequent western blot analysis. Immunoblot image (left), statistical grayscale plot (right). n = 3. (D, E). Cells overexpressing or silencing CREG1 were infected with ALV-J and subjected to subcellular fractionation. The cytosolic fraction was collected for DNA extraction, and the cytosolic mtDNA levels were quantified by RT-qPCR. n = 6. (F, G). CREG1-overexpressing cells were left untreated or treated with ethidium bromide (EtBr) for 5 (d) Mitochondria and nuclei were labeled with MitoTracker Red and DAPI, respectively (I). Scale bars, 100 µm. RT-qPCR detection of total cellular mtDNA expression levels (J). n = 6. (H). The level of IFN-β in the supernatant of untreated or EtBr-treated CREG1-overexpressing cells was measured by ELISA. n = 6. (I). The expression levels of OASL, IFIT5, and ISG12–2 in untreated or EtBr-treated CREG1-overexpressing cells were quantified by RT-qPCR. n = 6. For (A-E, G, H) the values are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student’s t test.

Notably, upon revisiting the proteomics data, we found that the antiviral response of CREG1 was associated with certain mitochondrial proteins, such as heat shock protein family D (Hsp60) member 1 (HSPD1, also known as cpn60), a mitochondrial chaperonin involved in protein folding. HSPD1 is upregulated in response to cellular stress and may serve as an early indicator of mitochondrial damage (26). Overall, these data indicate that during antiviral responses, CREG1 induces mitochondrial morphological damage, disrupts electron transport, ATP production, and membrane potential, and promotes mitophagy.

Mitochondrial dysfunction triggers innate immune signaling by releasing mtDNA into the cytosol. To investigate whether CREG1 affects the release of mitochondrial mtDNA, we analyzed the accumulation of mtDNA in the cytosol of OE-CREG1 cells. Our results demonstrate that ectopic expression of CREG1 leads to a significant accumulation of mitochondrial genomic DNA in the cytosol, without affecting the overall mitochondrial DNA content (Figures 4D, E, Supplementary Figures S4C, D). Concurrently, an accumulation of mitochondrial transcription factor A (TFAM) was also detected in the cytosol (Figure 4A).

Next, to determine whether the innate immune response observed in OE-CREG1 cells depends on mtDNA, we employed ethidium bromide (EtBr) to deplete mitochondrial nucleic acids. The results showed that EtBr effectively removed mitochondrial nucleic acids from the cells and markedly suppressed the expression of I-IFN and ISGs in CREG1-overexpressing cells (Figures 4F–I). These results suggest that mtDNA in the cytosol may be key mediators of the CREG1-induced innate immune response.

3.4 The pro-apoptotic effect of CREG1 is essential for its antiviral activity

Mitochondria act as a central regulator of multiple cell death pathways. Through the regulation of cytochrome c release, membrane potential, ROS generation, and mitochondrial permeability transition pore (mPTP) opening, they critically determine cell fate (27). We propose that CREG1-induced mitochondrial dysfunction may further trigger programmed cell death during antiviral responses. To this end, we measured intracellular levels of ROS and NO, as well as the release of cytochrome c into the cytosol. The results showed that ectopic expression of CREG1 enhanced ROS production (indicated by increased DCFDA fluorescence) and NO generation (Supplementary Figures S5A, B), and promoted the accumulation of cytosolic cytochrome c (Figures 5A, B). In contrast, knockdown of CREG1 reversed these effects, suggesting that CREG1 may induce cell death (Figures 5C, D). Our initial hypothesis was that CREG1 induced disease-associated inflammatory pyroptosis. Unexpectedly, we did not observe an upregulation of IL-1β and TNF-α in the supernatant of OE-CREG1 cells, suggesting that inflammatory pyroptosis did not occur (Supplementary Figures S5C, D). Subsequently, apoptosis was assessed by flow cytometry. The results demonstrated that ectopic expression of CREG1 significantly enhanced cell apoptosis under viral infection (Figures 5E, F). Consistently, analysis of the apoptosis-related proteins BCL-2, BAX, and caspase-3 further confirmed that CREG1 promotes apoptosis during antiviral responses (Figures 5G, H). Conversely, the absence of CREG1 significantly attenuated apoptosis (Supplementary Figures S5E–G). Taken together, these findings suggest that CREG1 plays a pro-apoptotic role.

Figure 5

Figure 5. CREG1-mediated pro-apoptotic activity contributes to its antiviral function. (A–D). Western blot was performed to detect cytochrome c levels in mitochondrial and cytosolic fractions of CREG1-overexpressing (A, B) or -knockdown cells (C, D) at 48 hpi with ALV-J. n = 3. (E, F). Apoptosis was analyzed by Annexin V/PI staining and flow cytometry in CREG1-overexpressing and control cells infected with ALV-J. (E) Flow cytometry dot plots illustrating apoptotic cell distribution. (F) Statistical analysis of early and late apoptosis. n = 5. (G, H). Western blot analysis of apoptosis-related proteins (BCL2, BAX, and cleaved caspase-3) in CREG1-overexpressing and control cells upon ALV-J infection. Immunoblot images (G) and quantification of relative protein levels (H). n = 3. (I, J). Cells were transfected to overexpress CREG1 for 24 h and then pretreated with Z-VAD-FMK (50 μM) or DMSO for 1 h prior to ALV-J infection. Apoptosis was analyzed by flow cytometry 24 hpi. (I) Representative flow cytometry dot plots. (J) Quantification of early and late apoptotic cells. n = 6. (K, L). CREG1-overexpressing cells were pretreated with Z-VAD-FMK (50 μM) or DMSO for 1 h prior to ALV-J infection following 24 h of transfection. Viral envelope protein env expression was analyzed by Western blot at 24 hpi. Immunoblot images (K) and quantification of relative protein levels (L). n = 3. For (B, D, F, H, J, L) the values are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student’s t test.

Subsequently, to investigate whether the antiviral activity of CREG1 is linked to its pro-apoptotic effect, we treated OE-CREG1 cells with an apoptosis inhibitor, Z-VAD-FMK, prior to ALV-J infection. Treatment with Z-VAD-FMK significantly inhibited CREG1-induced cell apoptosis but had no effect on the activation of I-IFN (Figures 5I, J, Supplementary Figure S5H). Next, we investigated the impact of CREG1 on viral replication when apoptosis was suppressed. The results demonstrated that the antiviral activity of CREG1 was markedly compromised under conditions of apoptosis inhibition (Figures 5K, L).

3.5 CREG1 interacts with HSPD1, and its regulatory effect on mitochondrial function is contingent upon HSPD1

To further investigate the mechanism by which CREG1 regulates mitochondrial dysfunction, we employed co-immunoprecipitation and mass spectrometry to screen for proteins interacting with CREG1 under viral infection (Supplementary Figure S6). Notably, mass spectrometry analysis revealed that many of the potential CREG1-interacting proteins are involved in mitochondrial function, including molecular chaperones (e.g., HSPD1, HSPA9), transporter-related proteins (e.g., SLC25A3, SLC25A4), and structural stability proteins (e.g., PHB, PHB2) (Supplementary Table 4). Previous reports demonstrated an interaction between CREG1 and HSPD1 in mouse skeletal muscle (16). In our previous proteomic analysis, HSPD1 was identified as upregulated. Consistently, western blot analysis confirmed that overexpression of CREG1 led to an increase in HSPD1 expression, whereas knockdown of CREG1 resulted in its downregulation (Supplementary Figures S7A, B). These findings imply a potential interaction between CREG1 and HSPD1. To this end, we transfected Flag-CREG1 alone or co-transfected it with HA-HSPD1 into DF-1 cells to determine whether an interaction between the two proteins occurs in the presence or absence of ALV-J infection. As expected, co-immunoprecipitation assays confirmed that CREG1 interacts with either endogenous HSPD1 or exogenously expressed HA-HSPD1 under both infected and uninfected conditions (Figures 6A, B). Therefore, these findings indicate that CREG1 interacts with HSPD1.

Figure 6

Figure 6. CREG1 interacts with HSPD1, and HSPD1 also exhibits antiviral activity. (A, B). Co-IP analysis of CREG1-HSPD1 interaction with or without ALV-J infection. (C). ALV-J expression was detected by western blot or RT-qPCR at 48 hpi following HSPD1 overexpression. n = 3/6. (D–F). ELISA detection of IFN-β in the supernatant (D), and RT-qPCR analysis of ISGs (OASL, IFIT5, ZNFX1, ISG12-2) expression (E) and cytosolic mtDNA levels (F) in HSPD1-overexpressing cells at 48 hpi with ALV-J. n = 6. (G–L). In ALV-J-infected cells with HSPD1 overexpression or knockdown, the fluorescence intensities of JC1 (G), TMRE (H), and MitoSOX (I) were measured using a microplate reader at 48 hpi. The ratio of red fluorescence to green fluorescence (J). Statistical graphs of relative fluorescence intensities for TMRE (K) and MitoSOX (L). n = 6 (H, I) or n = 4 (J). (M) In ALV-J-infected cells overexpressing HSPD1, western blotting was performed to detect the expression levels of Parkin, TFAM, TOMM20, cytochrome c, and VDAC1 in mitochondrial and cytosolic fractions. n = 3. For (C-F, J-M), the values are shown as the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 by unpaired Student’s t test.

Next, to determine whether HSPD1 has antiviral effects similar to CREG1, we independently assessed its impact on viral replication. The results of viral replication assays showed that HSPD1 overexpression inhibited viral replication, whereas HSPD1 knockdown significantly enhanced viral replication (Figure 6C and Supplementary Figures S7C–E). HSPD1 similarly promotes the expression of I-IFN and ISGs during the antiviral response (Figures 6D, E). In addition, under viral infection conditions, we investigated the effects of HSPD1 on mitochondrial function. Our results demonstrated that ectopic expression of HSPD1 markedly affected multiple aspects of mitochondrial biology, including increased levels of cytosolic mitochondrial DNA (Figure 6F), a reduction in mitochondrial membrane potential, decreased MitoSOX fluorescence (Figures 6G–L), and elevated cytochrome c levels (Figure 6M).

In contrast, knockdown of HSPD1 led to opposite effects compared to its overexpression (Supplementary Figures S7F–J). Although ectopic expression of HSPD1, similar to CREG1, increased cytosolic mtDNA and cytochrome c, its impact on apoptosis during the antiviral response remained minimal, regardless of whether HSPD1 was overexpressed or knocked down (Supplementary Figures S7K, L).

Finally, to determine whether the antiviral effect of CREG1 depends on HSPD1, we co-transfected Flag-CREG1 with either si-HSPD1 or HA-HSPD1, and assessed the effects of CREG1 on viral replication and mitochondrial function under conditions of HSPD1 knockdown or overexpression. The results showed that knockdown of HSPD1 reversed the decrease in mitochondrial membrane potential and the reduction in superoxide levels induced by CREG1 overexpression (Figures 7A–F), but did not affect CREG1-mediated antiviral activity or apoptosis (Figures 7G, H and Supplementary Figures S8A–C). This suggests that the regulation of mitochondrial function by CREG1 is dependent on its interaction with HSPD1. Interestingly, compared with single transfection of either gene, co-expression of CREG1 and HSPD1 produced a stronger antiviral effect, which was attributed to the enhanced induction of I-IFN and ISGs (Supplementary Figures S8D, S7H). Collectively, these data indicate that HSPD1 is required for CREG1-mediated regulation of mitochondrial function, but not for antiviral activity and apoptosis.

Figure 7

Figure 7. HSPD1 is required for CREG1-mediated regulation of mitochondrial function. (A–H). In cells with HSPD1 knockdown and CREG1 overexpression, CREG1 knockdown and HSPD1 overexpression, or co-expression of CREG1 and HSPD1, JC-1 (A), TMRE (C), and MitoSOX (E) fluorescence intensities were measured using a microplate reader at 48 hpi following ALV-J infection. Expression of the viral envelope protein Env was detected by western blot (G). The ratio of red fluorescence to green fluorescence (B). Statistical graphs of relative fluorescence intensities for TMRE (D) and MitoSOX (F). The relative protein quantification chart (H). n = 4 (B, D, F) or n = 3 (H). For (B, D, F, H), the data are presented as mean ± SD. Statistical comparisons were conducted between CREG1 + si-NC vs. CREG1 + si-HSPD1, HSPD1 + si-NC vs. HSPD1 + si-CREG1, and among the CREG1 + Flag, HSPD1 + Flag, and CREG1 + HSPD1 groups. Statistical significance was determined using unpaired Student’s t-test, with ns indicating no significant difference, *p < 0.05, **p < 0.01, and ***p < 0.001.

4 Discussion

Mitochondria orchestrate innate immune defenses against RNA viruses through dynamic regulation of antiviral signaling cascades (8). In this study, we identified CREG1 as a novel antiviral gene. The antiviral activity of CREG1 primarily derives from its regulation of mitochondrial function. Overexpression of CREG1 leads to a decrease in mitochondrial membrane potential, triggers mitophagy, reduces ATP production and respiratory chain activity, increases ROS generation, and promotes the release of mtDNA and cytochrome c into the cytoplasm. On one hand, the release of mtDNA into the cytoplasm activates the cGAS-STING signaling pathway, resulting in increased I-IFN production and upregulation of interferon-stimulated genes. On the other hand, the elevated intracellular levels of ROS and the release of cytochrome c into the cytoplasm, which induce apoptosis, also exert detrimental effects on viral replication. Importantly, the regulatory effect of CREG1 on mitochondrial function is mediated through its interaction with the mitochondrial chaperone HSPD1. The co-expression of CREG1 and HSPD1 confers enhanced antiviral activity.

A striking observation in our study is that CREG1-induced mitochondrial dysfunction triggers activation of the cGAS-STING signaling pathway and enhances innate immune responses via cytosolic release of mtDNA. Meanwhile, the activation of the cGAS-STING pathway in suppressing ALV-J replication was also an unexpected finding in our study. In general, the innate immune response mediated by the cGAS-STING pathway predominantly detects double-stranded DNA (dsDNA) originating from DNA viruses. However, RNA viruses can also antagonize cGAS-STING. For example, SARS-CoV-2 3CL protease disrupts the assembly of the functional STING signaling complex and downstream signaling by inhibiting K63-linked ubiquitination of STING (28). However, the precise role of the cGAS-STING signaling pathway during ALV infection remains unclear. For instance, it is not known whether the lack of cGAS-STING activation is due to active antagonism by ALV viral proteins, similar to the mechanism observed in SARS-CoV-2 infection. In this study, activation of the cGAS-STING signaling pathway was found to influence ALV replication to a certain extent. Given the central role of innate immune responses in restricting ALV-J replication (29, 30), modulation of the cGAS-STING signaling pathway holds promise as a potential intervention strategy to suppress ALV infection.

Viruses often reprogram mitochondrial respiratory activity and apoptotic signaling to create a cellular environment conducive to their replication and persistence (6). Following SARS-CoV-2 infection, mitochondrial morphology undergoes significant alterations, characterized by matrix condensation and cristae swelling, which are associated with reduced oxidative phosphorylation (OXPHOS) polypeptides, impaired import of inner mitochondrial membrane proteins, increased mitochondrial reactive oxygen species (mROS) production, and suppression of core mitochondrial gene expression (31). However, studies on the interaction between ALV and mitochondria during infection remain limited. ALV-J is a retrovirus capable of inducing tumorigenesis in chicken tissues. We speculate that mitochondrial activity is inevitably involved in the course of viral infection, as tumorigenesis is often accompanied by mitochondrial metabolic dysfunction. Such alterations include impaired OXPHOS and elevated mROS production. The accumulation of ROS can lead to DNA damage and genomic instability, and subsequently activate oncogenic signaling pathways such as NF-κB and MAPK, thereby facilitating virus-mediated cellular transformation and tumor development (32). Previous reports have indicated that ALV-J can activate signaling cascades such as PI3K/Akt, Wnt/β-catenin, and ERK/MAPK, which directly or indirectly regulate cell apoptosis, thereby promoting viral replication (33). Moreover, ALV replicates slowly and establishes long-term latency within host cells, relying on the survival of infected cells to support viral replication or reactivation. Thus, the inhibition of apoptosis is essential for efficient ALV replication. In this study, CREG1 induced robust apoptosis by promoting mitochondrial dysfunction, which led to the release of cytochrome c into the cytosol. Notably, treatment with an apoptosis inhibitor partially attenuated the antiviral effect of CREG1. Future studies should further explore the interplay between ALV infection, mitochondrial function, and apoptosis, which may provide new strategies for combating ALV.

Whether through activation of the cGAS-STING signaling pathway or induction of apoptosis to initiate cellular immunity, the antiviral activity of CREG1 stems from its regulation of mitochondrial function. Although CREG1 has been reported to localize to mitochondria (16), our results indicate that it is primarily a cytoplasmic or nuclear protein. Its ability to modulate mitochondrial function fundamentally relies on its interaction with the mitochondrial chaperone protein HSPD1. As a mitochondrial chaperone, HSPD1 assists in the import and proper folding of proteins within mitochondria, playing a critical role in metabolic reprogramming (34). In this study, similar to CREG1, overexpression of HSPD1 also exhibited the ability to inhibit ALV-J replication, primarily by promoting the expression of IFN-β and ISGs. Importantly, previous studies have reported that HSPD1 interacts with IRF3 to promote the induction of IFN-β (35). Our results indicate that HSPD1 overexpression leads to mitochondrial dysfunction, which in turn promotes IFN-β expression. Nevertheless, whether HSPD1 interacts with members of the IRF family in this context remains to be elucidated.

Furthermore, knockdown of HSPD1 reversed mitochondrial dysfunction induced by CREG1, clearly demonstrating that the regulation of mitochondrial function by CREG1 depends on its interaction with HSPD1. Regrettably, despite the dependence of mitochondrial regulation by CREG1 on HSPD1, the underlying mechanism explaining why the effect of CREG1 on apoptosis remains largely unaffected following HSPD1 knockdown remains unclear. We speculate that CREG1 may regulate apoptosis by influencing other mitochondria-related proteins, as our Co-IP analysis of potential CREG1-interacting proteins identified multiple mitochondrial-associated proteins, such as SLC25A4. In a recent report, SLC25A4 was identified as a novel regulator of mitochondrial dysfunction and apoptosis-related cardiomyocyte subpopulations. Moreover, downregulation of SLC25A4 effectively improved mitochondrial function and reduced apoptosis (36). These observations suggest that CREG1 may trigger apoptosis through a broader mitochondrial regulatory network beyond its interaction with HSPD1.

Finally, we were surprised to find that co-expression of CREG1 and HSPD1 resulted in a more potent antiviral effect, accompanied by higher expression levels of IFN-β and ISGs. Interestingly, a previous study using a lentiviral library expressing 383 individual ISGs identified 69 ISGs that synergized with ZAP to exert antiviral effects. Further analyses demonstrated that 31 of these ISGs exhibited significant synergy with ZAP, and quantitative data showed that co-expression of these ISGs with ZAP led to a substantial reduction in viral infection rates, markedly outperforming the effect of either factor alone (37). Previous studies have demonstrated the antiviral effects of multiple host genes, such as ACSL1 (38), CH25H (39), and TRIM45 (40), and the potential synergistic interactions among these antiviral effectors in enhancing host defense warrant further investigation.

5 Conclusions

In summary, our study reveals CREG1 as a novel antiviral gene. By interacting with HSPD1, CREG1 regulates mitochondrial dysfunction to trigger both I-IFN activation and apoptosis, thereby suppressing viral replication (Figure 8). We propose that further investigation into the crosstalk among viral infection, mitochondrial function, innate immune responses, and apoptotic pathways may provide new strategies for controlling ALV-J.

Figure 8

Figure 8. Diagram of the antiviral mechanism of CREG1.

Data availability statement

The data presented in the study are deposited in the GSA at the National Genomics Data Center, provisional accession number is [subCRA060703].

Ethics statement

The animal study was approved by South China Agriculture University Institutional Animal Care and Use Committee. The study was conducted in accordance with the local legislation and institutional requirements.

Author contributions

QZ: Writing – review & editing, Writing – original draft, Conceptualization, Project administration, Data curation. MW: Investigation, Data curation, Resources, Writing – original draft. MP: Data curation, Formal Analysis, Writing – original draft, Investigation. JX: Investigation, Resources, Methodology, Writing – original draft. TX: Writing – original draft, Conceptualization, Resources. WL: Writing – review & editing, Funding acquisition. XZ: Funding acquisition, Writing – review & editing.

Funding

The author(s) declared that financial support was received for this work and/or its publication. This work was supported by the National Natural Science Foundation of China (grant numbers 31801030 and 31571269), the China Agriculture Research System (grant number CARS-41).

Conflict of interest

The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Generative AI statement

The author(s) declared that generative AI was not used in the creation of this manuscript.

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Supplementary material

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fimmu.2025.1760120/full#supplementary-material

References

1. Feng M, Xie T, Li Y, Zhang N, Lu Q, Zhou Y, et al. A balanced game: chicken macrophage response to ALV-J infection. Vet Res. (2019) 50:20. doi: 10.1186/s13567-019-0638-y

PubMed Abstract | Crossref Full Text | Google Scholar

2. Feng M, Dai M, Xie T, Li Z, Shi M, and Zhang X. Innate immune responses in ALV-J infected chicks and chickens with hemangioma in vivo. Front Microbiol. (2016) 7:786. doi: 10.3389/fmicb.2016.00786

PubMed Abstract | Crossref Full Text | Google Scholar

3. Dou W, Li H, Cheng Z, Zhao P, Liu J, Cui Z, et al. Maternal antibody induced by recombinant gp85 protein vaccine adjuvanted with cpG-ODN protects against ALV-J early infection in chickens. Vaccine. (2013) 31:6144–9. doi: 10.1016/j.vaccine.2013.06.058

PubMed Abstract | Crossref Full Text | Google Scholar

4. Zhang L, Cai D, Zhao X, Cheng Z, Guo H, Qi C, et al. Liposomes containing recombinant gp85 protein vaccine against ALV-J in chickens. Vaccine. (2014) 32:2452–6. doi: 10.1016/j.vaccine.2014.02.091

PubMed Abstract | Crossref Full Text | Google Scholar

5. Nunnari J and Suomalainen A. Mitochondria: in sickness and in health. Cell. (2012) 148:1145–59. doi: 10.1016/j.cell.2012.02.035

PubMed Abstract | Crossref Full Text | Google Scholar

6. Spinelli JB and Haigis MC. The multifaceted contributions of mitochondria to cellular metabolism. Nat Cell Biol. (2018) 20:745–54. doi: 10.1038/s41556-018-0124-1

PubMed Abstract | Crossref Full Text | Google Scholar

7. Sun L, Wu J, Du F, Chen X, and Chen ZJ. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science. (2013) 339:786–91. doi: 10.1126/science.1232458

PubMed Abstract | Crossref Full Text | Google Scholar

8. Foo J, Bellot G, Pervaiz S, and Alonso S. Mitochondria-mediated oxidative stress during viral infection. Trends Microbiol. (2022) 30:679–92. doi: 10.1016/j.tim.2021.12.011

PubMed Abstract | Crossref Full Text | Google Scholar

9. Orzalli MH and Kagan JC. Apoptosis and necroptosis as host defense strategies to prevent viral infection. Trends Cell Biol. (2017) 27:800–9. doi: 10.1016/j.tcb.2017.05.007

PubMed Abstract | Crossref Full Text | Google Scholar

10. Czabotar PE and Garcia-Saez AJ. Mechanisms of BCL-2 family proteins in mitochondrial apoptosis. Nat Rev Mol Cell Biol. (2023) 24:732–48. doi: 10.1038/s41580-023-00629-4

PubMed Abstract | Crossref Full Text | Google Scholar

11. McArthur K, Whitehead LW, Heddleston JM, Li L, Padman BS, Oorschot V, et al. BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science. (2018) 359:6378. doi: 10.1126/science.aao6047

PubMed Abstract | Crossref Full Text | Google Scholar

12. Veal E, Eisenstein M, Tseng ZH, and Gill G. A cellular repressor of E1A-stimulated genes that inhibits activation by E2F. Mol Cell Biol. (1998) 18:5032–41. doi: 10.1128/mcb.18.9.5032

PubMed Abstract | Crossref Full Text | Google Scholar

13. Song H, Yan C, Tian X, Zhu N, Li Y, Liu D, et al. CREG protects from myocardial ischemia/reperfusion injury by regulating myocardial autophagy and apoptosis. Biochim Biophys Acta Mol Basis Dis. (2017) 1863:1893–903. doi: 10.1016/j.bbadis.2016.11.015

PubMed Abstract | Crossref Full Text | Google Scholar

14. Liu D, Tian X, Liu Y, Song H, Cheng X, Zhang X, et al. CREG ameliorates the phenotypic switching of cardiac fibroblasts after myocardial infarction via modulation of CDC42. Cell Death Dis. (2021) 12:355. doi: 10.1038/s41419-021-03623-w

PubMed Abstract | Crossref Full Text | Google Scholar

15. Liu D, Xing R, Zhang Q, Tian X, Qi Y, Song H, et al. The CREG1-FBXO27-LAMP2 axis alleviates diabetic cardiomyopathy by promoting autophagy in cardiomyocytes. Exp Mol Med. (2023) 55:2025–38. doi: 10.1038/s12276-023-01081-2

PubMed Abstract | Crossref Full Text | Google Scholar

16. Song H, Tian X, Liu D, Liu M, Liu Y, Liu J, et al. Creg1 improves the capacity of the skeletal muscle response to exercise endurance via modulation of mitophagy. Autophagy. (2021) 17:4102–18. doi: 10.1080/15548627.2021.1904488

PubMed Abstract | Crossref Full Text | Google Scholar

17. Zhang Q, Mo G, Xie T, Zhang Z, Fu H, Wei P, et al. Phylogenetic analysis of ALV-J associated with immune responses in yellow chicken flocks in South China. Mediators Inflammation. (2021) 2021:6665871. doi: 10.1155/2021/6665871

PubMed Abstract | Crossref Full Text | Google Scholar

18. Lee SH, Jung JH, Park TK, Moon CE, Han K, Lee J, et al. Proteome alterations in the aqueous humor reflect structural and functional phenotypes in patients with advanced normal-tension glaucoma. Sci Rep. (2022) 12:1221. doi: 10.1038/s41598-022-05273-0

PubMed Abstract | Crossref Full Text | Google Scholar

19. Hashiguchi K and Zhang-Akiyama QM. Establishment of human cell lines lacking mitochondrial DNA. Methods Mol Biol. (2009) 554:383–91. doi: 10.1007/978-1-59745-521-3_23

PubMed Abstract | Crossref Full Text | Google Scholar

20. Liu X and Fagotto F. A method to separate nuclear, cytosolic, and membrane-associated signaling molecules in cultured cells. Sci Signal. (2011) 4:pl2. doi: 10.1126/scisignal.2002373

PubMed Abstract | Crossref Full Text | Google Scholar

21. Qu X, Li X, Li Z, Liao M, and Dai M. Chicken peripheral blood mononuclear cells response to avian leukosis virus subgroup J infection assessed by single-cell RNA sequencing. Front Microbiol. (2022) 13:800618. doi: 10.3389/fmicb.2022.800618

PubMed Abstract | Crossref Full Text | Google Scholar

22. Vasiyani H, Mane M, Rana K, Shinde A, Roy M, Singh J, et al. DNA damage induces STING mediated IL-6-STAT3 survival pathway in triple-negative breast cancer cells and decreased survival of breast cancer patients. Apoptosis. (2022) 27:961–78. doi: 10.1007/s10495-022-01763-8

PubMed Abstract | Crossref Full Text | Google Scholar

23. Mo G, Fu H, Hu B, Zhang Q, Xian M, Zhang Z, et al. SOCS3 promotes ALV-J virus replication via inhibiting JAK2/STAT3 phosphorylation during infection. Front Cell Infect Microbiol. (2021) 11:748795. doi: 10.3389/fcimb.2021.748795

PubMed Abstract | Crossref Full Text | Google Scholar

24. Killarney ST, Washart R, Soderquist RS, Hoj JP, Lebhar J, Lin KH, et al. Executioner caspases restrict mitochondrial RNA-driven type I IFN induction during chemotherapy-induced apoptosis. Nat Commun. (2023) 14:1399. doi: 10.1038/s41467-023-37146-z

PubMed Abstract | Crossref Full Text | Google Scholar

25. Glover HL, Schreiner A, Dewson G, and Tait SWG. Mitochondria and cell death. Nat Cell Biol. (2024) 26:1434–46. doi: 10.1038/s41556-024-01429-4

PubMed Abstract | Crossref Full Text | Google Scholar

26. Kumar R, Chaudhary AK, Woytash J, Inigo JR, Gokhale AA, Bshara W, et al. A mitochondrial unfolded protein response inhibitor suppresses prostate cancer growth in mice via HSP60. J Clin Invest. (2022) 132:13. doi: 10.1172/jci149906

PubMed Abstract | Crossref Full Text | Google Scholar

27. Lartigue L, Kushnareva Y, Seong Y, Lin H, Faustin B, and Newmeyer DD. Caspase-independent mitochondrial cell death results from loss of respiration, not cytotoxic protein release. Mol Biol Cell. (2009) 20:4871–84. doi: 10.1091/mbc.e09-07-0649

PubMed Abstract | Crossref Full Text | Google Scholar

28. Rui Y, Su J, Shen S, Hu Y, Huang D, Zheng W, et al. Unique and complementary suppression of cGAS-STING and RNA sensing- triggered innate immune responses by SARS-cov-2 proteins. Signal Transduct Target Ther. (2021) 6:123. doi: 10.1038/s41392-021-00515-5

PubMed Abstract | Crossref Full Text | Google Scholar

29. Chen S, Wang J, Wang Q, Ping Y, Yang T, Ma K, et al. Autophagy-mediated TET2 degradation by ALV-J env protein suppresses innate immune activation to promote viral replication. J Virol. (2025) 99:e02003–24. doi: 10.1128/jvi.02003-24

PubMed Abstract | Crossref Full Text | Google Scholar

30. Chen S, Wang D, Liu Y, Zhao R, Wu T, Hu X, et al. Targeting the histone methyltransferase disruptor of telomeric silencing 1-like restricts avian leukosis virus subgroup J replication by restoring the innate immune response in chicken macrophages. Front Microbiol. (2020) 11:603131. doi: 10.3389/fmicb.2020.603131

PubMed Abstract | Crossref Full Text | Google Scholar

31. Cortese M, Lee JY, Cerikan B, Neufeldt CJ, Oorschot VMJ, Köhrer S, et al. Integrative imaging reveals sars-cov-2-induced reshaping of subcellular morphologies. Cell Host Microbe. (2020) 28:853–66.e5. doi: 10.1016/j.chom.2020.11.003

PubMed Abstract | Crossref Full Text | Google Scholar

32. Zong WX, Rabinowitz JD, and White E. Mitochondria and cancer. Mol Cell. (2016) 61:667–76. doi: 10.1016/j.molcel.2016.02.011

PubMed Abstract | Crossref Full Text | Google Scholar

33. Tang S, Li J, Chang YF, and Lin W. Avian leucosis virus-host interaction: the involvement of host factors in viral replication. Front Immunol. (2022) 13:907287. doi: 10.3389/fimmu.2022.907287

PubMed Abstract | Crossref Full Text | Google Scholar

34. Parma B, Wurdak H, and Ceppi P. Harnessing mitochondrial metabolism and drug resistance in non-small cell lung cancer and beyond by blocking heat-shock proteins. Drug Resist Update. (2022) 65:100888. doi: 10.1016/j.drup.2022.100888

PubMed Abstract | Crossref Full Text | Google Scholar

35. Lin L, Pan S, Zhao J, Liu C, Wang P, Fu L, et al. HSPD1 interacts with IRF3 to facilitate interferon-beta induction. PloS One. (2014) 9:e114874. doi: 10.1371/journal.pone.0114874

PubMed Abstract | Crossref Full Text | Google Scholar

36. Zhou T, Pan J, Xu K, Yan C, Yuan J, Song H, et al. Single-cell transcriptomics in MI identify slc25a4 as a new modulator of mitochondrial malfunction and apoptosis-associated cardiomyocyte subcluster. Sci Rep. (2024) 14:9274. doi: 10.1038/s41598-024-59975-8

PubMed Abstract | Crossref Full Text | Google Scholar

37. Karki S, Li MM, Schoggins JW, Tian S, Rice CM, and MacDonald MR. Multiple interferon stimulated genes synergize with the zinc finger antiviral protein to mediate anti-alphavirus activity. PloS One. (2012) 7:e37398. doi: 10.1371/journal.pone.0037398

PubMed Abstract | Crossref Full Text | Google Scholar

38. Zhang Q, Xie T, Mo G, Zhang Z, Lin L, and Zhang X. Acsl1 inhibits ALV-J replication by IFN-I Signaling and PI3K/AKT pathway. Front Immunol. (2021) 12:774323. doi: 10.3389/fimmu.2021.774323

PubMed Abstract | Crossref Full Text | Google Scholar

39. Xie T, Feng M, Zhang X, Li X, Mo G, Shi M, et al. Chicken CH25H inhibits ALV-J replication by promoting cellular autophagy. Front Immunol. (2023) 14:1093289. doi: 10.3389/fimmu.2023.1093289

PubMed Abstract | Crossref Full Text | Google Scholar

40. Wang J, Wang Q, Ping Y, Huang X, Yang T, Bi Y, et al. Identification and characterization of chicken TRIM45 and its role as a negative regulator of ALV-J replication in vitro. Avian Pathol. (2025) 54:255–64. doi: 10.1080/03079457.2024.2419039

PubMed Abstract | Crossref Full Text | Google Scholar